Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Method The recombinant plasmid pCD-rbFGF was mixed with collagen and was implanted in the necrotic femoral head. Expression ...Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Method The recombinant plasmid pCD-rbFGF was mixed with collagen and was implanted in the necrotic femoral head. Expression of basic fibroblast growth factor (bFGF) was examined by RT-PCR and immunohistochemical method. Re-pair of the femoral head was observed by histological and histomorphometric analysis. Result Expression of bFGF was detected in the femoral head transfected with bFGF gene, indicating significant increase of angiogenesis 2 weeks after gene transfection and increased new bone formation 8 weeks after gene transfection on histom-orphometric analysis (P< 0.01). Conclusion Transfection of bFGF gene enhances bone tissue angiogenesis. Repair in osteonecrosis would be accelerated accordingly.展开更多
In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization I...In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization IncRNA-malat1l,qPCR was used to detect the expression of IncRNA-malat1 before and after transfection of the interference vector.It was found that malat1 was not silenced by IncRNA Smart silencers plasmid.qPCR results showed no significant difference in expression level of malat1 mRNA before and after transfection smart silencers plasmid of malat1 at 24 h,48h,3 d and7 d.The subcellular localization analysis found that malat1 was mainly located in the nucleus.It is speculated that it is difficult to achieve silencing of lncRNA located in the nucleus by applying interference technology of siRNA plasmid vector.qPCR results showed that the expression level of malat1 mRNA before and after malat1 transfection was significantly decreased at 24 h and 3 d compared with the normal group and the no-load group(P<0.05).In conclusion,it was suggested that the SGCs of DRG belonged to primary cells.展开更多
Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications,such as CAR T cell-based therapie...Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications,such as CAR T cell-based therapies.mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral-or DNA transposon-based approaches for the generation of adoptive T cells.However,limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells.In this study,we demonstrate that gold nanoparticle-mediated vapor nanobubble(VNB)photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells.Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration,while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%.Importantly,compared to electroporation,which is the reference technology for nonviral transfection of T cells,a fivefold increase in the number of transfected viable Jurkat T cells was observed.Altogether,our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells,with promising potential for the future engineering of cells in therapeutic and fundamental research applications.展开更多
To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector...To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.展开更多
In order to determine the effect of foreign genes on a transgenic parasite,the pathogenicity and development in chickens of transgenic E.tenella strain TE1,which expresses yellow fluorescent protein (YFP) and dihydrof...In order to determine the effect of foreign genes on a transgenic parasite,the pathogenicity and development in chickens of transgenic E.tenella strain TE1,which expresses yellow fluorescent protein (YFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii(TgDHFR-TS), were compared with that of the parental strain BJ.Results indicated that the fecundity of the transgenic parasite(TE1) was reduced at least 4 times relative to that of the BJ strain.Low dosage of the TE1 strain induced less pathogenesis in chickens than did the BJ strain,but chickens inoculated with a higher dosage of TE1 oocysts displayed severe pathogenicity and mortality as did the BJ strain.In addition,trophozoites, the first generation and the second generation meronts and merozoites,microgamonts and macrogamonts of the transgenic parasite TE1 were seen by fluorescence microscopy.More interestingly,not all four sporonts in the sporulating transgenic oocysts express YFP.These findings suggest that the expression of YFP and TgDHFR-TS genes to some extent reduced pathogenicity and reproductive potential of transgenic E.tenella.Recombination between homologous or non-homologous chromosomes occurred during zygotic meiosis in E.tenella strain TE1.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 construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by P...Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.展开更多
The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and M...The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and Mab BuGR1(recognizing the DNA binding domain of GR_H).The specific binding peak of 0.98μmol/L[~3H]triamcinolone acetonide(TA)was higher than that of 52.50nmol/L[~3H]TA.The re-sult suggests that the antigenic determinants of GR_L are similar to those of GR_H.ThecDNA of rat liver GR_H was introduced into GR_H-and GR_L-negative mouse fibroblast cellline E82.A3 by calcium phosphate coprecipitation.A number of clones which expressGR_H were selected with G418(400μg/ml).The results of radioligand binding assay indicatethat GR_H gene is expressed successfully and GR_L also may be encoded by GR_H gene.展开更多
