Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factor...Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.展开更多
BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high dose...BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high doses of drug are required and the success rate of model induction is low. It is necessary to develop an improved method to establish a temporal lobe epilepsy （TLE） animal model. OBJECTIVE： To explore an economic, stable and efficient method of establishing a TLE animal model. DESIGN, TIME AND SETTING： A completely randomized, controlled study. The experiments were performed in the Cellular Function Laboratory of the Physiology Department, Anhui Medical University from March to July 2007. MATERIALS： Twenty adult male Wistar rats, weighing 230-260 g, were provided by the Experimental Animal Centre of Nanjing Medical University. Kainic acid was purchased from Sigma in USA. Type SN-2 stereotaxic apparatus was made by Narishge in Japan. METHODS： Wistar rats were randomly divided into a kainic acid （KA） group （n = 12） and a normal saline （NS） group （n = 8）. For intrahippocampal microinjection, a burr hole was drilled in the skull at the following stereotaxic coordinates： anteroposterior （AP） 4.1 mm caudal to bregma; lateral （ML） 4.2 mm right lateral to the midline. Rats in the KA group were injected with 2.5 μL KA （0.4 g/L） into the center of the CA3 region, while in the NS group the same volume of NS was injected into the same site. MAIN OUTCOME MEASURES： Both groups were monitored under a video capture system for 12 weeks to record spontaneous seizures. Intracranial eletroencepholograph （IEEG） recordings in vivo were performed after the behavioral observations. After the IEEG recordings, hippocampi were processed into coronal sections. Nissl and Timm stainings were then performed to observe and confirm pathology. RESULTS： Twenty rats were involved in the final analysis. Behavioral observations： the eadiest spontaneous onset of epilepsy appeared 2 weeks after injection of KA. Eight rats had spontaneous onset of epilepsy 3-12 weeks after treatment. None of rats in the NS group had spontaneous onset of epilepsy. IEEG recordings： Epileptic-form waves, such as sharp waves and spike waves, were calculated by artificial analysis The number of epileptic-form waves in the KA group increased significantly compared to those of the NS group （P 〈 0.01）. Morphology results： In the KA group, Nissl staining and Timm staining revealed typical pathology in the hippocampal temporosphenoid lobe. In the NS group, no pathology was observed. CONCLUSION： Intrahippocampal microinjection of KA is a reliable method to establish a temporal lobe epilepsy animal model, requiring low doses of kainic acid and giving a high rate of success.展开更多
The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we...The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.展开更多
Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (onl...Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.展开更多
The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select b...The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.展开更多
Daidzein (DA) was microinjected into mediobasal hypothalamus (MBH) and nucleus ventrome-dialis thalami (VM) of the castrated male Goettingen Mini pigs (n = 9, 10 μl·animal-1, 8 pgDA·μl-1 and plasma luteini...Daidzein (DA) was microinjected into mediobasal hypothalamus (MBH) and nucleus ventrome-dialis thalami (VM) of the castrated male Goettingen Mini pigs (n = 9, 10 μl·animal-1, 8 pgDA·μl-1 and plasma luteinizing hormone (LH) levels were compared between the prior and the posterior treatment. The result showed that LH levels after the cerebral administration (ad) tended to increase compared to the levels before ad. In MBH, LH levels in 4 cases (4/5), rose and were not changed in 1 case at 0.5 - 2 hours after ad compared to those before ad. There were no significant changes at 2.5 hours after ad. When it was injected in VM, LH levels in 3 cases (3/4) rose, and were not changed in 1 case after ad compared to those before ad. In the control, there were no changes in plasma LH levels between the pre-and post-treatment except 1 case in MBH. This study suggested that DA could up-regulate LH secretion through hypothalamus level in the castrated boars.展开更多
Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard met...Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.展开更多
The microinjection of Zebrafish embryos is significant to life science and biomedical research.In this article,a novel automated system is developed for cell microinjection.A sophisticated microfluidic chip is designe...The microinjection of Zebrafish embryos is significant to life science and biomedical research.In this article,a novel automated system is developed for cell microinjection.A sophisticated microfluidic chip is designed to transport,hold,and inject cells continuously.For the first time,a microinjector with microforce perception is proposed and integrated within the enclosed microfluidic chip to judge whether cells have been successfully punctured.The deep learning model is employed to detect the yolk center of zebrafish embryos and locate the position of the injection needle within the yolk,which enables enhancing the precision of cell injection.A prototype is fabricated to achieve automatic batch microinjection.Experimental results demonstrated that the injection efficiency is about 20 seconds per cell.Cell puncture success rate and cell survival rate are 100%and 84%,respectively.Compared to manual operation,this proposed system improves cell operation efficiency and cell survival rate.The proposed microinjection system has the potential to greatly reduce the workload of the experimenters and shorten the relevant study period.展开更多
