人胚神经干细胞移植治疗大鼠脑缺血的实验研究

Experimental study of human neural stem cells transplantation for treatment of cerebral ischemia in rats

目的研究人胚神经干细胞(hNSCs)移植治疗脑缺血大鼠的效果及其在缺血大鼠脑内的状况.方法从自然流产的孕10～13周的人胚脑组织中分离、培养神经干细胞.采用线栓法制作大鼠脑缺血模型,1 d后经尾静脉移植未分化的hNSCs入脑缺血大鼠体内,对移植后大鼠进行神经损害严重程度评分(NSS),用免疫组化方法观察移植后hNSCs的存活、迁徙、分化状况.结果从人胎脑中成功培养出hNSCs,培养条件下呈悬浮状态生长,形成神经球,绝大多数的细胞表达神经干细胞的标记物神经巢蛋白(nestin).hNSCs移植组大鼠自移植后3周末起其NSS显著低于对照组(P＜0.05);移植后2、3、4、5周脑组织切片中均可见5-溴脱氧嘧啶尿苷(Brdu)染色阳性细胞,缺血侧明显多于对侧(P＜0.05),移植后3、4、5周末明显多于移植后2周(均P＜0.05);移植组各时间点脑组织切片中均可见nestin染色阳性细胞;在Brdu阳性细胞群中,73.8%为胶质纤维酸性蛋白(GFAP)染色阳性的星形胶质细胞,16.7%为2,3-环核苷酸磷酸二脂酶(CNPase)染色阳性的少突胶质细胞,9.5%为神经元特异性烯醇化酶(NSE)染色阳性的神经元.结论经静脉移植hNSCs能有效改善脑梗死动物的神经功能,hNSCs体内体外均具有多向分化潜能,受缺血部位微环境信号的影响分化成3种主要类型的神经细胞.

Objective To explore the effect of human neural stem cells(hNSCs) transplantation to treat cerebral ischemic rats and the status of transplanted hNSCs in the ischemic brain tissue of these rats.Methods Human neural stem cells were separated from 10~13 weeks brains of human embryo and were cultured and induced to differentiate. The middle cerebral artery occlusion rat models were made and the human neural stem cells were transplanted through tail vein 1 day later. Neurological Severity Scores (NSS) tests were undertaken in two groups after transplantation. Immunohistochemistry was used to check the differentiation and migration of human neural stem cells in vitro and vivo.Results Neural stem cells from human embryonic brains had been successfully cultured. These cells formed typical neurospheres in suspension, and the majorities expressed nestin. Three weeks later, the neurological function of rats that received transplantation recovered much better than the rats without transplantation, as evidenced by NSS ( P<0.05). Within the brain tissues, 5-bromodeoxyuridine (Brdu) reactive hNSCs were distributed throughout the lesions in recipient rats 2th, 3th, 4th and 5th week after transplantation. The vast majority of cells localized in the ischemic hemisphere and few cells were observed in the contralateral hemisphere ( P<0.05). More Brdu reactive cells were found in the brain tissues at 3th, 4th and 5th week than at 2th week (all P<0.05). Nestin reactive cells were found within the lesions at all observing time point after transplantation. Among all the Brdu reactive cells, 73.8% were astrocytes which were positive for the marker glial fibrillary acidic protein (GFAP), 16.7% were oligodendrocytes confirmed by cyclic nucleotide 3′phosphohydrolase (CNPase) stain, and 9.5% were neurons which presented positive of neuron specific enolase (NSE). Conclusions Intravenous transplantation of hNSCs may effectively improve neurological function of rats with cerebral ischemia. The transplanted cells have multipotential differentiation properties in vitro and vivo and differentiate into three main types of neurocytes induced by microenvironmental signals in the ischemic lesion.