转染脑源性神经营养因子的骨髓间充质干细胞移植治疗大鼠脑缺血损伤的实验研究

目的： 探讨移植转染脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)基因的大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在宿主脑缺血区的基因表达情况，以及对神经损伤的修复作用。方法： 将pcDNA 3.1-BDNF转染到大鼠MSCs中,并检测其表达情况。建立大鼠大脑中动脉梗塞(middle cerebral artery occlusion,MCAO)模型,并将大鼠随机分成对照组、PBS移植组、MSCs移植组和转基因MSCs移植组,每组均8只。将PBS悬液、5-溴脱氧尿嘧啶核苷(bromodeoxyuridine，BrdU)标记的MSCs悬液、BrdU标记的转基因MSCs悬液分别移植到PBS移植组，MSCs移植组和转基因MSCs移植组大鼠的纹状体缺血区半暗带，对照组不移植。移植后1～30 d对各组大鼠进行神经损害严重程度评分(neurological severity score, NSS)并相互比较。移植后30 d，检测各组大鼠脑缺血区BDNF、神经元特异性烯醇化酶(neuron specific enolasw,NSE)和胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达情况。结果： 大鼠脑缺血区移植转基因MSCs 24 h后，检测到BDNF的表达。移植后1 d各组NSS评分无显著差异，移植后7、14、21、30 d均显示转基因MSCs移植组低于其他组。移植后30 d, 转基因MSCs组BDNF阳性细胞数较MSCs组明显增多；NSE、GFAP阳性细胞数较其他组显著增多。结论： 大鼠脑缺血区移植转基因MSCs后表达基因产物，同时其表达的NSE、GFAP蛋白增加，对宿主的神经损伤具有修复作用。转染BDNF基因的MSCs移植是治疗脑缺血性疾病的可行途径。

Research on transplantation of brain-derived neurotrophic factor-transfected bone marrow mesenchymal stem cells in therapy of rats with temporary middle cerebral artery occlusion

Objective: To detect the expression of brain-derived neurotrophic factor(BDNF) transfected bone marrow mesenchymal stem cells(MSCs) in rat brain, which was subjected to ischemia. Methods MSCs were transfected with BDNF gene by using lipofect technique and its gene expression was detected by immunocytochemistry. Temporary middle cerebral artery occlusion (MCAO) model was established in adult male SD rats, which were randomly divided into control, PBS transplantation,MSCs transplantation and BDNF-transfected MSCs transplantation groups,with 8 rats in each group. At 3 d after modeling, rats in PBS transplantation, MSCs transplantation and BDNF-transfected MSCs transplantation groups were received with phosphate buffered saline, BrdU(bromodeoxyuridine)-labelled MSCs and BDNF transfected MSCs injection, respectively, whereas control group with no transplantation. Evaluation of nerve function with neurological injury severity score(NSS) was performed after transplantation. The expression of BDNF, neuron-specific enolase and glial fibrillary acidic protein(GFAP) at 30 d after modeling was determined in all groups. Results The expression of BDNF in labelled MSCs, at 24 h after transfection, was demonstrated by immunohistochhemistry. There were no significant differences in NSS between all groups at 1 d after transplantation. However, the values of NSS in BDNF-transfected MSCs transplantation group were significantly lower than that in other groups at 7, 14, 21, 30 d after transplantation(P＜0.05). At 30 d after transplantation, the number of positive cells of BDNF in BDNF-transfected MSCs transplantation group was higher than that in MSCs transplantation group, and the number of positive cells of NSE and GFAP in BDNF-tansfected group was higher than that in the other three groups. Conclusion There was expression of BDNF in cerebral ischemia rat, transplanted with BDNF-transfected MSCs, which reduced brain injury by incerasing the expression of NSE and GFAP secreted by nerve cells and glia cells. Transplantation of BDNF-transfected MSCs might be a novel method for treating cerebral ischemia.