骨髓间充质干细胞移植治疗缺血再灌注脑损伤

Experimental study of bone marrow mesenchymal stem cell transplantation for cerebral ischemic reperfusion injuries

目的探讨骨髓间充质干细胞（MSC）移植对缺血再灌注脑损伤的治疗效果及机制。方法采用线栓法制备缺血再灌注脑损伤SD大鼠模型。96只大鼠随机分为4组：模型组、MSC移植组、PBS对照组和假手术组，每组24只。取体外培养至第6代的骨髓MSC，经烟酸己可碱33342标记后，于造模24h后经阴茎静脉进行移植，对照组注射等量PBS。分别于实验第2、3、4、8、15、22天对各组大鼠进行神经损伤严重程度（NSS）评分，通过荧光显微镜观察MSC在脑组织内的定植情况，并对脑组织进行2，3，5-氯化三苯四唑（TTC）染色及神经干细胞、神经元、星形胶质细胞的特异性标志物[神经上皮干细胞蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、胶质纤维酸性蛋白（GFAP）]的免疫组织化学染色。结果MSC移植后2d在受损的脑组织局部便可见蓝色荧光分布，并于15d时达到高峰。免疫组织化学结果显示，与模型组和对照组相比，MSC移植组大鼠受损脑组织局部Nestin、NSE、GFAP阳性细胞的数量均明显增多，差异均有统计学意义（P均〈0．05）。MSC移植组自第8天受损部位脑梗死的范围即出现显著缩小，NSS评分也明显下降，且与模型组及对照组比较，差异均有统计学意义（P均〈0．05）。结论骨髓MSC移植可有效增加缺血再灌注脑损伤局部神经干细胞、神经元、星形胶质细胞的数量，对神经损伤的修复起一定的促进作用。

Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cell （MSC） transplantation on cerebral ischemie reperfusion injury. Methods Sprague-Dawleye rat models of cerebral ischemia- reperfusion injury were established with suture method. Ninety-six rats were divided into 4 groups ： model group, MSC transplantation group, control group （injection of phosphate buffer saline, PBS） and sham operation group,with 24 rats in each group. The 6th generation of bone marrow MSCs cultured in vitro were selected and labeled with hoechst 33342, and then transplanted into rats through Penile vein 24 hours after the establishment of model. Equivalent PBS was injec- ted into the control group. Neurological severity score （NSS） was assessed on the 2^nd day,3^rd day,4th day,8th day, 15^th day, and the 22^th day in each group. The plantation of MSC in the brain tissue was observed by fluorescence microscope. Furthermore,serial paraffin sections were subsequently stained with 2,3,5-triphenyl tetrazolium chloride, and immuno- histochemistry stain with Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （ GFAP）, which could be used as the specific markers of neural stem cells, neurons and astrocytes. Results The distribution of blue fluorescent in the impaired brain tissue could be seen at 2 days after transplantation of MSC, and it reached the peak at the 15th day. The result of immunohistochemistry showed that the cells positive for Nestin, NSE or GFAP in the impaired local brain tissue were increased greatly in the MSC transplantation group compared with the model group and control group, and the differences were statistically significant （ all P 〈 0.05）. The scope of cerebral infarction in the impaired brain tissue diminished from the 8^th day in the MSC transplantation group,the score of NSS was also decreased evident- ly, the differences were statistically significant compared with the model group and control group （ all P 〈 0.05 ）. Con- cluslons Bone marrow MSC transplantation can increase the number of neural stem cells ,neurons and astrocytes in the location of cerebral ischemic reperfusion injuries ,which may promote the repairment of nerve injury.