objective: To construct a human Bax eukaryotic expression vector and detect its expression in hu man drug resistant gastric cancer cells. Methods: Bax gene was first inserted into polyclonal sites of pBK CMV vector. T...objective: To construct a human Bax eukaryotic expression vector and detect its expression in hu man drug resistant gastric cancer cells. Methods: Bax gene was first inserted into polyclonal sites of pBK CMV vector. Then the recombinant plasmid vector was trans fected into vincristine resistant human gastric cancer cells SGC7901/VCR by lipofectamine. G418 was used to select resistant clones. Lastly, Western blot was used to observe the expression of Bax protein. Results: Recombinant plasmids were successfully con structed. When the plasmids were trans fected into SGC7901/VCR cellsl stable trans fected cell clones were obtained 30 d after G418 selection. The Western blotting demonstrated a significant expression of Bax protein in pBK-Bax cDNA trans fected cells. Conclusion: This construction of Bax gene transfectant will be conducive to a further study of the role of Bax in gastric cancer multi-drug-resistance.展开更多
Objective: To identify eukaryotic expression vector of human bone morphogenetic protein 2 pcDNA3/BMP2, verify its expression in transfected human mesenchymal stem cells (hMSCs) and the effect on hMSCs differentiation....Objective: To identify eukaryotic expression vector of human bone morphogenetic protein 2 pcDNA3/BMP2, verify its expression in transfected human mesenchymal stem cells (hMSCs) and the effect on hMSCs differentiation. Methods: The BMP2 gene was cloned into a eukaryotic expression vector pcDNA3. Transfected the recombinant into hMSCs by liposome. Immunnohistochemistry and in situ hybridization methods were used to identify the expression of BMP2 mRNA and protein; ALP and Von Kossa stains were performed to identify the BMP2 gene differentiated effect on the hMSCs. Results: The pcDNA3/BMP2 fragments were as large as theory. BMP2 mRNA and protein were expressed and synthesized both in 48 h and 4 weeks after transfection, the ALP and Ca deposit exhibition, which marked the osteogenic lineage of hMSCs, were enhanced and sped. Conclusion: Transfection of pcDNA3/BMP2 is able to provide transient and persistent expression in hMSCs, and promote the MSCs differentiation to osteogenic lineage.展开更多
OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression o...OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.展开更多
Breast cancer containing estrogen receptors (ER ) are responslve to antiestrogen treatment and have a better prognosis compared with ER-negative tumors. The loss of estrogen receptors appears to be associated with a p...Breast cancer containing estrogen receptors (ER ) are responslve to antiestrogen treatment and have a better prognosis compared with ER-negative tumors. The loss of estrogen receptors appears to be associated with a progression to less clifferentiated cells. We transfected the human ER into the ER-negative breast cancer cell line MDA MD 231 cells. We found that expresslon of adequate ER is strong associated with the ability of human breast cancer cell growth inhibition and progression. Compared with nontransfected or mock-trans fected cells, ER-transfected cells exhibited growth slower, forming smaller colonles in soft agar and growth inhibited by estrogen and tamoxifen. Therefore reactivation or transfection of the estrogen receptor gene can be considered as therapeutic approaches to hormone-independent breast cancer.展开更多
Objective To investigate the cleavage activities of ribozyme RCP in blocking HBV gene expression and replication in eukaryotic cells. Methods: The recombinant plasmid PCR/pSVL which can transiently express ribozyme RC...Objective To investigate the cleavage activities of ribozyme RCP in blocking HBV gene expression and replication in eukaryotic cells. Methods: The recombinant plasmid PCR/pSVL which can transiently express ribozyme RCP in eukaryotic cells was constructed and introduced into HepG2215 cell line with the technique of lipofectamine-mediated gene transfer. The vector pSVL transfection group served as the control. HB-sAg and HBsAg from culture medium of the cells were tested with solid-phase radioimmunoassay. Results:The cpm obtained from the medium samples showed that the inhibition rate of ribozyme RCP for HBsAg andHBeAg was 26. 7% and 24. 8% respectively in this transient expression system. Conclusion:Hammerhead ribozyme RCP can inhibit to some extent the expression of HBV gene.展开更多
Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and ce...Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth. Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spec-trophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCTl was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.展开更多
基金Supported by the National Natural Sciences Foundation of China(30170945).