infection model for studying inflammation and the innate immune response.We investigated the genes and signaling pathways activated by static immersion and caudal vein microinjection infection using transcriptome prof...infection model for studying inflammation and the innate immune response.We investigated the genes and signaling pathways activated by static immersion and caudal vein microinjection infection using transcriptome profiling and reverse-transcription quantitative PCR(RT-qPCR)to compare the innate immune response in 3 days post-fertilization(dpf)zebrafish larvae infected by Vibrio parahaemolyticus Vp13 strain.The median lethal dose(LD50)values at 96 h following immersion and microinjection were 3.63×107 CFU/mL and 5.76×102 CFU/nL,respectively.An innate immune response was initiated after 2 h of incubation with the respective LD50 for each infection method.Six hundred and two genes in the immersion group and 359 genes in the microinjection group were activated and differentially expressed post-infection.Sixty-three Gene Ontology(GO)terms and four Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were significantly enriched in the immersion group,compared with only three GO terms and no KEGG pathways in the microinjection group.Two genes,tnfb and ccl20a.3,were significantly up-regulated in both groups.We speculated that immersion infection may affect initial dorsal determination,cytochromes,and fatty acid-binding proteins,as well as inflammation,while microinjection infection may mainly directly affect the immune response.Infection with doses>LD50(1.09×109 CFU/mL and 1.09×103 CFU/nL by immersion and microinjection,respectively)caused more significant up-regulation of il11a,tnfa,tnfb,il1b,ccl34a.4,ccl20a.3,irak3,cxcl18b,and ccl35.1,suggesting that in addition to the classical innate immunity genes tnfa,tnfb,il1b,and il6,the genes il11a,ccl34a.4,ccl20a.3,cxcl18b,and ccl35.1 were also important for defending against Vp13 infection.These findings highlight the genes involved in the responses of zebrafish to Vp13 infection via different routes and doses,and thus provide the basis for further analyses of immune response signaling pathways.展开更多
Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods S...Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods Superovulation and synchronization of estrus were induced in female Japanese White Rabbits by injecting hormone, then mating with male. After collected the fertilized eggs, the human apoA-Ⅱ gene was microinjected into the male pronucleus of eggs. The injected eggs were transferred into recipient female rabbits. Last, extract DNA from the new borns ear and determine whether the newborns were transgenic by polymerase chain reaction (PCR) or Southern blot analysis. Results A total of 822 embryos with microinjection of human apoAⅡ gene were implanted into 28 recipient rabbits. The number of surviving newborns was 37. 3 transgenic positive surviving founders were found with human apoA-Ⅱ.展开更多
Polyunsaturated fatty acids （PUFAs） are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease a...Polyunsaturated fatty acids （PUFAs） are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease and diabetes. In this study, we aimed to investigate whether the sFat-1 gene from Caenorhabditis briggsae could be functionally expressed in transgenic pigs, and whether the transgenic could synthesize high quality ω-3 PUFAs endogenously. In this study, a gene construct consisting of CMV promoter and 1.9 kb cDNA of ω-3 fatty acid desaturase gene （sFat-1） from C. briggsae was injected into the male pronucleus of pig embryos by microinjection. The piglets were screened for the transgene by PCR, Southern blot and reverse transcription-PCR analysis. Pigs that give positive results were mated with wild-type pigs to produce the next generation and the transmission of transgene was examined by PCR analysis. Fatty acids compositions of various tissues in the transgenic pigs were then analyzed by gas chromatograph. A total of 878 embryos were transferred into 42 recipients, among which 29 successfully got pregnant and gave birth to a total of 162 piglets, and 8 of them were identified to be transgenic. Fatty acid compositions in the transgenic pigs were altered, and the levels of ω-6：ω-3 ratios were decreased from 14.53 in the control to 2.62 in Fat-1 transgenic pigs. A number of primary sFat-1-transgenic pigs were bred in this study, which lays the foundation for cultivation of new varieties of transgenic pigs.展开更多
