RNA interference(RNAi)is a post-transcriptional gene-silencing technique induced by the introduction of double-stranded RNA(dsRNA)or small interfering RNA(siRNA)[1].RNAi-based strategies have been extensively applied ...RNA interference(RNAi)is a post-transcriptional gene-silencing technique induced by the introduction of double-stranded RNA(dsRNA)or small interfering RNA(siRNA)[1].RNAi-based strategies have been extensively applied in the treatment of human diseases and crop protection against insect pests[2-4].With the availability of the full genome sequences of major mosquito vectors,RNAi has become increasingly used as a novel means of mosquito control[5].展开更多
Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,esp...Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,especially for the advanced forms of the disease. In the last year,short double stranded RNA molecules termed small interfering RNAs(si RNAs) and micro interfering RNAs(mi RNA),emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of si RNAs/mi RNAs molecular targets and on the development of suitable si RNA/mi RNAs delivery systems.展开更多
AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed a...AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay （ELISA）. Intracellular viral DNA and covalently closed circular DNA （cccDNA） were quantified by real-time polymerase chain reaction （PCR）. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR （RT-PCR）. RESULTS： siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION： Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.展开更多
Objective To construct an expression vector of small interfering RNA （siRNA） against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast can...Objective To construct an expression vector of small interfering RNA （siRNA） against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.展开更多
As a new treatment technique,photothermal therapy(PTT)has aroused worldwide attention in cancer treatment,mainly due to its excellent absorption ability,easy regulation,and biodegradability.Photothermal conversion mat...As a new treatment technique,photothermal therapy(PTT)has aroused worldwide attention in cancer treatment,mainly due to its excellent absorption ability,easy regulation,and biodegradability.Photothermal conversion materials with enhanced permeability and retention effect can be targeted easily to tumor tissue.They can accumulate efficiently to tumor tissues and allow normal tissues and organs not to be affected by temperature,thus significantly helping to reduce the systemic toxicity and improve the antitumor effect.However,PTT alo ne often suffers from the rapeutic resistance and reduced therapeutic efficacy,due to photothermal nanomaterial-mediated fundamental cellular defense mechanism of heat shock response,which could be inhibited by small interfering RNA(siRNA).Nevertheless,photothermal conversion materials as an excellent siRNA delivery carrier may conside rably enhance the delivery efficiency of siRNA.Therefore,photothermal and RNA interfering(RNAi)synergistic therapy has recently aroused extensive attention in tumor treatment.In this review,we mainly summarize the recent advances of photothermal and RNAi synergistic therapy,including some synergistic therapeutic nanoplatforms of inorganic and organic photothermal materials and other combined therapies such as combining with small molecular antitumor agents or PDT/imaging.The combination of various treatment techniques may considerably improve the synergistic therapeutic effect of PTT and RNAi in the treatment of cancers.展开更多
RNA interference,widely regarded as a key mechanism for cells to regulate gene expression,is a natural gene silencing phenomenon.It can be used as the gene knockdown to reverse the multidrug resistance of tumor cells ...RNA interference,widely regarded as a key mechanism for cells to regulate gene expression,is a natural gene silencing phenomenon.It can be used as the gene knockdown to reverse the multidrug resistance of tumor cells and has been applied in the field of biomedicine,exhibiting huge potential in drug target identification,optimization of drug targets,multidrug resistance,etc.This paper first introduces the mechanism of RNA interference and the formation mechanism of multidrug resistance of tumor cells,on the basis of which it reviews the application and challenges of RNA interference technology in reversing multidrug resistance.Additionally,the development of the siRNA delivery system is illustrated.展开更多
AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was ...AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.展开更多
Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side...Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs(si RNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and si RNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited sideeffects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is overexpressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and nonviral si RNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic si RNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein, ganglioside GM1 cell surface ligand, epidermal growth factor receptor receptors, monoclonal antibodies, retinoic acid receptors, integrin receptors targeted by Arg-Gly-Asp peptide, folate, and transferrin receptors are the most widely studied cell surface receptors which are used for the site specific delivery of drugs and si RNA-based therapeutics in HCC and discussed in detail in this article.展开更多
