脑源性神经营养因子基因重组慢病毒转染骨髓间质干细胞移植治疗大鼠脑出血的实验研究

Curative effect of transplantation of mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene on intracerebral hemorrhage in rats

目的观察脑源性神经营养因子（BDNF）基因重组慢病毒转染骨髓间质干细胞（MSCs）移植治疗大鼠脑出血的疗效。方法分离培养大鼠MSCs；将带有BDNF的慢病毒载体转染MSCs；RT—PCR、蛋白质印迹检测转基因MSCsBDNF基因及蛋白水平表达。制备大鼠脑出血模型，采用抽签法随机分为磷酸盐缓冲液（PBS）组、MSCs组、空病毒转染骨髓间质干细胞（MSCs—EGFP）组、脑源性神经营养因子基因重组慢病毒转染骨髓问质干细胞（MSCs—EGFP．BDNF）组，每组15只。72h后细胞移植，记录各组细胞移植后7、14、21d神经功能缺损改善程度；免疫荧光双标检测MSCs脑内迁移和分化情况。结果MSCs—EGFP—BDNF组BDNF基因及蛋白水平表达明显高于MSCs组及MSCs—EGFP组；与PBS组（7d：2．0±0．4，14d：1．7±0．2，21d：1．3±0．2）相比，MSCs组（7d：1．6±0．2，14d：1．2±0．3，21d：0．8±0．2）、MSCs—EGFP组（7d：1．6±0．3，14d：1．1±0．2，21d：0．8±0．3）及MSCs-EGFP—BDNF组（7d：1．2±0．3，14d：0．6±0．1，21d：0．2±0．2）大鼠神经功能评分均有不同程度改善（F=6．667、18．417、20．882，均P〈0．05），其中MSCs—EGFP．BDNF组改善最为显著；免疫荧光双标显示MSCs-EGFP—BDNF组胶原纤维酸性蛋白、神经元特异性核蛋白、环核苷酸磷酸二酯酶阳性率明显高于MSCs组及MSCs—EGFP组，而MSCs组与MSCs—EGFP组比较差异无统计学意义。结论BDNF基因重组慢病毒修饰的MSCs基因及蛋白水平表达均增高；修饰的MSCs移植后可迁移至脑出血灶周围存活并分化表达神经细胞标志物，促进脑出血后神经功能修复。

[ Abstract] Objective To observe the curative effect of transplantation of mesenchymal stem cells （MSCs） transfeeted with recombinant lentiviral vectors carrying brain-derived neurotrophic factor （BDNF） gene on intraeerebral hemorrhage in rats. Methods MSCs were isolated from the rat bone marrow, cultured and transfeeted by recombinant lentiviral vectors carrying BDNF gene. Intracerebral hemorrhagic models were constructed and randomly divided into 4 groups： phosphate buffered saline transplanted （PBS） group, MSCs group, mesenchymal stem cells transfected with empty lentiviral vectors transplanted （MSCs-EGFP） group and mesenchymal stem cells transfected with recombinant lentiviral vectors carrying brain-derived neurotrophic factor gene transplanted （MSCs-EGFP-BDNF） group. PBS and MSCs were transplanted according to the groups 72 hours after the establishment of models. The improvements of the neurological function were recorded of each group 7 d, 14 d, and 21 d after the transplantation. Double labeling immunofluorescent staining were used to detect the migration and the differentiation of transplanted MSCs. Results MSCs-EGFP-BDNF group had significant higher levels of BDNF gene and protein expression than MSCs group and MSCs-EGFP group. All MSCs transplanted groups （ MSCs groups ： 7 d ： 1.6 ± 0. 2, 14 d ： 1.2 ±0. 3, 21 d：0. 8 ±0. 2; MSCs-EGFP groups：7 d：1. 6±0. 3, 14 d：1. 1 ±0. 2, 21 d：0. 8 ±0. 3; MSCs- EGFP-BDNFgroup：7 d：l.2 ±0.3, 14 d： 0.6±0.1, 21 d：0.2±0.2） had more improvements in the neural function （F =6. 667, 18.417, 20. 882,all P 〈0. 05） than PBS group（7 d：2. 0 ＋0. 4, 14 d：l. 7 ± 0. 2, 21 d：1. 3 ± 0. 2）, and MSCs-EGFP-BDNF group had the most significant improvement. With double labeling immunofluorescent staining, the MSCs-EGFP-BDNF group had significantly higher positive rates of glial fibrilary acidicprotein, neuron specific nuclear protein ,2＇ ,3＇-cyclic nucleotide 3 ＇-phosphodiesterase than MSCs group and MSCs-EGFP group, while there was no significant differences between the latter two. Conclusions The expression levels of gene and protein are higher for the MSCs modified with recombinant lentiviral vectors carrying BDNF gene. The modified MSCs can migrate to the perihematomal brain issue of intracerebral hemorrhage, express the characteristic molecules of neurons and improve the neural function after intracerebral hemorrhage.