The deleterious effect of vasculature damage on the outcome of spinal cord injury has long been recognized, and numerous clinical studies have shown that the presence of hemorrhage into the spinal cord is directly ass...The deleterious effect of vasculature damage on the outcome of spinal cord injury has long been recognized, and numerous clinical studies have shown that the presence of hemorrhage into the spinal cord is directly associated with a poorer neurological outcome. Vascular damage leads to de- creased blood flow to the cord and the release of potentially toxic blood-borne components. Here we consider the mechanisms that may be contributing to hemorrhage-induced damage and discuss the utility of a new model of spinal cord hemorrhage, which was urgently required as most of our current understanding has been extrapolated from intracerebral hemorrhage studies.展开更多
Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to inf...Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/ or openers of these ion channels could interfere with sperm function. In this study, in vivo electmporation （EP） and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower （P〈0.05） hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca2＋ peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hvoeractivation.展开更多
Over the past few decades, the determination of antioxidant activity by chemical probe has been widely reported, but in vivo evaluation via model organisms of Drosophila melanogaster and rapid discovery system has not...Over the past few decades, the determination of antioxidant activity by chemical probe has been widely reported, but in vivo evaluation via model organisms of Drosophila melanogaster and rapid discovery system has not been studied adequately. In this study, we determined the antioxidant activity of lees and demonstrated the ability of compounds in lees to scavenge H_2O_2 in vitro and in vivo by different chemical probes. Lees increased the ability of Drosophila against oxidative stress and antioxidant enzyme activity in vivo. Five ingredients of organic acids and flavones in lees extract that rapidly scavenged H_2O_2 were revealed by a post-column-derived HPLC-UV-FLD system based on 4-hydroxyphenylacetic acid（PHPAA）chemiluminescence. Additionally, another fluorescent probe,N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine（NBCD）, was selected to evaluate the reactive oxygen species（ROS） scavenging capacity of lees in living Drosophila melanogaster using a microfluidic injection test coupled with microscopic imaging analysis, and similar effects were observed in flies when they were treated with tartaric acid and caffeic acid. The results demonstrated that the novel integrated system was suitable for screening and evaluating antioxidant ingredients from natural products.展开更多
INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For...INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).展开更多
The globus pallidus is the relay nucleus of the basal ganglia,and changes in its electrical activity can cause motor impairment.Apelin-13 is widely distributed in the central and peripheral nervous systems.It has been...The globus pallidus is the relay nucleus of the basal ganglia,and changes in its electrical activity can cause motor impairment.Apelin-13 is widely distributed in the central and peripheral nervous systems.It has been demonstrated that apelin-13 plays important roles in the regulation of blood pressure and other non-motor functions.However,its role in motor function has rarely been reported.In the present study,apelin-13(10μM/100μM)was injected into the globus pallidus of rats.The results showed that apelin-13 increased the spontaneous discharges in the majority of pallidal neurons.However,an apelin-13-induced inhibitory effect on the firing rate was also observed in a few pallidal neurons.In postural tests,after the systemic administration of haloperidol,unilateral pallidal injection of apelin-13 caused a contralateral deflection.Together,these findings suggest that apelin-13 regulates the electrical activity of pallidal neurons and thus participates in central motor control in rats.The study was approved by the Animal Ethics Committee of Qingdao University(approval No.20200615Wistar0451003020)on June 15,2020.展开更多
Aim: To observe the effect of electroacupuncture (EA) on acute myocardial ischemia (AMI) induced changes of cardiac sympathetic discharges and the effects of some related receptors in the spinal cord. Methods: A total...Aim: To observe the effect of electroacupuncture (EA) on acute myocardial ischemia (AMI) induced changes of cardiac sympathetic discharges and the effects of some related receptors in the spinal cord. Methods: A total of 53 rabbits anesthetized with mixture solution of 25% urethane (420 mg/kg) and 1.5% chloralose (50 mg/kg) were used in this study. AMI was induced by occlusion of the ventricular branch of the left coronary artery. Discharges of the left cardiac sympathetic nerve were recorded by using a bipolar platinum electrode. Bilateral "Ximen"(PC 40) and "Kongzhui"(LU 6) were stimulated electrically by using an EA therapeutic apparatus or an electrical stimulator. DPDPE δ opiate receptor agonist, 20 nmol, 10 μL, n=8), Naltrindole Hydrochloride (δ opiate receptor antagonist, 20 nmol, 10 μL, n=8), DAP5 (NMDA receptor antagonist, 5 nmol, 10 μL, n=9) and CNQX (non NMDA receptor antagonist, 5 nmol, 