Objective:To observe the clinical manifestations of allergic rhinitis mice and the expression changes of the eosinophils CCR3 and the granule protein rnRNA in the bone marrow,peripheral blood and nasal lavage fluid.Mc...Objective:To observe the clinical manifestations of allergic rhinitis mice and the expression changes of the eosinophils CCR3 and the granule protein rnRNA in the bone marrow,peripheral blood and nasal lavage fluid.Mctliods:Twenty-four BALB/c mice were randomly divided into the control group.PBS therapy group.siKNA therapy group and the CCR3 siRNA therapy group(n=6).Allergic rhinitis model were sensitized and stimulated by ovalbunfin,and CCR3 siKNA therapy group were administered with CCH3 transnasally before stimulated.The levels of the eosinophils CCR3.MBP.ECP and EPO in bone marrow,peripheral blood and nasal lavage fluid were detected by RT-PCR.Results:Compared to the control group and CCR3 siR.NA therapy group,the nasal mucosa of the PBS therapy group and siRNA therapy group developed epithalaxy.goblet cells hyperplasia,squamous epithelium metaplasia,epithelium necrosis,lamina propria and submucosa gland hyperplasia,vasodilatation,tissue edema,and the characterized eosinophil infiltration.RT-PCR indicated that the CCR3 rnRNA,MBP.ECP and EPC)expression in bone marrow,peripheral blood and nasal lavage fluid of the CCR3 siKNA therapy group was lower than the PBS therapy group and siR.NA therapy group(P<0.05).Conclusions:The RNA interference therapy to CCR3 by local administration pernasal can suppress the process of the development,migration and invasion of the allergic rhinitis eosinophil,thus can reduce the effect of eosinophils and then reduce the inflammation effect of the allergic rhinitis.It may be a new treatment for respiratory tract allergic inflammation.展开更多
RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequenc...RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80％ at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0～60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.展开更多
The bearing capacity of interfering footings located near the slope face suffers from reduced bearing capacity due to the formation of the curtailed passive zone. Depending upon the position of the footing, their spac...The bearing capacity of interfering footings located near the slope face suffers from reduced bearing capacity due to the formation of the curtailed passive zone. Depending upon the position of the footing, their spacing and steepness of the slope different extents of bearing capacity reduction can be exhibited. A series of finite element investigation has been done with the aid of Plaxis 3 D v AE.01 to elucidate the influence of various geotechnical and geometrical parameters on the ultimate bearing capacity of interfering surface strip footings located at the crest of the natural soil slope. Based on the large database obtained from the numerical simulation, a6-8-1 Artificial Neural Network architecture has been considered for the assessment of the ultimate bearing capacity of interfering strip footings placed on the crest of natural soil slope. Sensitivity analyses have been conducted to establish the relative significance of the contributory parameters, which exhibited that for the stated problem, apart from shear strength parameters, the setback ratio and spacing of footing are the prime contributory parameters.展开更多
Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving inc...Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells(HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix(ECM). Transforming growth factor-beta(TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference(RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA(si RNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 si RNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 si RNA in liver fibrosis.展开更多
Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted ...Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5×106 IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.展开更多
AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).ME...AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.展开更多
To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, th...To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.展开更多
A solution culture experiment was conducted to investigate the effects of collection time and interferingions on separation and determination of low-molecular-weight organic acids in root exudates of soybeanusing the ...A solution culture experiment was conducted to investigate the effects of collection time and interferingions on separation and determination of low-molecular-weight organic acids in root exudates of soybeanusing the method for directly collecting root exudates. The suitable collection time of root exudates andthe interfering ions affecting organic acid determination were determined. The method for removing theinterfering ions was established and analyzed. The release amount of root exudates increased with theincrease of collection time from 0 to 120 min but decreased with increasing of collection time from 120 to 240min. The maximum exuding amounts of organic acids were observed in root exudates at the collection time of120 min. There was a significant difference of organic acid components between the treatments of collectiontime of 120 min and 240 min. Citric acid was found only in the treatment of 120 min collection time. NO3-was the main interfering ion in organic acid determination and had the same retention time as oxalic acid.Anion exchangs resin (SAX) properly treated by HPLC (high performance liquid chromatography) solventcould remove NO3- anion in sample solution of root exudates, thus enhancing the recoveries of organic acidsin root exudates. There was no significant effect of the chemicals added into sample solution such as H3PO4,SAX and KNO3 on the retention time of organic acids.展开更多
Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was repo...Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein （Aβ） and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA （siRNA） against the sphK1 gene （sphKl-siRNA） was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay （ELISA）. Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.展开更多
Tumor necrosis factor-alpha（TNF-α） has been found to be centrally involved in the development of ischemia-reperfusion injury（IRI）-induced inflammation and apoptosis. Knockdown of TNF-α gene using small interferi...Tumor necrosis factor-alpha（TNF-α） has been found to be centrally involved in the development of ischemia-reperfusion injury（IRI）-induced inflammation and apoptosis. Knockdown of TNF-α gene using small interfering RNA（si RNA） may protect renal IRI. Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. TNF-α si RNA was administered intravenously to silence the expression of TNF-α. The therapeutic effects of si RNA were evaluated in terms of renal function, histological examination, and overall survival following lethal IRI. A single systemic injection of TNF-α si RNA resulted in significant knockdown of TNF-α expression in ischemia-reperfusion injured kidney. In comparison with control mice, levels of BUN and serum creatinine were significantly reduced in mice treated with si RNA. Pathological examination demonstrated that tissue damage caused by IRI was markedly reduced as a result of TNF-α si RNA treatment. Furthermore, survival experiments showed that nearly 90% of control mice died from lethal IRI, whereas more than 50% of si RNApretreated mice survived until the end of the eight-day observation period. We have demonstrated for the first time that silencing TNF-α by specific si RNA can significantly reduce renal IRI and protect mice against lethal kidney ischemia, highlighting the potential for si RNA-based clinical therapy.展开更多
The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and S...The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.展开更多
Objective:To investigate the inhibition effect of siRNA interference on NGF induced by inflammatory factor IL-6,and JUL—1 so as to provide novel targets for clinical treatment of discogenic low back pain.Methods:The ...Objective:To investigate the inhibition effect of siRNA interference on NGF induced by inflammatory factor IL-6,and JUL—1 so as to provide novel targets for clinical treatment of discogenic low back pain.Methods:The intervertebral disc nucleus and annulus fibrosus cells of rats were separated-The cells were co-cultured with different concentrations(10 nmol/L,20nmol/L,50 nmol/L,100 nmol/L)of IL-6 and IL-1β.The NGF-siRNA was leaded into the cocultured cells with its import ability assessed by flow cytometry instrument tests,hefore and after which the NCF mRNA expression was detected by real-time Q-PCR and the NGF content was detected by ELISA.Results:Flow cytometry instrument test results showed that the NGFsiRNA cell conversion rate was 99.8%.Real-time Q-PCR detection results showed that compared with negative control group,the NGF mRNA expression of co-cultured cells treated by 10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L IL-6 and IL-1βwere respectively raised 3.4,3.7,4.7,3.7 times which were all significantly down-regulated after the import of NGF-siRNA.EILSA detection results showed that compared with negative control group,the NGF content of cocultured medium treated by 10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L I-L6 and IL-1βwere respectively raised 2.9,3.3,4.5,7.4 times which were all significantly decreased after the import of NGF-siRNA.Conclusions:These molecular biological results suggest that inflammatory factor IL-6 and IL-1βcould stimulate NCF on intervertebral disc cells in vitro culture model and its efficiency is concentration dependent,while siRNA interference can inhibit the stimulation effect of IL-6 and IL-1βon intervertebral disc cell,which provides a new targets for the clinical treatment of discogenic low back pain.展开更多
基金supported by grants from the National Key Research and Development Program(2023YFE0113600).
文摘RNA interference(RNAi)is a post-transcriptional gene-silencing technique induced by the introduction of double-stranded RNA(dsRNA)or small interfering RNA(siRNA)[1].RNAi-based strategies have been extensively applied in the treatment of human diseases and crop protection against insect pests[2-4].With the availability of the full genome sequences of major mosquito vectors,RNAi has become increasingly used as a novel means of mosquito control[5].
