Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se ...Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se requirements. We have continued to study Se regulation of GPX1 to better understand the underlying mechanism and to gain insight into how cells themselves regulate nutrient status. In progressive Se deficiency in rats, GPX1 activity,protein and mRNA all decrease in a dramatic, coordinated and exponential fashion such that Se-deficient GPX1 mRNA levels are 6-15% of Sexadequate levels. mRNA levels for other Sedependent proteins are far less decreased in the same animals. The mRNA levels for a second Se-dependent peroxidase, phospholipid hydroperoxide glutathione peroxidase (GPX4 ), are little affected by Se deficiency, demonstrating that Se regulation of GPX1 is unique. Se regulation of GPX1 activity in growing male and female rats shows that the Se requirernent is 100 ng/g diet, based on liver GPX1 activity; use of GPX1 mRNA as the parameter indicates that the Se requirement is nearer to 50 ng Se/g diet in both male and female rats. This approach will readily detect an altered dietary Se requirement, as shown by the incremental increases in dietary Se requirement by 150, 100 or 50 ng Se/g diet in Seudeficient rat pups repleted with Se for 3, 7 or 14 d, respectively. Studies with CHO cells stably transfected with recombinant GPX1 also show that overexpression of GPX1 does not alter the minimum level of media Se necessary for Se-adequate levels of GPX1 activity or mRNA. We hypothesize that classical GPX1 has an integral biological role in the mechanism used by cells to regulate Se status,making GPX1 an especially useful and effective parameter for determining Se requirements in animals展开更多
Objective Loss-of-function mutation of p53,a tumor suppressor gene,is an important mechanism for the development of human cancers. In this study we tried to transfect p53N15-based fusion peptide into H1299,a lung canc...Objective Loss-of-function mutation of p53,a tumor suppressor gene,is an important mechanism for the development of human cancers. In this study we tried to transfect p53N15-based fusion peptide into H1299,a lung cancer cell line,and evaluate the anti-tumor effects of the fusion peptide. Methods Adeno-associated virus (AAV) vectors were used for transfecting p53N15 fusion peptide into p53-null lung adenocarcinoma H1299 cells.展开更多
AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was ...AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in.three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.RESULTS： HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferationcapacity was obviously lower in HepG2/pcDNA3-X cells （0.08910±0.003164） than in HepG2 （0.14410±0.004927） and HepG2/pcDNA3 cells （0.12150±0.007159） （P〈0.05 and P〈0.01）. Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells （980/2 000） than HepG2 （420/2 000）, HepG2/pcDNA3 cells （520/2 000） （P〈0.05 and P〈0.01）. Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）. Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells （P〈0.05 and P〈0.01）. Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）.CONCLUSION： HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.展开更多
AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into...AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into incasingcells PA317 by lipofectamine mediated method,the clones of the cells transferred with hlL-6were selected by G418,and targeted HT-29 cellswere infected with the virus granules secretedfrom PA317 and also selected by G418.Test genetranscription and expression level byhybridization,ELISA and MTT assay,etc.Analyze tumor inhibitory effects according to thecell growth curve,plating forming rate andtumorigenicity in nude mice.RESULT Successfully constructed andtransfected recombinant expressing vectorspZIPIL-6 cDNA and got positive transfected celllines.The colon cancer cell line(HT-29 IL-5)transfected with the hlL-5 gene by retroviralvector was established.The log proliferationperiod and the doubling time of this cell line wasbetween 4 to 7 days and 2.5 days according tothe direct cell count,the cell proliferation wasobviously inhibited with MTT assay,the platinginhibitory rate was 50% by plating efficiencytest.When HT-29 IL-6 cells were inoculated intothe nude mice subcutaneously,carcinogenicactivity of the solid tumor was found superior tothe control group and the size of tumor was notsignificantly enlarged.Injection of combinationvirus fluid containing 11.-6 gene intotransplantation tumors could inhibit the growthand development of the tumor.CONCLUSION IL-6 could inhibit the growth andproliferation of colon cancer cells by retroviralvector-mediated transduction.展开更多