10 μL, n=8) were respectively injected into the thoracic subarachnoid space of the spinal cord in different groups, followed by observing their effects on changes of sympathetic activity evoked by EA of the abovementioned acupoints. Results: ① After AMI, sympathetic discharges increased (200.56±79.89%) in 10 cases and decreased (-59.34 ±7.06%) in other 9 cases in comparison with their individual basal values. After EA of "Ximen" (PC 4) and "Kongzhui"(LU 6), AMI induced increase and decrease changes of the sympathetic activity were suppressed significantly, but the effect of EA of LU 6 was weaker than that of EA of PC 4. ② Following EA of PC 4 and LU 6, sympathetic discharges increased significantly in 2 and 4 cases, decreased apparently in 7 and 3 cases, and had no striking changes in 1 and 3 cases respectively. The mean reaction threshold of sympathetic activity after EA of PC 4 and LU 6 were 2.1±0.65 mA and 3.28±1.13 mA separately. ③ After pre treatment with DPDPE, the reaction threshold of the cardiac sympathetic activity to EA of PC 4 elevated significantly (35.89±6.12%); while after pre treatment with Naltrindole, this reaction threshold decreased considerably (84.88±26.58%). Following intrathecal injection of DAP5 (n=9) and CNQX (n=9) , the reaction thresholds of the cardiac sympathetic activity to EA of PC 4 increased obviously (142.06±60.27% and 112.54±28.58% separately). It suggests that spinal δ opioid receptor, NMDA and non NMDA receptors are involved in EA induced changes of sympathetic activity. Conclusion: ① EA could regulate AMI induced changes of cardiac sympathetic activity; and ② spinal δ opioid receptors, NMDA and non NMDA receptors participate in the effect of EA on the cardiac sympathetic activity.展开更多
Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell a...Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.展开更多
Three main parameters were selected to study their importance in transformation by budmicroinjection in non-head Chinese cabbage [Brassica campestris ssp. chinensis (L.) Makinovar. communis Tsen et Lee]. The results s...Three main parameters were selected to study their importance in transformation by budmicroinjection in non-head Chinese cabbage [Brassica campestris ssp. chinensis (L.) Makinovar. communis Tsen et Lee]. The results showed that the developmental stage of floral bud, theconcentrations of sucrose and surfactant Silwet L-77 were critical for the successfultransformation by this new method. The suitable bud size is 2-3 mm in length, the favorableconcentration of sucrose and surfactant Silwet L-77 are 8 and 0.02% respectively. When thesucrose concentration was greater than 10% or that of Silwet L-77 was above 0.1%, the treatedbuds became yellow and finally blighted. 4/6 T1 seedlings resistant to kanamycin were positiveby PCR analysis, and T2 progeny of all these positive T1 plants have one or more hybridizingbands by Southern blot. Under 5% sucrose, 0.02% Silwet L-77 and grade 2 bud (2-3 mm in itslength) parameters, the most favorable transformation efficiency is about 0.56%, and meanefficiency reaches 0.16% in all experiments indicating that bud microinjection is potentialtransformation way in non-head Chinese cabbage.展开更多
Glioblastoma multiforme （GBM） is a highly aggressive glial brain tumor with an unfavorable prognosis despite all current therapies including surgery, radiation and chemotherapy. One characteristic of this tumor is a...Glioblastoma multiforme （GBM） is a highly aggressive glial brain tumor with an unfavorable prognosis despite all current therapies including surgery, radiation and chemotherapy. One characteristic of this tumor is a strong synthesis of vascular endothelial growth factor （VEGF）, an angiogenesis factor, followed by pronounced vascularization. VEGF became a target in the treatment of GBM, for example with bevacizumab or the tyrosine kinase inhibitor axitinib, which blocks VEGF receptors. To improve patients＇ prognosis, new targets in the treatment of GBM are under investigations. The role of gap junctions in GBM remains un- known, but some experimental therapies affect these intercellular channels to treat the tumor. Gap junctions are composed of connexins to allow the transport of small molecules between adjacent cells through gap junc- tional intercellular communication （GJIC）. Based on data derived from astrocytes in former studies, which show that VEGF is able to enhance GJIC, the current study analyzed the effects of VEGF, radiation therapy and VEGF receptor blockade by axitinib on GJIC in human GBM cell lines U-87 and U-251. While VEGF is able to induce GJIC in U-251 cells but not in U-87 cells, radiation enhances GJIC in both cell lines. VEGF reocptor blockade by axitinib diminishes radiation induced effects in U-251 partially, while increases GJIC in U-87 cells. Our data indicate that VEGF and radiation are both modifying components of GJ1C in pathologic brain tumor tissue.展开更多
基金supported by a grant from State Key Laboratory of Proteomics of China,No.SKLP-K201401(to SJL)the National Key Project of Basic Research of China,No.2009CB918301(to SJL)the National Natural Science Foundation of China,Nos.30430310,30140001,30370460(to SJL)
文摘Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.