基金Supported by"Fondazione Cassa di Risparmio of Trieste"the"Fondazione Benefica Kathleen Foreman Casali of Trieste"+2 种基金the"Beneficentia Stiftung"of Vaduz Liechtensteinthe Italian Minister of Instruction,UniversityResearch(MIUR),PRIN 2010-11,No.20109PLMH2(in part)
文摘Hepatocellular carcinoma(HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective,especially for the advanced forms of the disease. In the last year,short double stranded RNA molecules termed small interfering RNAs(si RNAs) and micro interfering RNAs(mi RNA),emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of si RNAs/mi RNAs molecular targets and on the development of suitable si RNA/mi RNAs delivery systems.
基金The Youth Foundation of Heilongjiang Province,No.QC06C061the Foundation of Education Department,Heilongjiang Province,No.11521089
文摘AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay （ELISA）. Intracellular viral DNA and covalently closed circular DNA （cccDNA） were quantified by real-time polymerase chain reaction （PCR）. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR （RT-PCR）. RESULTS： siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION： Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.
基金Supported by the Key Science and Technology Research Project ofShaanxi Province [2003K10-G35,2004K13-G11(1)].
文摘Objective To construct an expression vector of small interfering RNA （siRNA） against survivin and observe its effects on survivin expression and proliferation of human pancreatic cancer cell line PC-2 and breast cancer cell line MCF-7. Methods Constructed an expression vector of siRNA against survivin and transfected it into PC-2 and MCF-7 cells using lipofectamine^TM 2000. The expression of survivin was detected by semi-quanfifive RT-PCR and immunohistochemistry, and its effects on proliferation of PC-2 and MCF-7 cells were detected by MTT assay. Results The introduction of sequence-specific siRNA could efficiently suppress survivin expression at both mRNA and protein levels in the two cancer cell lines. In PC-2 cells, the expression inhibition rates were 81.25% at mRNA level and 74.24% at protein level In MCF-7 cells, the expression inhibition rates were 64.91% at mRNA level and 79. 72% at protein level The proliferation of PC-2 and MCF-7 cells was also suppressed, and24 and 48 hours after the cells were reseeded, the proliferation inhibition rates of PC-2 cells were 28. 00% and 33. 38%, and that of MCF-7 cells were 31.58% and 33.02%, respectively. Conclusions The expression vector of siRNA against survivin can block survivin expression in PC-2 and MCF-7 cells efficiently and specifically. Down regulation of survivin expression can suppress proliferation of PC-2 and MCF-7 cells. Survivin RNAi may have potential value in gene therapy of human cancers.
基金supported by the Hunan Provincial Natural Science Foundation of China(No.2018JJ1019)the Hu-Xiang Young Talent Program(No.2018RS3094)+2 种基金the National Natural Science Foundation of China(No.31871003)the Beijing Institute of Technology Research Fund Program for Young Scholars and the Fundamental Research Funds for the Central Universities(No.2018CX01023)the Young Elite Scientist Sponsorship Program of Beijing Association for Science and Technology。
文摘As a new treatment technique,photothermal therapy(PTT)has aroused worldwide attention in cancer treatment,mainly due to its excellent absorption ability,easy regulation,and biodegradability.Photothermal conversion materials with enhanced permeability and retention effect can be targeted easily to tumor tissue.They can accumulate efficiently to tumor tissues and allow normal tissues and organs not to be affected by temperature,thus significantly helping to reduce the systemic toxicity and improve the antitumor effect.However,PTT alo ne often suffers from the rapeutic resistance and reduced therapeutic efficacy,due to photothermal nanomaterial-mediated fundamental cellular defense mechanism of heat shock response,which could be inhibited by small interfering RNA(siRNA).Nevertheless,photothermal conversion materials as an excellent siRNA delivery carrier may conside rably enhance the delivery efficiency of siRNA.Therefore,photothermal and RNA interfering(RNAi)synergistic therapy has recently aroused extensive attention in tumor treatment.In this review,we mainly summarize the recent advances of photothermal and RNAi synergistic therapy,including some synergistic therapeutic nanoplatforms of inorganic and organic photothermal materials and other combined therapies such as combining with small molecular antitumor agents or PDT/imaging.The combination of various treatment techniques may considerably improve the synergistic therapeutic effect of PTT and RNAi in the treatment of cancers.