AIM:To investigate the relationship between overexpression of urokinase plasminogen activator(uPA) and hepatitis B virus(HBV) related liver diseases in a transgenic mouse model.METHODS:Albumin-tetracycline reverse tra...AIM:To investigate the relationship between overexpression of urokinase plasminogen activator(uPA) and hepatitis B virus(HBV) related liver diseases in a transgenic mouse model.METHODS:Albumin-tetracycline reverse transcriptional activator and tetO-uPA transgenic mice were generated respectively through pronuclear injection and crossed to produce the double transgenic in-alb-uPA mice,for which doxycycline(Dox)-inducible and liver-specific over-expression of uPA can be achieved.Hydrodynamic transfection of plasmid adeno-associated virus(AAV)1.3HBV was performed through the tail veins of the Dox-induced in-alb-uPA mice.Expression of uPA and HBV antigens were analyzed through double-staining immunohistochemical assay.Cytokine production was detected by enzyme linked immunosorbent assay and α-fetoprotein(AFP) mRNA level was evaluated through real-time quantitative polymerase chain reaction.RESULTS:Plasmid AAV-1.3HBV hydrodynamic transfection in Dox-induced transgenic mice not only resulted in severe liver injury with hepatocarcinoma-like histological changes and hepatic AFP production,but also showed an increased serum level of HBV antigens and cytokines like interleukin-6 and tumor necrosis factor-α,compared with the control group.CONCLUSION:Over-expression of uPA plays a synergistic role in the development of liver injury,inflammation and regeneration during acute HBV infection.展开更多
Adult, male, Sprague-Dawley rats were injected with granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells （GM-CSF-BMSCs） into the ischemic boundary zone at 24 hours after onset of mi...Adult, male, Sprague-Dawley rats were injected with granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells （GM-CSF-BMSCs） into the ischemic boundary zone at 24 hours after onset of middle cerebral artery occlusion. Results showed reduced infarct volume, decreased number of apoptotic cells, improved neurological functions, increased angiogenic factor expression, and increased vascular density in the ischemic boundary zone in rats that underwent GM-CSF-BMSCs transplantation compared with the BMSCs group. Experimental findings suggested that GM-CSF-BMSCs could serve as a potential therapeutic strategy for ischemic stroke and are superior to BMSCs alone.展开更多
To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer dr...To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13 靏/mL, 10.95 靏/mL and 16.52 靏/mL, respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34 靏/mL, 7.48 靏/mL and 13.70 靏/mL, respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.展开更多
Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivatio...Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.展开更多
This study was designed to investigate the effects of local delivery of adipose-derived stem cells （ADSCs） transfected with transcription factor osterix （OSX） on bone formation during distraction osteogenesis. New...This study was designed to investigate the effects of local delivery of adipose-derived stem cells （ADSCs） transfected with transcription factor osterix （OSX） on bone formation during distraction osteogenesis. New Zealand white rabbits （n=54） were randomly divided into three groups （18 rabbits per group）. A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the dght mandible of all experimental rabbits, rabbits in group A were treated with ADSCs transfected with pEGFP-OSX, group B with ADSCs transfected with pEGFP-N1, and group C with physiological saline. Radiographic and histological examinations were processed after half of the animals within each group were humanely killed by injection of sodium pentothal at Week 2 or 6 after surgery. The distraction bone density was measured as its projectional bone mineral density （BMD）. Three parameters were measured, namely, the thickness of new trabeculae （TNT）, and the volumes of the newly generated cortical bone （NBV1） and the cancellous bone （NBV2） of the distracted regions. Good bone generation in the distraction areas was found in group A, which had the highest BMD, TNT, and NBV in the distraction zones among the groups. There was no significant difference in bone generation in the distraction areas between groups B and C. The results indicate that the transplantation of ADSCs transfected with pEGFP-OSX can effectively promote bone generation during distraction in vivo.展开更多
Macrophages(Mφs) are not only a kind of immune effector cells but also a kind of antigenpresenting cells(APC). In order to improve their antitumor effect, we transfected interferongamma(IFNγ) gene into Mφs by recom...Macrophages(Mφs) are not only a kind of immune effector cells but also a kind of antigenpresenting cells(APC). In order to improve their antitumor effect, we transfected interferongamma(IFNγ) gene into Mφs by recombinant adenovirus because IFNγis a kind of potent macrophageactivating factor(MAF). High level of IFNγ could be detected in the supernatants of Mφs after IFNγ gene transfection and IFNγ secretion peaked at 20 hour. The cytotoxicity of IFNγgenetransfected Mφs increased significantly. The secretion of TNF, IL1, nitric oxide(NO) also increased to some extent. The results demonstrated that recombinant adenovirusmediated IFNγ gene transfection could improve the effector functions of Mφs efficiently.展开更多