基金Funds for the Excellent Talent of Anhui Province of China, No.06043090National Century Excellent Talents in University of China, No.NCET-06-0557Natural Science Foundation of Anhui Province Department of Education, No. KJ2007A028
文摘BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high doses of drug are required and the success rate of model induction is low. It is necessary to develop an improved method to establish a temporal lobe epilepsy （TLE） animal model. OBJECTIVE： To explore an economic, stable and efficient method of establishing a TLE animal model. DESIGN, TIME AND SETTING： A completely randomized, controlled study. The experiments were performed in the Cellular Function Laboratory of the Physiology Department, Anhui Medical University from March to July 2007. MATERIALS： Twenty adult male Wistar rats, weighing 230-260 g, were provided by the Experimental Animal Centre of Nanjing Medical University. Kainic acid was purchased from Sigma in USA. Type SN-2 stereotaxic apparatus was made by Narishge in Japan. METHODS： Wistar rats were randomly divided into a kainic acid （KA） group （n = 12） and a normal saline （NS） group （n = 8）. For intrahippocampal microinjection, a burr hole was drilled in the skull at the following stereotaxic coordinates： anteroposterior （AP） 4.1 mm caudal to bregma; lateral （ML） 4.2 mm right lateral to the midline. Rats in the KA group were injected with 2.5 μL KA （0.4 g/L） into the center of the CA3 region, while in the NS group the same volume of NS was injected into the same site. MAIN OUTCOME MEASURES： Both groups were monitored under a video capture system for 12 weeks to record spontaneous seizures. Intracranial eletroencepholograph （IEEG） recordings in vivo were performed after the behavioral observations. After the IEEG recordings, hippocampi were processed into coronal sections. Nissl and Timm stainings were then performed to observe and confirm pathology. RESULTS： Twenty rats were involved in the final analysis. Behavioral observations： the eadiest spontaneous onset of epilepsy appeared 2 weeks after injection of KA. Eight rats had spontaneous onset of epilepsy 3-12 weeks after treatment. None of rats in the NS group had spontaneous onset of epilepsy. IEEG recordings： Epileptic-form waves, such as sharp waves and spike waves, were calculated by artificial analysis The number of epileptic-form waves in the KA group increased significantly compared to those of the NS group （P 〈 0.01）. Morphology results： In the KA group, Nissl staining and Timm staining revealed typical pathology in the hippocampal temporosphenoid lobe. In the NS group, no pathology was observed. CONCLUSION： Intrahippocampal microinjection of KA is a reliable method to establish a temporal lobe epilepsy animal model, requiring low doses of kainic acid and giving a high rate of success.
基金This work was supported by the National Natural Science Foundation of China(32070502,31730074,32072419 and 31501905).
文摘The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.
文摘Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.
基金the National Natural Sci-ence Foundation of China(Nos.31672636,31772834,and 31972774)the National Key R&D Program of China(Nos.2018YFD0901202 and 2018YFD0900202)the Key Research and Development Program of Shandong Pro-vince,China(No.2019GHY1120070)。
文摘The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.
文摘Daidzein (DA) was microinjected into mediobasal hypothalamus (MBH) and nucleus ventrome-dialis thalami (VM) of the castrated male Goettingen Mini pigs (n = 9, 10 μl·animal-1, 8 pgDA·μl-1 and plasma luteinizing hormone (LH) levels were compared between the prior and the posterior treatment. The result showed that LH levels after the cerebral administration (ad) tended to increase compared to the levels before ad. In MBH, LH levels in 4 cases (4/5), rose and were not changed in 1 case at 0.5 - 2 hours after ad compared to those before ad. There were no significant changes at 2.5 hours after ad. When it was injected in VM, LH levels in 3 cases (3/4) rose, and were not changed in 1 case after ad compared to those before ad. In the control, there were no changes in plasma LH levels between the pre-and post-treatment except 1 case in MBH. This study suggested that DA could up-regulate LH secretion through hypothalamus level in the castrated boars.
文摘Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.
基金support from the National Natural Science Foundation of China(32101626)the Shandong Province Key R&D Plan Project(2022LZGC020)+1 种基金the Natural Science Foundation of Jiangsu Province of China(BK20220490)the Fundamental Research Funds for the Central Universities under Grant No.226-2024-00227.
文摘The microinjection of Zebrafish embryos is significant to life science and biomedical research.In this article,a novel automated system is developed for cell microinjection.A sophisticated microfluidic chip is designed to transport,hold,and inject cells continuously.For the first time,a microinjector with microforce perception is proposed and integrated within the enclosed microfluidic chip to judge whether cells have been successfully punctured.The deep learning model is employed to detect the yolk center of zebrafish embryos and locate the position of the injection needle within the yolk,which enables enhancing the precision of cell injection.A prototype is fabricated to achieve automatic batch microinjection.Experimental results demonstrated that the injection efficiency is about 20 seconds per cell.Cell puncture success rate and cell survival rate are 100%and 84%,respectively.Compared to manual operation,this proposed system improves cell operation efficiency and cell survival rate.The proposed microinjection system has the potential to greatly reduce the workload of the experimenters and shorten the relevant study period.
基金This work was supported by the Ministry of Education Returnees Research Fund(D-8002-15-0042)the China-US Ocean Research Center Fund(A1-3201-19-3013)the Shanghai First-Class Discipline Construction Fund.