基金the Doctoral Promotion Program Research Initiation Fund of Suzhou Polytechnic Institute of Agriculture(grant number:GSP20200066).
文摘RNA interference,widely regarded as a key mechanism for cells to regulate gene expression,is a natural gene silencing phenomenon.It can be used as the gene knockdown to reverse the multidrug resistance of tumor cells and has been applied in the field of biomedicine,exhibiting huge potential in drug target identification,optimization of drug targets,multidrug resistance,etc.This paper first introduces the mechanism of RNA interference and the formation mechanism of multidrug resistance of tumor cells,on the basis of which it reviews the application and challenges of RNA interference technology in reversing multidrug resistance.Additionally,the development of the siRNA delivery system is illustrated.
基金Supported by Tiantan Hospital Scientific Project Grant Fund
文摘AIM:To investigate the inhibitory effects of RNA interference (RNAi) on expression of matrix metalloproteinase-2 (MMP-2) gene and invasiveness and adhesion of human pancreatic cancer cell line,BxPC-3.METHODS:RNAi was performed using the vector (pGPU6)-based small interference RNA (siRNA) plasmid gene silence system to specifically knock down MMP-2 expression in pancreatic cancer cell line,BxPC-3. Four groups of different specific target sequence in coding region of MMP-2 and one non-specific sequence were chosen to construct four experimental siRNA plasmids of pGPU6-1,pGPU6-2,pGPU6-3 and pGPU6-4,and one negative control siRNA plasmid of pGPU6 (-). MMP-2 expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation and apoptosis were examined by methyl thiazolyl tetrazolium (MTT) and flow cytometry,respectively. The abilities of adhesion and invasion were detected by cell adhesion assay and cell invasion assay using Transwell chambers.RESULTS:The expression of MMP-2 was inhibited and the inhibitory effects of different sequence varied. pGPU6-1 group had the most efficient inhibitory effect,followed by pGPU6-2 and pGPU6-3 groups.Invasiveness and adhesion were more significantly reduced in pGPU6-1,pGPU6-2 and pGPU6-3 groups as compared with pGPU6 (-) and blank control groups. However,no difference concerning cell proliferation and apoptosis was observed after transfection between experiment groups and control groups.CONCLUSION:RNAi against MMP-2 successfully inhibited the mRNA and protein expression of MMP-2 in the pancreatic cancer cell line,BxPC-3,leading to a potent suppression of tumor cell adhesion and invasion without affecting cell proliferation and apoptosis. These findings suggest that the RNAi approach towards MMP-2 may be an effective therapeutic strategy for the clinical management of pancreatic tumor.
文摘Hepatocellular carcinoma(HCC) is the 5th most common malignancy which is responsible for more than half million annual mortalities; also, it is the third leading cause of cancer related death. Unfavorablesystemic side-effects of chemotherapeutic agents and susceptibility to the degradation of small interfering RNAs(si RNAs), which can knock down a specific gene involved in the disease, have hampered their clinical application. So, it could be beneficial to develop an efficient carrier for the stabilization and specific delivery of drugs and si RNA to cells. Targeted nanoparticles have gained considerable attention as an efficient drug and gene delivery system, which is due to their capability in achieving the highest accumulation of cytotoxic agents in tumor tissue, modifiable drug pharmacokinetic- and bio-distribution, improved effectiveness of treatment, and limited sideeffects. Recent studies have shed more light on the advantages of novel drug loaded carrier systems vs free drugs. Most of the animal studies have reported improvement in treatment efficacy and survival rate using novel carrier systems. Targeted delivery may be achieved passively or actively. In passive targeting, no ligand as homing device is used, while targeting is achieved by incorporating the therapeutic agent into a macromolecule or nanoparticle that passively reaches the target organ. However, in active targeting, the therapeutic agent or carrier system is conjugated to a tissue or cell-specific receptor which is overexpressed in a special malignancy using a ligand called a homing device. This review covers a broad spectrum of targeted nanoparticles as therapeutic and nonviral si RNA delivery systems, which are developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and their characteristics and opportunities for the clinical applications of drugs and therapeutic si RNA are discussed in this article. Asialoglycoprotein receptors, low-density lipoprotein, ganglioside GM1 cell surface ligand, epidermal growth factor receptor receptors, monoclonal antibodies, retinoic acid receptors, integrin receptors targeted by Arg-Gly-Asp peptide, folate, and transferrin receptors are the most widely studied cell surface receptors which are used for the site specific delivery of drugs and si RNA-based therapeutics in HCC and discussed in detail in this article.
