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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,1d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 investigate the effect of human embryonic neural stem cells (hNSCs) transplantation on cerebral ischemia in rats and its status in the brain of ischemic rats. Methods Neural stem cells (NSCs) were isolated and cultured from human embryonic brain tissue of 10 ~ 13 weeks after spontaneous abortion. The rat model of cerebral ischemia was established by thread occlusion method. After 1 day, the hNSCs were transplanted into the ischemic rat via caudal vein, and the severity of neurological damage (NSS) was evaluated after transplanted. Immunohistochemistry The survival, migration and differentiation of hNSCs after transplantation were observed. Results The hNSCs were successfully cultured from the human fetal brain and grown in suspension in culture to form neurospheres. Most of the cells expressed neural stem cell marker nestin. The NSS of hNSCs transplantation group was significantly lower than that of the control group (P <0. 05) 3 weeks after transplantation. Brdudu (UB) ) Were significantly higher in the ischemic group than those in the contralateral group (P <0.05). At 3, 4, and 5 weeks after transplantation, they were significantly more than those at 2 weeks after transplantation (all P <0.05) Nestin positive cells were found in brain sections. In Brdu positive cells, 73.8% were glial fibrillary acidic protein (GFAP) -positive astrocytes, 16.7% were 2, 3 cyclic nucleotides Oligodendrocytes stained positive with phosphodiesterase (CNPase) and 95% neurons stained with neuron specific enolase (NSE). Conclusion The transplanted hNSCs can effectively improve the neurological function of the animals with cerebral infarction. The hNSCs have multi-directional differentiation potential in vitro and in vivo, and differentiate into three main types of neural cells under the influence of microenvironment signal of ischemic site.