It’s well known that interleukin-2 (IL-2) plays animportant role in eliciting antitumor immunityparticularly mediated by T cells. In addition, theexpression of MHC can be enhanced by IL-2 genetransfection in tumor ce...It’s well known that interleukin-2 (IL-2) plays animportant role in eliciting antitumor immunityparticularly mediated by T cells. In addition, theexpression of MHC can be enhanced by IL-2 genetransfection in tumor cells. Recent studies have indicatedthat at least two signals are required for the activation ofnaive T cells by antigen-bearing target cells: an antigen-sopific signal and a crystimulatory signal. B7-l is展开更多
The prognosis for patients with primarymalignant glioma remains poor, in spite of a varietyof different forms of treatments. Broad suppressionof humoral and cell-mediated immunity is found inpatients with malignant gl...The prognosis for patients with primarymalignant glioma remains poor, in spite of a varietyof different forms of treatments. Broad suppressionof humoral and cell-mediated immunity is found inpatients with malignant gliomas. Interleukin-2 (IL-2)production and IL-2 receptor expression are展开更多
Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR...Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5’ and 3’ untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdr1 mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.展开更多
Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofec...Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofectamine. The expression of Cx43 was identified by Northern blot analyses, in situ hybridization and immunohistochemistry. MTT assay and average number of AgNORs (Argyrophlic nuclear organizer regions) were used to determine the cell proliferation. TUNEL method was used for detection of cell apoptosis, and scrape loading and dye tranfer method for examination of GJIC. Results: The Cx43 expression was greatly upregulated when Cx43 gene was transfected into TJ905 glioma cells. The cell proliferation was inhibited while the cell apoptosis was not increased and GJIC was significantly restored in the glioma cells transfected with Cx43 gene. Conclusion: Cx43 gene has an inhibitory effect on the glioma cell proliferation, but no effect on induction of cell apoptosis. The restoration of GJIC may be the major mechanism involved in its effect. Cx43 gene can be the candidate for gene therapy of gliomas.展开更多
OBJECTIVE: To study the function of costimulation sign in tumor immunology and construct the new cell line B7^+ Smmc7721. METHODS: The B7 gene was transfected into the hepatocarcinoma cell Smmc7721 by liposma. Polymer...OBJECTIVE: To study the function of costimulation sign in tumor immunology and construct the new cell line B7^+ Smmc7721. METHODS: The B7 gene was transfected into the hepatocarcinoma cell Smmc7721 by liposma. Polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR) were applied to test the result. MTT colorimetric assay was used to value the killing effect of lymphokine activated killer cells (LAK) activated by IL-2 on the transfected cell line and the original line. RESULT: B7^+ Smmc7721 was improved to be steadily expressed B7 molecule and LAK cells could more effectively act on the B7^+ Smmc7721 cells. CONCLUSION: The B7 gene can be transfected to hepatocarcinoma cells and can be expressed steadily in vitro, thus increasing the efficiency of LAK cells activated by IL-2 on them.展开更多
Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma tr...Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μl) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.展开更多
To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β ...To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β 1 and Lipofectamine Reagent in vitro . The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF β 1 gene could promote the osteogenic potential of cultured BMSCs.展开更多
文摘Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se requirements. We have continued to study Se regulation of GPX1 to better understand the underlying mechanism and to gain insight into how cells themselves regulate nutrient status. In progressive Se deficiency in rats, GPX1 activity,protein and mRNA all decrease in a dramatic, coordinated and exponential fashion such that Se-deficient GPX1 mRNA levels are 6-15% of Sexadequate levels. mRNA levels for other Sedependent proteins are far less decreased in the same animals. The mRNA levels for a second Se-dependent peroxidase, phospholipid hydroperoxide glutathione peroxidase (GPX4 ), are little affected by Se deficiency, demonstrating that Se regulation of GPX1 is unique. Se regulation of GPX1 activity in growing male and female rats shows that the Se requirernent is 100 ng/g diet, based on liver GPX1 activity; use of GPX1 mRNA as the parameter indicates that the Se requirement is nearer to 50 ng Se/g diet in both male and female rats. This approach will readily detect an altered dietary Se requirement, as shown by the incremental increases in dietary Se requirement by 150, 100 or 50 ng Se/g diet in Seudeficient rat pups repleted with Se for 3, 7 or 14 d, respectively. Studies with CHO cells stably transfected with recombinant GPX1 also show that overexpression of GPX1 does not alter the minimum level of media Se necessary for Se-adequate levels of GPX1 activity or mRNA. We hypothesize that classical GPX1 has an integral biological role in the mechanism used by cells to regulate Se status,making GPX1 an especially useful and effective parameter for determining Se requirements in animals