基金supported by the National Natural Science Fund Project in China(grant No.:81060084)Jiangxi Provincial Natural Science Fund Project in China(grant No.:2010gzy0251)+1 种基金Jiangxi Provincial Health Department Project in China(grant No.:20131059)Jiangxi Provincial Department of Science and Technology Project in China(grant No.:20133BBG70071)
文摘Objective:To observe the clinical manifestations of allergic rhinitis mice and the expression changes of the eosinophils CCR3 and the granule protein rnRNA in the bone marrow,peripheral blood and nasal lavage fluid.Mctliods:Twenty-four BALB/c mice were randomly divided into the control group.PBS therapy group.siKNA therapy group and the CCR3 siRNA therapy group(n=6).Allergic rhinitis model were sensitized and stimulated by ovalbunfin,and CCR3 siKNA therapy group were administered with CCH3 transnasally before stimulated.The levels of the eosinophils CCR3.MBP.ECP and EPO in bone marrow,peripheral blood and nasal lavage fluid were detected by RT-PCR.Results:Compared to the control group and CCR3 siR.NA therapy group,the nasal mucosa of the PBS therapy group and siRNA therapy group developed epithalaxy.goblet cells hyperplasia,squamous epithelium metaplasia,epithelium necrosis,lamina propria and submucosa gland hyperplasia,vasodilatation,tissue edema,and the characterized eosinophil infiltration.RT-PCR indicated that the CCR3 rnRNA,MBP.ECP and EPC)expression in bone marrow,peripheral blood and nasal lavage fluid of the CCR3 siKNA therapy group was lower than the PBS therapy group and siR.NA therapy group(P<0.05).Conclusions:The RNA interference therapy to CCR3 by local administration pernasal can suppress the process of the development,migration and invasion of the allergic rhinitis eosinophil,thus can reduce the effect of eosinophils and then reduce the inflammation effect of the allergic rhinitis.It may be a new treatment for respiratory tract allergic inflammation.
基金supported by the Grant No.2003AA208215 from the National High Technology Programs of Chinathe Grant No.30270311 from the National Natural Science Foundation of China.
文摘RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80％ at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0～60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
文摘The bearing capacity of interfering footings located near the slope face suffers from reduced bearing capacity due to the formation of the curtailed passive zone. Depending upon the position of the footing, their spacing and steepness of the slope different extents of bearing capacity reduction can be exhibited. A series of finite element investigation has been done with the aid of Plaxis 3 D v AE.01 to elucidate the influence of various geotechnical and geometrical parameters on the ultimate bearing capacity of interfering surface strip footings located at the crest of the natural soil slope. Based on the large database obtained from the numerical simulation, a6-8-1 Artificial Neural Network architecture has been considered for the assessment of the ultimate bearing capacity of interfering strip footings placed on the crest of natural soil slope. Sensitivity analyses have been conducted to establish the relative significance of the contributory parameters, which exhibited that for the stated problem, apart from shear strength parameters, the setback ratio and spacing of footing are the prime contributory parameters.
基金Supported by National Natural Science Foundation of China(81373465)
文摘Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells(HSCs) remains a central event in fibrosis. HSCs are the main source of extracellular matrix(ECM). Transforming growth factor-beta(TGF-β), which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference(RNAi) is a novel gene disruption technology. Studies have shown that small interfering RNA(si RNA) targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 si RNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 si RNA in liver fibrosis.
