BDNF基因重组慢病毒转染MSCs诱导分化神经样细胞的实验研究

目的 将BNDF基因重组慢病毒转染MSCs,观察其体外诱导及脑内移植后分化神经样细胞的情况。方法 分离培养大鼠MSCs; 将携带有BDNF的慢病毒转染MSCs; 镜下观察诱导后MSCs的形态变化; 制备大鼠脑出血模型,并随机分为PBS组、MSCs组、MSCs-EGFP组、MSCs-EGFP-BDNF组,记录各组细胞移植7、14、21 d神经功能缺损评分、脑组织切片免疫荧光单标检测MSCs的脑内迁移、免疫荧光双标检测MSCs脑内分化神经样细胞的情况。结果 镜下观察经BDNF基因重组慢病毒诱导的MSCs由均匀一致的长梭形逐渐呈现出有单极、双极甚至多极的类神经样细胞,而空病毒组及未转染组MSCs细胞形态无明显变化; 移植入脑出血模型脑内后MSCs组、MSCs-EGFP组及MSCs-EGFP-BDNF组大鼠的神经功能评分与PBS组比较均有不同程度改善,其中MSCs-EGFP-BDNF组改善最为显著(P<0.05); MSCs-EGFP-BDNF组迁移至脑出血灶周围组织的MSCs明显多于MSCs组及MSCs-EGFP组(P<0.05),MSCs组与MSCs-EGFP组除7 d外其余比较无明显差异(P>0.05); MSCs-EGFP-BDNF组胶原纤维酸性蛋白阳性率明显高于MSCs组及MSCs-EGFP组(P<0.01),而MSCs组与MSCs-EGFP组比较无明显差异(P>0.05)。结论 BDNF基因重组慢病毒修饰的MSCs体外可诱导其向神经样细胞分化; 移植后迁移至脑出血灶周围组织的细胞数较其它组明显增多; 可促进脑出血大鼠的神经功能修复并检测到其分化为神经细胞标志物的表达。

Experimental study on differentiation of neuron-like cells induced by BDNF gene recombinant lentivirus transfected MSCs

ObjectiveTo observe the differentiation of MSCs to neuron-like cells after lentivirus transfection applied with BNDF gene recombination in vitro and intracerebral transplantation.Methods Rat MSCs were isolated and cultured.Lentivirus carried with BDNF was transfected into MSCs.Morphological changes of MSCs were observed under microscope after induction.The rat cerebral hemorrhage models were prepared and randomly divided into PBS group,MSCs group,MSCs-EGFP group and MSCs-EGFP-BDNF group.The neurological function deficit scores of cells in each group were recorded at the 7,14 and 21^th day after transplantation.The intracerebral migration of MSCs was detected through brain tissue section single-labeled immunofluorescence.And the differentiation of intracerebral neuron-like cells from MSCs was defected by double-labeled immunofluorescence.Results It was observed that the MSCs induced by BDNF gene recombinant lentivirus were gradually and differentially expressed in long fusiform neuron-like cells with monopolar,bipolar or even multipolar.While the cellular morphology of MSCs in the empty virus group and the untransfected group had no significant changes.The neurological function scores of rats in the MSCs group,the MSCs-EGFP group,the MSCs-EGFP-BDNF group and the PBS group were improved in different degrees after transplantation into the cerebral hemorrhage models,and the improvement in the MSCs-EGFP-BDNF group was the most significant(P<0.05).The number of MSCs around the lesion tissue in the MSCs-EGFP-BDNF group was significantly more than that in the MSCs group and the MSCs-EGFP group at the 7,14 and 21^th day after transplantation(P<0.05),there was no significant differences between MSCs group and the MSCs-EGFP group at the 14 and 21^th day except at the 7^th day(P>0.05).The positive rate of glial fibrillary acidic protein in the MSC-EGCs-EGFP group was significantly higher than that in the MSCs group and the MSCs-EGFP group(P< 0.05),while there was no between MSCs group and the MSCs-EGFP group(P>0.05).Conclusion BDNF recombinant lentivirus modified MSCs could induce itself to differentiate into neural-like cells in vitro; the number of cells migrated to the surrounding cerebral hemorrhage focal tissue after transplantation was significantly increased; the BDNF recombinant lentivirus modified MSCs could promote the repair of neurological function in rats with intracerebral hemorrhage and detect the expression of the differentiated neurocyte marker.