基金This work was supported by grantsfromThe Int .Cooperation Projectfor National &Abroad Lab.of the National Natural Sciences Foundation ofChina(2002008) and The Science &Technology Foundation of Liaoningprovince (20022140)
文摘Objective Loss-of-function mutation of p53,a tumor suppressor gene,is an important mechanism for the development of human cancers. In this study we tried to transfect p53N15-based fusion peptide into H1299,a lung cancer cell line,and evaluate the anti-tumor effects of the fusion peptide. Methods Adeno-associated virus (AAV) vectors were used for transfecting p53N15 fusion peptide into p53-null lung adenocarcinoma H1299 cells.
文摘AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in.three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.RESULTS： HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferationcapacity was obviously lower in HepG2/pcDNA3-X cells （0.08910±0.003164） than in HepG2 （0.14410±0.004927） and HepG2/pcDNA3 cells （0.12150±0.007159） （P〈0.05 and P〈0.01）. Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells （980/2 000） than HepG2 （420/2 000）, HepG2/pcDNA3 cells （520/2 000） （P〈0.05 and P〈0.01）. Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）. Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells （P〈0.05 and P〈0.01）. Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）.CONCLUSION： HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.
文摘AIM To observe the tumor inhibitory effects bytransfecting IL-6 cDNA into colon cancer cell lineHT-29 with retroviral vector pZIP cDNA.METHODS Human IL-6 gene was reconstructedin retrovirus vector and transfected into incasingcells PA317 by lipofectamine mediated method,the clones of the cells transferred with hlL-6were selected by G418,and targeted HT-29 cellswere infected with the virus granules secretedfrom PA317 and also selected by G418.Test genetranscription and expression level byhybridization,ELISA and MTT assay,etc.Analyze tumor inhibitory effects according to thecell growth curve,plating forming rate andtumorigenicity in nude mice.RESULT Successfully constructed andtransfected recombinant expressing vectorspZIPIL-6 cDNA and got positive transfected celllines.The colon cancer cell line(HT-29 IL-5)transfected with the hlL-5 gene by retroviralvector was established.The log proliferationperiod and the doubling time of this cell line wasbetween 4 to 7 days and 2.5 days according tothe direct cell count,the cell proliferation wasobviously inhibited with MTT assay,the platinginhibitory rate was 50% by plating efficiencytest.When HT-29 IL-6 cells were inoculated intothe nude mice subcutaneously,carcinogenicactivity of the solid tumor was found superior tothe control group and the size of tumor was notsignificantly enlarged.Injection of combinationvirus fluid containing 11.-6 gene intotransplantation tumors could inhibit the growthand development of the tumor.CONCLUSION IL-6 could inhibit the growth andproliferation of colon cancer cells by retroviralvector-mediated transduction.
基金Supported by National Program of Infection Diseases,No. 2012ZX10004-502The National High Technology Program ("863" Program) of China,No.2007AA02Z151
文摘AIM:To investigate the relationship between overexpression of urokinase plasminogen activator(uPA) and hepatitis B virus(HBV) related liver diseases in a transgenic mouse model.METHODS:Albumin-tetracycline reverse transcriptional activator and tetO-uPA transgenic mice were generated respectively through pronuclear injection and crossed to produce the double transgenic in-alb-uPA mice,for which doxycycline(Dox)-inducible and liver-specific over-expression of uPA can be achieved.Hydrodynamic transfection of plasmid adeno-associated virus(AAV)1.3HBV was performed through the tail veins of the Dox-induced in-alb-uPA mice.Expression of uPA and HBV antigens were analyzed through double-staining immunohistochemical assay.Cytokine production was detected by enzyme linked immunosorbent assay and α-fetoprotein(AFP) mRNA level was evaluated through real-time quantitative polymerase chain reaction.RESULTS:Plasmid AAV-1.3HBV hydrodynamic transfection in Dox-induced transgenic mice not only resulted in severe liver injury with hepatocarcinoma-like histological changes and hepatic AFP production,but also showed an increased serum level of HBV antigens and cytokines like interleukin-6 and tumor necrosis factor-α,compared with the control group.CONCLUSION:Over-expression of uPA plays a synergistic role in the development of liver injury,inflammation and regeneration during acute HBV infection.