基金Project supported by the National Natural Science Foundation of China (Nos. 30560160, 30560048, and 30560149)the Program for the New Century Excellent Talents in University of China (No. NCET-05-0757)the Key Scientific Project of Hainan Province, China (No. 081013)
文摘Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5×106 IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.
基金Supported by National Natural Science Fundation of China(No.81000387)
文摘AIMTo determine whether small interfering RNA (siRNA) of PGC-1α could inhibit vascular endothelial growth factor (VEGF) expression and tube formation in human retinal vascular endothelial cells (hRVECs).METHODShRVECs transfected with peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) siRNA were incubated for 24h and then placed into a normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) environment for another 16h. PGC-1α mRNA and protein levels were detected by real-time PCR and Western blot. VEGF mRNA and protein levels were detected by real-time PCR and ELISA. Cell proliferation was evaluated by BrdU incorporation assay. Forty-eight hours after siRNA transfection, hRVECs were planted into Matrigel-coated plates and cultured under normoxic (20%, O<sub>2</sub>) or hypoxic (1%, O<sub>2</sub>) conditions for another 48h. The tube formation of hRVECs was observed under an optical microscope and quantified by counting the number of branch points and calculating the total tube length.RESULTSPGC-1α mRNA and protein levels were significantly reduced by PGC-1α siRNA, and VEGF mRNA and protein levels also decreased significantly. The percentage of BrdU-labeled cells in siPGC-1α groups were significantly decreased compared with control siRNA groups under normoxia and hypoxia in cell proliferation assay. In the tube formation assay, PGC-1α siRNA treated cells formed significantly fewer tubes.CONCLUSIONBlocking PGC-1α expression can inhibit VEGF expression in hRVECs and inhibit their ability to form tubes under both normoxic and hypoxic conditions.
基金Supported by the Ministry of Agricultural Nuarture of New Varieties Genetically Modified Organisms Significant Special Funding (2008ZX08007-002)
文摘To examine the effect of myogenin gene on the differentiation of bovine skeletal muscle satellite cell, we constructed small interfering RNA plasmid vector to obtain myogenin knockdown bovine skeletal muscle cells, then used cell transfection, real time RCR and Western Blot to detect the influence of myogenin to cell differentiation. Results showed that the knockdown of myogenin significantly decreased its expression and other muscle-specific genes. Compared to the control, it could differentiate into few myotubes when challenged by low serum in the medium. These findings provided an important theoretical basis for further explore of the genetic mechanism in adult skeletal muscle, the remedy of muscle injuries and the cultivation of high-yield transgenic cattle.
文摘A solution culture experiment was conducted to investigate the effects of collection time and interferingions on separation and determination of low-molecular-weight organic acids in root exudates of soybeanusing the method for directly collecting root exudates. The suitable collection time of root exudates andthe interfering ions affecting organic acid determination were determined. The method for removing theinterfering ions was established and analyzed. The release amount of root exudates increased with theincrease of collection time from 0 to 120 min but decreased with increasing of collection time from 120 to 240min. The maximum exuding amounts of organic acids were observed in root exudates at the collection time of120 min. There was a significant difference of organic acid components between the treatments of collectiontime of 120 min and 240 min. Citric acid was found only in the treatment of 120 min collection time. NO3-was the main interfering ion in organic acid determination and had the same retention time as oxalic acid.Anion exchangs resin (SAX) properly treated by HPLC (high performance liquid chromatography) solventcould remove NO3- anion in sample solution of root exudates, thus enhancing the recoveries of organic acidsin root exudates. There was no significant effect of the chemicals added into sample solution such as H3PO4,SAX and KNO3 on the retention time of organic acids.