文摘infection model for studying inflammation and the innate immune response.We investigated the genes and signaling pathways activated by static immersion and caudal vein microinjection infection using transcriptome profiling and reverse-transcription quantitative PCR(RT-qPCR)to compare the innate immune response in 3 days post-fertilization(dpf)zebrafish larvae infected by Vibrio parahaemolyticus Vp13 strain.The median lethal dose(LD50)values at 96 h following immersion and microinjection were 3.63×107 CFU/mL and 5.76×102 CFU/nL,respectively.An innate immune response was initiated after 2 h of incubation with the respective LD50 for each infection method.Six hundred and two genes in the immersion group and 359 genes in the microinjection group were activated and differentially expressed post-infection.Sixty-three Gene Ontology(GO)terms and four Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were significantly enriched in the immersion group,compared with only three GO terms and no KEGG pathways in the microinjection group.Two genes,tnfb and ccl20a.3,were significantly up-regulated in both groups.We speculated that immersion infection may affect initial dorsal determination,cytochromes,and fatty acid-binding proteins,as well as inflammation,while microinjection infection may mainly directly affect the immune response.Infection with doses>LD50(1.09×109 CFU/mL and 1.09×103 CFU/nL by immersion and microinjection,respectively)caused more significant up-regulation of il11a,tnfa,tnfb,il1b,ccl34a.4,ccl20a.3,irak3,cxcl18b,and ccl35.1,suggesting that in addition to the classical innate immunity genes tnfa,tnfb,il1b,and il6,the genes il11a,ccl34a.4,ccl20a.3,cxcl18b,and ccl35.1 were also important for defending against Vp13 infection.These findings highlight the genes involved in the responses of zebrafish to Vp13 infection via different routes and doses,and thus provide the basis for further analyses of immune response signaling pathways.
文摘Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods Superovulation and synchronization of estrus were induced in female Japanese White Rabbits by injecting hormone, then mating with male. After collected the fertilized eggs, the human apoA-Ⅱ gene was microinjected into the male pronucleus of eggs. The injected eggs were transferred into recipient female rabbits. Last, extract DNA from the new borns ear and determine whether the newborns were transgenic by polymerase chain reaction (PCR) or Southern blot analysis. Results A total of 822 embryos with microinjection of human apoAⅡ gene were implanted into 28 recipient rabbits. The number of surviving newborns was 37. 3 transgenic positive surviving founders were found with human apoA-Ⅱ.
基金supported by the National Transgenic Breeding Project, China (2008ZX08010-004)the National Natural Science Foundation of China (30830080)+1 种基金the National Basic Research Program of China (G2006CB102105, 2009CB941604)the National High Technology Research and Development Program of China (20060110Z1039, 2008AA10Z143)
文摘Polyunsaturated fatty acids （PUFAs） are essential for normal growth in mammals, especially the ω-3 PUFAs, which play important roles in preventing several life-threatening diseases, such as coronary heart disease and diabetes. In this study, we aimed to investigate whether the sFat-1 gene from Caenorhabditis briggsae could be functionally expressed in transgenic pigs, and whether the transgenic could synthesize high quality ω-3 PUFAs endogenously. In this study, a gene construct consisting of CMV promoter and 1.9 kb cDNA of ω-3 fatty acid desaturase gene （sFat-1） from C. briggsae was injected into the male pronucleus of pig embryos by microinjection. The piglets were screened for the transgene by PCR, Southern blot and reverse transcription-PCR analysis. Pigs that give positive results were mated with wild-type pigs to produce the next generation and the transmission of transgene was examined by PCR analysis. Fatty acids compositions of various tissues in the transgenic pigs were then analyzed by gas chromatograph. A total of 878 embryos were transferred into 42 recipients, among which 29 successfully got pregnant and gave birth to a total of 162 piglets, and 8 of them were identified to be transgenic. Fatty acid compositions in the transgenic pigs were altered, and the levels of ω-6：ω-3 ratios were decreased from 14.53 in the control to 2.62 in Fat-1 transgenic pigs. A number of primary sFat-1-transgenic pigs were bred in this study, which lays the foundation for cultivation of new varieties of transgenic pigs.
基金supported by a grant from Medical Research Council,No.MRCG0300456
文摘The deleterious effect of vasculature damage on the outcome of spinal cord injury has long been recognized, and numerous clinical studies have shown that the presence of hemorrhage into the spinal cord is directly associated with a poorer neurological outcome. Vascular damage leads to de- creased blood flow to the cord and the release of potentially toxic blood-borne components. Here we consider the mechanisms that may be contributing to hemorrhage-induced damage and discuss the utility of a new model of spinal cord hemorrhage, which was urgently required as most of our current understanding has been extrapolated from intracerebral hemorrhage studies.
