Transfection Of Rat Bone Marrow Mesenchymal Stem Cells With Adenovirus Vector Carrying HBCL-2 And HVEGF₁₆₅ Genes

Objective:To establish the experimental method for isolating, culturing and amplifying bone marrow mesenchymal stem cells (BMSCs) in vitro in rats. BMSCs were transfected using a recombinant adenovirus vector with hBCL-2/hVEGF₁₆₅ co-expression genes. The expression level of genes and their biological effects were detected, which offered research data for further animal experiments and clinical studies.Methods:1. Bilateral femora and tibiae were extracted from SD rats, and the cavity of bone marrow was washed repetitively using H-DMEM culture cells. The lavage fluid was added into the culture bottles. And BMSCs were isolated, cultured and proliferated using the wall-adhering method. Changes in cellular morphology and the growth status were observed under microscopic visions.2. Identification on surface antigen-determining cluster (CD) on the collected third-generation cells (P3) was performed using flow cytometry. BMSCs were transfected using the recombinant adenovirus solution with different volume but the same concentration, and the optimal multiplication of infection (MOI) was obtained through detecting the expression of green fluorescent protein by flow cytometry.3. After obtaining BMSCs with the optimal MOI, the cells were lysated to extract the total RNA, hBCL-2/hVEGF₁₆₅-specific primers were designed, and the expression of target genes could be verified by PCR in mesenchymal stem cells in the mRNA level. Then the supernatant of transfected cells was collected, and the concentration of secretion protein VEGF₁₆₅ was detected using enzyme linked immunosorbent assay (ELISA). Meanwhile, the transfected BMSCs were lysated to extract the protein, and Western Blot was adopted to detect the expression level of the intracellular protein BCL-2, thereby measuring the expression of both genes in protein expression level.4. Finally, the biological effect of VEGF₁₆₅ and BCL-2 in transfected BMSCs was observed through MTT and apoptosis detection.Results:1. Bone marrow mesenchymal stem cells have been separated and purified successfully. Spindle or polygonal primary cells could be observed, and the cell proliferation rate accelerated significantly after passage. The coverage of cells reached 90% of the flask bottom. The cells in good conditions and high quantity were obtained to complete the following experiment.2. CD29/CD44/CD90 positive and CD34/CD45 negative cells were revealed through detecting the surface markers of BMSCs by flow cytometry, which was consistent with the character of BMSCs. BMSCs were transfected by recombinant adenovirus solution with different volumes. Through flow cytometry detection of the green fluorescent protein, the optimal multiplication of infection was 400.3. And then BMSCs were transfected by recombinant adenovirus according to the optimal MOI, and two foreign genes were detected after transfection. For protein expression, it was detected that the peak concentration of VEGF₁₆₅ in the supernatant of transfected cells reached (924.3±56.5) pg/ml by Elisa Kit. There was a statistical significance (p<0.05) in the concentration in multiple time points, and it was also detected by Western Bolt that the expression of BCL-2 protein increased significantly compared with the control group.4. Proliferation of BMSCs was promoted when observing the VEGF₁₆₅-containing cell supernatant using MTT assay, which indicated that the supernatant had significant biological effects (p<0.05). There was a statistical significance (p<0.05) in the apoptotic rate of transfected BMSCs when using annexin V-PE/7-AAD for detection.Conclusions:1. Our laboratory had constructed the co-expression recombinant adenovirus vector carrying hBCL-2/hVEGF₁₆₅ genes, which could transfect BMSCs successfully.2. Transfected BMSCs could express target genes -- hBCL-2 and hVEGF₁₆₅, whose expression products had significant biological effects.