摘要
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 Bone marrow mesenchymal stem cells（BMSCs） were transfected using recombinant adenovirus vector carring hBcl-2/hVEGF165 co-expression genes for assessment of expression and biological effects of these.Methods Rat BMSCs were isolated and purified by attachment culture methed,subsequently transfected with recombinant adenovirus vector.Targeted mRNA expression was quantified by PCR.Western Blot and Elisa were applied to assess protein expression of Bcl-2 and hVEGF165 in transfected cells.Finally,the biological effects of hVEGF165 and Bcl-2 in were observed via MTT and apoptosis detection.Results Bone marrow mesenchymal stem cells were separated and purified and transfected by recombinant adenovirus successfully.The mRNA of hBcl-2 and hVEGF165 were detected after transfection.hVEGF165 in the supernatant of transfected cells was detected,with peak concentration of（924.3±56.5）pg/mL,by Elisa Kit,with significant differences comparing to those in control group in at different time spots（P0.05）.Meanwhile,significant increase of Bcl-2 protein was detected in transfected group by Western（P0.05）.Proliferation of BMSCs was significantly promoted with hVEGF165-containing supernatant using MTT assay（P0.05）.Significant reduction of apoptosis was also observed in transfected BMSCs using annexin V-PE/7-AAD detection（P0.05）.Conclusion Transfected BMSCs could express functional target genes,hBcl-2 and hVEGF165.
出处
《广东医学》
CAS
CSCD
北大核心
2011年第5期548-551,共4页
Guangdong Medical Journal