文摘Interest in ion channels as drug targets for contraception has grown with the realization that certain ion channel subunits are located exclusively in sperm. Selective knockdown of ion channel subunits can lead to infertility without ill effects, and selective inhibitors and/ or openers of these ion channels could interfere with sperm function. In this study, in vivo electmporation （EP） and rete testis microinjection-mediated plasmid DNA were adopted to silence CatSper2 expression, which is essential in sperm hyperactivation. The results showed that high transfection efficiency and expression were achieved by plasmid DNA that was directly injected into the rete testis. As a result of the expression of CatSper2 being blocked, the treatment group showed significantly lower （P〈0.05） hyperactivation rate, fertilization rate in vitro, migration motility in viscoelastic solution and intracellular Ca2＋ peak. The low hyperactivation and fertilization rates lasted for 60 days. Meanwhile, analysis of the sperm survival rate and testis histology indicated that in vivo EP had no significant effect on the function of the testis, spermatogenesis or sperm activity. The present study demonstrated that it was feasible to achieve male contraception by silencing the expression of CatSper2, the key protein involved in sperm hvoeractivation.
基金financially supported by the Collaborative Innovation Center of Research on Functional Ingredients from Agricultural Residues (Guangxi University of Chinese medicine,No. CICAR 2015-B2)
文摘Over the past few decades, the determination of antioxidant activity by chemical probe has been widely reported, but in vivo evaluation via model organisms of Drosophila melanogaster and rapid discovery system has not been studied adequately. In this study, we determined the antioxidant activity of lees and demonstrated the ability of compounds in lees to scavenge H_2O_2 in vitro and in vivo by different chemical probes. Lees increased the ability of Drosophila against oxidative stress and antioxidant enzyme activity in vivo. Five ingredients of organic acids and flavones in lees extract that rapidly scavenged H_2O_2 were revealed by a post-column-derived HPLC-UV-FLD system based on 4-hydroxyphenylacetic acid（PHPAA）chemiluminescence. Additionally, another fluorescent probe,N-borylbenzyloxycarbonyl-3,7-dihydroxyphenoxazine（NBCD）, was selected to evaluate the reactive oxygen species（ROS） scavenging capacity of lees in living Drosophila melanogaster using a microfluidic injection test coupled with microscopic imaging analysis, and similar effects were observed in flies when they were treated with tartaric acid and caffeic acid. The results demonstrated that the novel integrated system was suitable for screening and evaluating antioxidant ingredients from natural products.
基金This work was supported by Projects of Tackling Key Problems in ScienceTechnology from the State Science+2 种基金Technology Ministry (TJ99-LA01) Shanghai ScienceTechnology Commission (994919033 )
文摘INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).
基金supported by the National Natural Science Foundation of China,Nos.31671076(to LC),81200872(to YX)Taishan Scholars Construction Project of China(to LC).
文摘The globus pallidus is the relay nucleus of the basal ganglia,and changes in its electrical activity can cause motor impairment.Apelin-13 is widely distributed in the central and peripheral nervous systems.It has been demonstrated that apelin-13 plays important roles in the regulation of blood pressure and other non-motor functions.However,its role in motor function has rarely been reported.In the present study,apelin-13(10μM/100μM)was injected into the globus pallidus of rats.The results showed that apelin-13 increased the spontaneous discharges in the majority of pallidal neurons.However,an apelin-13-induced inhibitory effect on the firing rate was also observed in a few pallidal neurons.In postural tests,after the systemic administration of haloperidol,unilateral pallidal injection of apelin-13 caused a contralateral deflection.Together,these findings suggest that apelin-13 regulates the electrical activity of pallidal neurons and thus participates in central motor control in rats.The study was approved by the Animal Ethics Committee of Qingdao University(approval No.20200615Wistar0451003020)on June 15,2020.