基金supported by a grant from "135 Project" Foundation of the Public Health Department of Jiangsu Province,ChinaNanjing Medical Science and Technique Development Foundation
文摘Adult, male, Sprague-Dawley rats were injected with granulocyte-macrophage colony-stimulating factor-transfected bone marrow stromal cells （GM-CSF-BMSCs） into the ischemic boundary zone at 24 hours after onset of middle cerebral artery occlusion. Results showed reduced infarct volume, decreased number of apoptotic cells, improved neurological functions, increased angiogenic factor expression, and increased vascular density in the ischemic boundary zone in rats that underwent GM-CSF-BMSCs transplantation compared with the BMSCs group. Experimental findings suggested that GM-CSF-BMSCs could serve as a potential therapeutic strategy for ischemic stroke and are superior to BMSCs alone.
基金the National Natural Science Foundation of China.
文摘To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13 靏/mL, 10.95 靏/mL and 16.52 靏/mL, respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34 靏/mL, 7.48 靏/mL and 13.70 靏/mL, respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.
基金supported by the National Natural Science Foundation of China,No.30970995the Postgraduate Science Research Innovation Project of Higher Learning University of Jiangsu Province in China,No.CXLX11_0735
文摘Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA （siRNA）-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.
基金Project supported by the Seed Fund of the Second Hospital of Shandong University (No.S2013010024),China
文摘This study was designed to investigate the effects of local delivery of adipose-derived stem cells （ADSCs） transfected with transcription factor osterix （OSX） on bone formation during distraction osteogenesis. New Zealand white rabbits （n=54） were randomly divided into three groups （18 rabbits per group）. A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the dght mandible of all experimental rabbits, rabbits in group A were treated with ADSCs transfected with pEGFP-OSX, group B with ADSCs transfected with pEGFP-N1, and group C with physiological saline. Radiographic and histological examinations were processed after half of the animals within each group were humanely killed by injection of sodium pentothal at Week 2 or 6 after surgery. The distraction bone density was measured as its projectional bone mineral density （BMD）. Three parameters were measured, namely, the thickness of new trabeculae （TNT）, and the volumes of the newly generated cortical bone （NBV1） and the cancellous bone （NBV2） of the distracted regions. Good bone generation in the distraction areas was found in group A, which had the highest BMD, TNT, and NBV in the distraction zones among the groups. There was no significant difference in bone generation in the distraction areas between groups B and C. The results indicate that the transplantation of ADSCs transfected with pEGFP-OSX can effectively promote bone generation during distraction in vivo.
文摘Macrophages(Mφs) are not only a kind of immune effector cells but also a kind of antigenpresenting cells(APC). In order to improve their antitumor effect, we transfected interferongamma(IFNγ) gene into Mφs by recombinant adenovirus because IFNγis a kind of potent macrophageactivating factor(MAF). High level of IFNγ could be detected in the supernatants of Mφs after IFNγ gene transfection and IFNγ secretion peaked at 20 hour. The cytotoxicity of IFNγgenetransfected Mφs increased significantly. The secretion of TNF, IL1, nitric oxide(NO) also increased to some extent. The results demonstrated that recombinant adenovirusmediated IFNγ gene transfection could improve the effector functions of Mφs efficiently.
文摘It’s well known that interleukin-2 (IL-2) plays animportant role in eliciting antitumor immunityparticularly mediated by T cells. In addition, theexpression of MHC can be enhanced by IL-2 genetransfection in tumor cells. Recent studies have indicatedthat at least two signals are required for the activation ofnaive T cells by antigen-bearing target cells: an antigen-sopific signal and a crystimulatory signal. B7-l is
文摘The prognosis for patients with primarymalignant glioma remains poor, in spite of a varietyof different forms of treatments. Broad suppressionof humoral and cell-mediated immunity is found inpatients with malignant gliomas. Interleukin-2 (IL-2)production and IL-2 receptor expression are
文摘Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5’ and 3’ untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdr1 mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.