基金supported by the National Natural Science Foundation of China (No. 81070879)
文摘Summary： Alzheimer＇s disease （AD） is an age-related, progressive neurodegenerative disorder that occurs gradually and results in memory, behavior, and personality changes. Abnormal sphingolipid metabolism was reported in AD previously. This study aimed to investigate whether sphK1 could exacerbate the accumulation of amyloid protein （Aβ） and sharpen the learning and memory ability of the animal model of AD using siRNA interference. An adenovirus vector expressing small interfering RNA （siRNA） against the sphK1 gene （sphKl-siRNA） was designed, and the effects of sphKl-siRNA on the APP/PS1 mouse four weeks after treatment with sphKl-siRNA hippocampal injection were examined. SphK1 protein expression was confirmed by using Western blotting and ceramide content coupled with SIP secretion was evaluated by enzyme-linked immunosorbent assay （ELISA）. Aβ load was detected by immunohistochemical staining and ELISA. Morris water maze was adopted to test the learning and memory ability of the APP/PS 1 mice. A significant difference in the expression of sphK1 protein and mRNA was observed between the siRNA group and the control group. Aβ load in transfected mice was accelerated in vivo, with significant aggravation of the learn- ing and memory ability. The sphKl gene modulation in the All load and the learning and memory ability in the animal model of AD may be important for the treatment of AD.
文摘Tumor necrosis factor-alpha（TNF-α） has been found to be centrally involved in the development of ischemia-reperfusion injury（IRI）-induced inflammation and apoptosis. Knockdown of TNF-α gene using small interfering RNA（si RNA） may protect renal IRI. Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. TNF-α si RNA was administered intravenously to silence the expression of TNF-α. The therapeutic effects of si RNA were evaluated in terms of renal function, histological examination, and overall survival following lethal IRI. A single systemic injection of TNF-α si RNA resulted in significant knockdown of TNF-α expression in ischemia-reperfusion injured kidney. In comparison with control mice, levels of BUN and serum creatinine were significantly reduced in mice treated with si RNA. Pathological examination demonstrated that tissue damage caused by IRI was markedly reduced as a result of TNF-α si RNA treatment. Furthermore, survival experiments showed that nearly 90% of control mice died from lethal IRI, whereas more than 50% of si RNApretreated mice survived until the end of the eight-day observation period. We have demonstrated for the first time that silencing TNF-α by specific si RNA can significantly reduce renal IRI and protect mice against lethal kidney ischemia, highlighting the potential for si RNA-based clinical therapy.
基金supported by National Natural Science Foundation of China(No.81441126)
文摘The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.
基金supported by Shandong Province Natural Science Foundation(28172a2)
文摘Objective:To investigate the inhibition effect of siRNA interference on NGF induced by inflammatory factor IL-6,and JUL—1 so as to provide novel targets for clinical treatment of discogenic low back pain.Methods:The intervertebral disc nucleus and annulus fibrosus cells of rats were separated-The cells were co-cultured with different concentrations(10 nmol/L,20nmol/L,50 nmol/L,100 nmol/L)of IL-6 and IL-1β.The NGF-siRNA was leaded into the cocultured cells with its import ability assessed by flow cytometry instrument tests,hefore and after which the NCF mRNA expression was detected by real-time Q-PCR and the NGF content was detected by ELISA.Results:Flow cytometry instrument test results showed that the NGFsiRNA cell conversion rate was 99.8%.Real-time Q-PCR detection results showed that compared with negative control group,the NGF mRNA expression of co-cultured cells treated by 10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L IL-6 and IL-1βwere respectively raised 3.4,3.7,4.7,3.7 times which were all significantly down-regulated after the import of NGF-siRNA.EILSA detection results showed that compared with negative control group,the NGF content of cocultured medium treated by 10 nmol/L,20 nmol/L,50 nmol/L,100 nmol/L I-L6 and IL-1βwere respectively raised 2.9,3.3,4.5,7.4 times which were all significantly decreased after the import of NGF-siRNA.Conclusions:These molecular biological results suggest that inflammatory factor IL-6 and IL-1βcould stimulate NCF on intervertebral disc cells in vitro culture model and its efficiency is concentration dependent,while siRNA interference can inhibit the stimulation effect of IL-6 and IL-1βon intervertebral disc cell,which provides a new targets for the clinical treatment of discogenic low back pain.
