| OBJECTIVES: Tissue-type plasminogen activator(tPA) gene was transduced into neonatal rat myocardial fibroblasts using newly constructed pseudotyped MuLV retroviral vector( VSV-G/MuLV) ,Determining the expression of tPA gene in myocardial fibroblasts and investigating whether neonatal rat myocardial fibroblasts could be the novel target cells of genetic therapy of anti-thrombosis.METHODS: (1) The envelope G glycoprotein from the vesicular stomatitis virus (VSV-G) could completely replace the env protein of MuLV to construct VSV-G pseudotyped MuLV retroviral vector ( VSV-G/MuLV ) which was originally produced from transient transfection of 293T/17 cell line with 3 plasmids with the method of calcium phosphate precipitation: CnBgSN is a viral vector plasmid carrying the lacZ report gene and neor drug resistant gene. HIT60 isplasmid carrying gag andpol viral genes. CVG is the plasmid for VSV-G envelope expression; The producer cell 293/GPG for VSV-G/MuLV was generated; The supernatant of VSV-G/MuLV was collected and its titer was determined. (2) Neonatal rat myocardial fibroblasts were isolated and cultured with the technique of differential anchoring velocity for two times, 60 minutes each time; Morphologic characters of those cells were observed with the inverted microscope; Immunocytochemical staining was used to evaluate and confirm myocardial fibroblasts. (3) Pseudotyped MuLV retroviral vector carrying tPA gene( VSV-G/tPA) was constructed using the way of transient transfection of 293T cell line with 3 plasmids: HIT60 is plasmid carrying gag and pol viral genes, CVG for VSV-G envelope expression, LtSN for tPA gene; The Collection of the supernatant of VSV-G/tPA and the determination of virus titer were performed after 293/GPG cell line was transduced with VSV-G/tPA; After neonatal rat myocardial fibroblasts were transduced with VSV-G/tPA and selected with G418, Clones of neonatal rat myocardial fibroblasts were observed; The dynamic change of tPA activity of the supernatant of cultured myocardial fibroblasts after tPA gene transduction was measured with chromogenic peptide substrate assay; The expression of tPA gene was determined using immunocytochemical staining.RESULTS: (1) The virus titer of VSV-G/MuLV was 6 106. (2) The model of cell culture of neonatal rat myocardial fibroblasts wassuccessfully established using the technique of differential anchoring velocity for two times. The purity of cells cultured was more than 95%; The character of cardiac fibroblasts cultured was evaluated and confirmed by using morphological observation, and immunocytochemical staining using antibody against vimentin indicated that the immunoreaction was strongly positive, meaning the percent of positive cells in which nucleus was surrounded by brown particles was more than 95% of all cells; Immunocytochemical staining using antibody against actin showed the immunoreaction was negative, those positive cells whose nucleus was surrounded by brown particles was less than 5% of all cells. (3) The virus titer of VSV-G/tPA was 4 106; Clones of neonatal rat myocardial fibroblasts transduced with VSV-G/tPA were formed about 2 weeks later after transduction; The tPA activity of the supernatant of gene-transduced myocardial fibroblasts was increased 1 day later, more obvious 2 days later and the highest during 3 to 5 days after transduction. The difference of tPA activity between the 1th or 2th day and the 3th, 4th, 5th day was significant separatly, no notable difference was found between the 1th day and 2th day, neither was among the 3th, 4th, 5th day. Immunocytochemical staining using antibody against tPA antigen in myocardial fibroblasts transduced with tPA gene indicated that the immunoreaction was strongly positive, but negative in those not transduced with tPA gene.CONCLUSIONS: tPA gene was successfully transduced into neonatal rat myocardial fibroblasts using vector VSV-G/MuLV, and the effective expression of tPA gene was determined in myocardial fibroblasts transduced with... |
PDF Full Text Request