文摘Objective To explore a new method for the therapy of avascular necrosis of the femoral head. Method The recombinant plasmid pCD-rbFGF was mixed with collagen and was implanted in the necrotic femoral head. Expression of basic fibroblast growth factor (bFGF) was examined by RT-PCR and immunohistochemical method. Re-pair of the femoral head was observed by histological and histomorphometric analysis. Result Expression of bFGF was detected in the femoral head transfected with bFGF gene, indicating significant increase of angiogenesis 2 weeks after gene transfection and increased new bone formation 8 weeks after gene transfection on histom-orphometric analysis (P< 0.01). Conclusion Transfection of bFGF gene enhances bone tissue angiogenesis. Repair in osteonecrosis would be accelerated accordingly.
文摘In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization IncRNA-malat1l,qPCR was used to detect the expression of IncRNA-malat1 before and after transfection of the interference vector.It was found that malat1 was not silenced by IncRNA Smart silencers plasmid.qPCR results showed no significant difference in expression level of malat1 mRNA before and after transfection smart silencers plasmid of malat1 at 24 h,48h,3 d and7 d.The subcellular localization analysis found that malat1 was mainly located in the nucleus.It is speculated that it is difficult to achieve silencing of lncRNA located in the nucleus by applying interference technology of siRNA plasmid vector.qPCR results showed that the expression level of malat1 mRNA before and after malat1 transfection was significantly decreased at 24 h and 3 d compared with the normal group and the no-load group(P<0.05).In conclusion,it was suggested that the SGCs of DRG belonged to primary cells.
基金Funding by the European Research Council(ERC)under the European Union’s Horizon 2020 research and innovation program(Grant No.648124)is acknowledged with gratitudeS.S.acknowledges the support of a VLAIO Grant(Grant Number:HBC.2017.0542.)+2 种基金J.C.F.(FWO Grant 1210120 N)J.V.H(FWO-SB grant 1S62519N)and R.X.(FWO Grants 1500418 N and 12Q8718N)gratefully acknowledge the financial support by the Flemish Research FoundationL.V.H.is a junior assistant of the Department of Biomedical Molecular Biology.
文摘Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications,such as CAR T cell-based therapies.mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral-or DNA transposon-based approaches for the generation of adoptive T cells.However,limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells.In this study,we demonstrate that gold nanoparticle-mediated vapor nanobubble(VNB)photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells.Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration,while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%.Importantly,compared to electroporation,which is the reference technology for nonviral transfection of T cells,a fivefold increase in the number of transfected viable Jurkat T cells was observed.Altogether,our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells,with promising potential for the future engineering of cells in therapeutic and fundamental research applications.
文摘To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.
基金supported by the National High Technology Research and Development Program of China(Project No. 2006AA02Z458)the Doctor Startup Foundation of Henan University of Science and Technology(09001350)
文摘In order to determine the effect of foreign genes on a transgenic parasite,the pathogenicity and development in chickens of transgenic E.tenella strain TE1,which expresses yellow fluorescent protein (YFP) and dihydrofolate reductase thymidylate synthase derived from Toxoplasma gondii(TgDHFR-TS), were compared with that of the parental strain BJ.Results indicated that the fecundity of the transgenic parasite(TE1) was reduced at least 4 times relative to that of the BJ strain.Low dosage of the TE1 strain induced less pathogenesis in chickens than did the BJ strain,but chickens inoculated with a higher dosage of TE1 oocysts displayed severe pathogenicity and mortality as did the BJ strain.In addition,trophozoites, the first generation and the second generation meronts and merozoites,microgamonts and macrogamonts of the transgenic parasite TE1 were seen by fluorescence microscopy.More interestingly,not all four sporonts in the sporulating transgenic oocysts express YFP.These findings suggest that the expression of YFP and TgDHFR-TS genes to some extent reduced pathogenicity and reproductive potential of transgenic E.tenella.Recombination between homologous or non-homologous chromosomes occurred during zygotic meiosis in E.tenella strain TE1.
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.
文摘Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-K by PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results: The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing. RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.
文摘The homogeneity between the high-and low-affinity glucocorticoid receptors(GR_H and GR_L)was testified by immunoaffinity chromatography using Mab N250(recognizing the immunogenic domain at the N terminal of GR_H)and Mab BuGR1(recognizing the DNA binding domain of GR_H).The specific binding peak of 0.98μmol/L[~3H]triamcinolone acetonide(TA)was higher than that of 52.50nmol/L[~3H]TA.The re-sult suggests that the antigenic determinants of GR_L are similar to those of GR_H.ThecDNA of rat liver GR_H was introduced into GR_H-and GR_L-negative mouse fibroblast cellline E82.A3 by calcium phosphate coprecipitation.A number of clones which expressGR_H were selected with G418(400μg/ml).The results of radioligand binding assay indicatethat GR_H gene is expressed successfully and GR_L also may be encoded by GR_H gene.
