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目的将hBcl-2和hVEGF165双基因共表达重组腺病毒载体转染大鼠骨髓间充质干细胞(BMSCs),检测目的基因的表达及表达产物的生物学效应。方法贴壁培养法分离纯化大鼠骨髓间充质干细胞;重组腺病毒转染BMSCs,用PCR法验证目的基因mRNA在间充质干细胞中的表达;用ELISA及Western Blot检测双基因在转染后细胞的蛋白表达量;用MTT法及Annexin V-PE/7-AAD法分别观察对转染后BMSCs增殖及凋亡的影响。结果大鼠BMSCs分离纯化成功。在mRNA水平,转染后的BMSCs可表达这两个外源基因;转染后细胞培养上清液hVEGF165峰浓度达到(924.3±56.5)pg/mL,多个时间点与对照组相比,浓度差异有统计学意义(P<0.05);Western Blot也检测到转染后细胞Bcl-2蛋白表达量明显比对照组增加(P<0.05)。MTT比色法观察到,转染细胞分泌的hVEGF165上清液可促进BMSCs的增殖(P<0.05);用Annexin V-PE/7-AAD检测转染后间充质干细胞的凋亡率下降,与对照组相比差异有统计学意义(P<0.05)。结论转染重组腺病毒载体的BMSCs能够表达hBcl-2及hVEGF165,并且表达产物具有生物学效应。
Objective To transfect rat bone marrow mesenchymal stem cells (BMSCs) with recombinant adenovirus vector expressing hBcl-2 and hVEGF165 genes to detect the expression of the target gene and the biological effects of the expressed product. Methods Bone marrow mesenchymal stem cells (MSCs) were isolated and purified by adherent culture. The recombinant adenovirus was transfected into BMSCs. The expression of target gene mRNA was detected by PCR in mesenchymal stem cells. The double gene was detected by ELISA and Western Blot The effects of MTT assay and Annexin V-PE / 7-AAD method on the proliferation and apoptosis of BMSCs were observed. Results Rat BMSCs were isolated and purified successfully. At the mRNA level, the transfected BMSCs could express the two exogenous genes; the peak concentration of hVEGF165 in the cell culture supernatant reached (924.3 ± 56.5) pg / mL after transfection. Compared with the control group, (P <0.05). Western Blot also detected that the expression of Bcl-2 protein in transfected cells was significantly higher than that in control cells (P <0.05). The MTT assay showed that the hVEGF165 supernatant secreted by transfected cells could promote the proliferation of BMSCs (P <0.05). The apoptosis rate of mesenchymal stem cells after transfection was detected by Annexin V-PE / 7-AAD, Compared with the control group, the difference was statistically significant (P <0.05). Conclusion BMSCs transfected with recombinant adenovirus can express hBcl-2 and hVEGF165, and the expressed products have biological effects.