文摘Aim: To observe the effect of electroacupuncture (EA) on acute myocardial ischemia (AMI) induced changes of cardiac sympathetic discharges and the effects of some related receptors in the spinal cord. Methods: A total of 53 rabbits anesthetized with mixture solution of 25% urethane (420 mg/kg) and 1.5% chloralose (50 mg/kg) were used in this study. AMI was induced by occlusion of the ventricular branch of the left coronary artery. Discharges of the left cardiac sympathetic nerve were recorded by using a bipolar platinum electrode. Bilateral "Ximen"(PC 40) and "Kongzhui"(LU 6) were stimulated electrically by using an EA therapeutic apparatus or an electrical stimulator. DPDPE δ opiate receptor agonist, 20 nmol, 10 μL, n=8), Naltrindole Hydrochloride (δ opiate receptor antagonist, 20 nmol, 10 μL, n=8), DAP5 (NMDA receptor antagonist, 5 nmol, 10 μL, n=9) and CNQX (non NMDA receptor antagonist, 5 nmol, 10 μL, n=8) were respectively injected into the thoracic subarachnoid space of the spinal cord in different groups, followed by observing their effects on changes of sympathetic activity evoked by EA of the abovementioned acupoints. Results: ① After AMI, sympathetic discharges increased (200.56±79.89%) in 10 cases and decreased (-59.34 ±7.06%) in other 9 cases in comparison with their individual basal values. After EA of "Ximen" (PC 4) and "Kongzhui"(LU 6), AMI induced increase and decrease changes of the sympathetic activity were suppressed significantly, but the effect of EA of LU 6 was weaker than that of EA of PC 4. ② Following EA of PC 4 and LU 6, sympathetic discharges increased significantly in 2 and 4 cases, decreased apparently in 7 and 3 cases, and had no striking changes in 1 and 3 cases respectively. The mean reaction threshold of sympathetic activity after EA of PC 4 and LU 6 were 2.1±0.65 mA and 3.28±1.13 mA separately. ③ After pre treatment with DPDPE, the reaction threshold of the cardiac sympathetic activity to EA of PC 4 elevated significantly (35.89±6.12%); while after pre treatment with Naltrindole, this reaction threshold decreased considerably (84.88±26.58%). Following intrathecal injection of DAP5 (n=9) and CNQX (n=9) , the reaction thresholds of the cardiac sympathetic activity to EA of PC 4 increased obviously (142.06±60.27% and 112.54±28.58% separately). It suggests that spinal δ opioid receptor, NMDA and non NMDA receptors are involved in EA induced changes of sympathetic activity. Conclusion: ① EA could regulate AMI induced changes of cardiac sympathetic activity; and ② spinal δ opioid receptors, NMDA and non NMDA receptors participate in the effect of EA on the cardiac sympathetic activity.
基金supported by grants from National Transgenic Creature Breeding Grand Project (2014ZX08008-005)
文摘Background： Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons（breeding and non-breeding season） on superovulation and the imported exogenous gene on growth.Results： In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep.Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons（P 〉 0.05）. The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group（P 〈 0.05）. The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference.Conclusions： Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.
文摘Three main parameters were selected to study their importance in transformation by budmicroinjection in non-head Chinese cabbage [Brassica campestris ssp. chinensis (L.) Makinovar. communis Tsen et Lee]. The results showed that the developmental stage of floral bud, theconcentrations of sucrose and surfactant Silwet L-77 were critical for the successfultransformation by this new method. The suitable bud size is 2-3 mm in length, the favorableconcentration of sucrose and surfactant Silwet L-77 are 8 and 0.02% respectively. When thesucrose concentration was greater than 10% or that of Silwet L-77 was above 0.1%, the treatedbuds became yellow and finally blighted. 4/6 T1 seedlings resistant to kanamycin were positiveby PCR analysis, and T2 progeny of all these positive T1 plants have one or more hybridizingbands by Southern blot. Under 5% sucrose, 0.02% Silwet L-77 and grade 2 bud (2-3 mm in itslength) parameters, the most favorable transformation efficiency is about 0.56%, and meanefficiency reaches 0.16% in all experiments indicating that bud microinjection is potentialtransformation way in non-head Chinese cabbage.
文摘Glioblastoma multiforme （GBM） is a highly aggressive glial brain tumor with an unfavorable prognosis despite all current therapies including surgery, radiation and chemotherapy. One characteristic of this tumor is a strong synthesis of vascular endothelial growth factor （VEGF）, an angiogenesis factor, followed by pronounced vascularization. VEGF became a target in the treatment of GBM, for example with bevacizumab or the tyrosine kinase inhibitor axitinib, which blocks VEGF receptors. To improve patients＇ prognosis, new targets in the treatment of GBM are under investigations. The role of gap junctions in GBM remains un- known, but some experimental therapies affect these intercellular channels to treat the tumor. Gap junctions are composed of connexins to allow the transport of small molecules between adjacent cells through gap junc- tional intercellular communication （GJIC）. Based on data derived from astrocytes in former studies, which show that VEGF is able to enhance GJIC, the current study analyzed the effects of VEGF, radiation therapy and VEGF receptor blockade by axitinib on GJIC in human GBM cell lines U-87 and U-251. While VEGF is able to induce GJIC in U-251 cells but not in U-87 cells, radiation enhances GJIC in both cell lines. VEGF reocptor blockade by axitinib diminishes radiation induced effects in U-251 partially, while increases GJIC in U-87 cells. Our data indicate that VEGF and radiation are both modifying components of GJ1C in pathologic brain tumor tissue.