基金This work was supported by the National Natural Science Foundation of China (No. 39870815).
文摘Objective: To evaluate the effect of Cx43 gene on gap junction intercellular communication (GJIC) and proliferation of glioma cells. Methods: Cx43 cDNA was transfected into TJ905 human glioblastoma cells using lipofectamine. The expression of Cx43 was identified by Northern blot analyses, in situ hybridization and immunohistochemistry. MTT assay and average number of AgNORs (Argyrophlic nuclear organizer regions) were used to determine the cell proliferation. TUNEL method was used for detection of cell apoptosis, and scrape loading and dye tranfer method for examination of GJIC. Results: The Cx43 expression was greatly upregulated when Cx43 gene was transfected into TJ905 glioma cells. The cell proliferation was inhibited while the cell apoptosis was not increased and GJIC was significantly restored in the glioma cells transfected with Cx43 gene. Conclusion: Cx43 gene has an inhibitory effect on the glioma cell proliferation, but no effect on induction of cell apoptosis. The restoration of GJIC may be the major mechanism involved in its effect. Cx43 gene can be the candidate for gene therapy of gliomas.
文摘OBJECTIVE: To study the function of costimulation sign in tumor immunology and construct the new cell line B7^+ Smmc7721. METHODS: The B7 gene was transfected into the hepatocarcinoma cell Smmc7721 by liposma. Polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR) were applied to test the result. MTT colorimetric assay was used to value the killing effect of lymphokine activated killer cells (LAK) activated by IL-2 on the transfected cell line and the original line. RESULT: B7^+ Smmc7721 was improved to be steadily expressed B7 molecule and LAK cells could more effectively act on the B7^+ Smmc7721 cells. CONCLUSION: The B7 gene can be transfected to hepatocarcinoma cells and can be expressed steadily in vitro, thus increasing the efficiency of LAK cells activated by IL-2 on them.
文摘Summary: The expression of protein vascular endothelial growth factor (VEGF) in osteosarcoma cells transfected with adeno-associated virus (rAAV)-antisense VEGF was studied to provide the foundation of osteosarcoma treatment through antivascularization. The rAAV-antisense VEGF at different doses (0, 20, 50, 100, 200, 240 μl) was transfected into osteosarcoma MG-63 cell. The cells and culture supernatants were collected before and after tansfection. The expression of VEGF protein was detected by using immunohistochemical staining (SP) and Western blot. SP and Western-blot tests revealed that the MG-63 Cells transfected with rAAV-antisense VEGF had less staining than those without transfection with rAAV-antisense VEGF, and the staining intensity was negatively correlated with the doses of genes. The corresponding A values of transfected genes with different doses of rAAV-antisense VEGF (0, 20, 50, 100, 200, 240 μl) were 86 614±13 776, 73 245±15 414, 61 078±12 124, 54 657±10 953, 39 802±11 308, 32 014±15 057 respectively, with the difference being significant (P<0.05). It was concluded that the expression of VEGF protein in MG-63 cells could be inhibited by rAAV-antisense VEGF.
文摘To study the osteogenic potential of cultured bone marrow stromal cells transfected with transforming growth factor β 1 gene in vitro , cultured BMSCs were transfected with the complexes of pcDNA 3 TGF β 1 and Lipofectamine Reagent in vitro . The cell proliferation was detected by MTT method and the morphological features of transfected BMSCs was observed. ALP stains and PNP method were used to measure ALP activity. In addition, the collagen type Ⅰ propeptides and mineralized matrixes were examined by immunohistochemical staining and tetracycline fluorescence labeling respectively. The morphological and biological characters of the transfected BMSCs were similar to those of osteoblasts and the cell proliferation was promoted. The cell layer displayed strong positive reaction for ALP stains and immunohistochemical staining. ALP activity and collagen type Ⅰ expression increased remarkably after transfection. Mineralized matrixes formed earlier and more in transfected BMSCs as compared with control group. It is concluded that transfecting with TGF β 1 gene could promote the osteogenic potential of cultured BMSCs.
