VEGF_(165)质粒转染骨髓间充质干细胞立体定向移植对局灶性缺血大鼠脑血管新生的影响

Influence of VEGF_(165) plasmid transfection BMSCs stereotactic transplantation in angiogenesis of focal ischemia rats

目的观察pEGFP-VEGF165质粒转染骨髓间充质干细胞（BMSCs）可否促进脑缺血大鼠血管新生。方法全骨髓贴壁法分离获取第三代BMSCs,当BMSCs生长汇合率为80%-90%时以脂转法转染pEGFP-VEGF165质粒。制作大鼠大脑中动脉闭塞（MCAO）模型,分为模型组、BMSCs移植组（BMSCs组）、转血管内皮生长因子（VEGF）基因BMSCs移植组（BMSCs/VEGF组）。于造模后7、14、21 d进行神经行为学评分,免疫组织化学染色检测脑缺血边缘区血管内皮细胞标志物CD34和VEGF的表达,激光共聚焦显微镜观察血管生成情况。结果 BMSCs组与BMSCs/VEGF组大鼠神经功能（BRS）评分较模型组明显降低（P〈0.01）,BMSCs/VEGF组BRS评分低于BMSCs组,但差异无统计学意义（P〉0.05）。与非缺血侧比较,BMSCs组和BMSCs/VEGF组缺血侧微血管直径、微血管面积均较其显著增加（P〈0.05或P〈0.01）;BMSCs组和BMSCs/VEGF组微血管直径、微血管面积均较模型组显著增加（P〈0.05）,但BMSCs组与BMSCs/VEGF组比较,差异无统计学意义（P〉0.05）。7 d时BMSCs/VEGF组VEGF阳性细胞较BMSCs组VEGF表达显著增加（P〈0.05）,其他时间点两组比较差异无统计学意义（P〉0.05）;BMSCs/VEGF组CD34阳性表达在各时间点较BMSCs组有所增加,但差异无统计学意义（P〉0.05）。结论经VEGF基因修饰的BMSCs移植可提高缺血区血管生成,仅在移植后7 d比单纯BMSCs移植VEGF表达升高有显著性差异,在神经行为学评分、微血管新生、CD34表达等方面与单纯BMSCs比较,无明显优势。原因可能与VEGF165质粒转染效率不高及脑血管新生机制复杂有关。

Objective To observe pEGFP-VEGF165 plasmid transfection BMSCs whether promote angiogenesis of focal ischemia rats. Methods The third generation of BMSCs were isolated by whole bone marrow adherence method. As BMSCs growth convergence rate reached 80%-90%, pEGFP-VEGF165 plasmid transferred by lipid transfer method. Rats were used to make MCAO model, including model group, BMSCs group, BMSCs/VEGF group. Neurological behavior score finished after model 7, 14, 21 d. Expression of vascular endothelial cell markers CD34 and VEGF were tested by immunohistochemistry staining. Angiogenesis was observed by confocal laser scanning microscopy. Results BRS scores in BMSCs group and BMSCs/VEGF group were decreased significantly than those that in model group（P〈 0.01）, BRS scores in BMSCs/VEGF group were lower than those in BMSCs group, but the difference was not statistically significant（P〉0.05）. Compared with non-ischemic side, microvessel diameter and area increased significantly in ischemic side in BMSCs group and BMSCs/VEGF group（P〈0.05 or P 0.01）. Microvessel diameter and area in BMSCs group and BMSCs/VEGF group were higher than those in model group（P〈0.05）, but there was no statistically significant difference between BMSCs group and BMSCs/VEGF group. At 7 d, the expression of VEGF and in BMSCs/VEGF group was significantly higher than that in BMSCs group（P〈0.05）. There was no statistically significant difference at other time;CD34 expression in BMSCs/VEGF group was higher than that in BMSCs group, but there was no statistically significant difference（P 〉0.05）. Conclusion BMSCs transplantation modified by VEGF gene can improve angiogenesis in ischemic area, VEGF expression in which have significant difference with pure BMSCs transplantation at post-transplant 7 d.Compared with pure BMSCs at neurological behavior score; angiogenesis; CD34 expression, there are no obvious advantage. Reason may be related to VEGF165 plasmid transfection rate low and complex mechanism of brain angiogenesis.