文摘objective: To construct a human Bax eukaryotic expression vector and detect its expression in hu man drug resistant gastric cancer cells. Methods: Bax gene was first inserted into polyclonal sites of pBK CMV vector. Then the recombinant plasmid vector was trans fected into vincristine resistant human gastric cancer cells SGC7901/VCR by lipofectamine. G418 was used to select resistant clones. Lastly, Western blot was used to observe the expression of Bax protein. Results: Recombinant plasmids were successfully con structed. When the plasmids were trans fected into SGC7901/VCR cellsl stable trans fected cell clones were obtained 30 d after G418 selection. The Western blotting demonstrated a significant expression of Bax protein in pBK-Bax cDNA trans fected cells. Conclusion: This construction of Bax gene transfectant will be conducive to a further study of the role of Bax in gastric cancer multi-drug-resistance.
文摘Objective: To identify eukaryotic expression vector of human bone morphogenetic protein 2 pcDNA3/BMP2, verify its expression in transfected human mesenchymal stem cells (hMSCs) and the effect on hMSCs differentiation. Methods: The BMP2 gene was cloned into a eukaryotic expression vector pcDNA3. Transfected the recombinant into hMSCs by liposome. Immunnohistochemistry and in situ hybridization methods were used to identify the expression of BMP2 mRNA and protein; ALP and Von Kossa stains were performed to identify the BMP2 gene differentiated effect on the hMSCs. Results: The pcDNA3/BMP2 fragments were as large as theory. BMP2 mRNA and protein were expressed and synthesized both in 48 h and 4 weeks after transfection, the ALP and Ca deposit exhibition, which marked the osteogenic lineage of hMSCs, were enhanced and sped. Conclusion: Transfection of pcDNA3/BMP2 is able to provide transient and persistent expression in hMSCs, and promote the MSCs differentiation to osteogenic lineage.
文摘OBJECTIVE: To explore the possibility of expression of exogenous gene in transduced bone marrow derived stromal cells (BMSCs). METHODS: The marker gene, pbLacZ, was transferred into cultured BMSCs and the expression of transduced gene by X-gal staining was examined. Then plasmid pcDNA3-rhBMP7 was delivered to cultured BMSCs. Through immunohistochemical staining and RT-PCR assay, the expression of rhBMP7 gene was detected. RESULTS: The exogenous gene could be expressed efficiently in transduced BMSCs. CONCLUSION: The present study provided a theoretical basis to gene therapy on the problems of bone and cartilage tissue.
文摘Breast cancer containing estrogen receptors (ER ) are responslve to antiestrogen treatment and have a better prognosis compared with ER-negative tumors. The loss of estrogen receptors appears to be associated with a progression to less clifferentiated cells. We transfected the human ER into the ER-negative breast cancer cell line MDA MD 231 cells. We found that expresslon of adequate ER is strong associated with the ability of human breast cancer cell growth inhibition and progression. Compared with nontransfected or mock-trans fected cells, ER-transfected cells exhibited growth slower, forming smaller colonles in soft agar and growth inhibited by estrogen and tamoxifen. Therefore reactivation or transfection of the estrogen receptor gene can be considered as therapeutic approaches to hormone-independent breast cancer.
文摘Objective To investigate the cleavage activities of ribozyme RCP in blocking HBV gene expression and replication in eukaryotic cells. Methods: The recombinant plasmid PCR/pSVL which can transiently express ribozyme RCP in eukaryotic cells was constructed and introduced into HepG2215 cell line with the technique of lipofectamine-mediated gene transfer. The vector pSVL transfection group served as the control. HB-sAg and HBsAg from culture medium of the cells were tested with solid-phase radioimmunoassay. Results:The cpm obtained from the medium samples showed that the inhibition rate of ribozyme RCP for HBsAg andHBeAg was 26. 7% and 24. 8% respectively in this transient expression system. Conclusion:Hammerhead ribozyme RCP can inhibit to some extent the expression of HBV gene.
文摘Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth. Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spec-trophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCTl was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.
