人胚神经干细胞移植治疗实验性自身免疫性脑脊髓炎大鼠的实验研究

目的:研究侧脑室注射人胚神经干细胞(hNSCs)移植治疗实验性自身免疫性脑脊髓炎(EAE)大鼠的有效性及hNSCs在EAE大鼠脑内的状况。方法:从自然流产的孕9～15周的人胚脑组织中分离、培养神经干细胞。用豚鼠全脊髓匀浆免疫Wistar大鼠制备EAE大鼠模型,分别在免疫后10、15、21天经侧脑室注射移植未分化的hNSCs入大鼠体内,观察hNSCs对EAE动物模型神经功能评分、脑内脱髓鞘病灶数目的影响,免疫组化方法观察移植后的hNSCs在EAE大鼠脑内存活、迁徙、分化的状况。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。绝大多数细胞表达神经干细胞的标记物神经巢蛋白(nestin)。hNSCs移植组各时间点大鼠脑组织切片中均可见Brdu、nestin、NSE、GFAP、CNPase染色阳性细胞,CNPase阳性的少突胶质细胞比例随时间的延长而逐渐增多;hNSCs移植组大鼠自免疫后30天起其神经功能评分显著低于对照组(P<0.05);移植后20、30天脑内脱髓鞘病灶数目明显少于对照组(P<0.05)。结论:hNSCs体内体外均具有多向分化潜能,受炎症部位微环境信号的影响分化成神经元、星型胶质细胞、少突胶质细胞。hNSCs移植能有效改善EAE动物的神经功能评分,减少病灶数目。

Experimental study on human neural stem cells transplantation for treating experimental autoimmune encephalomyelitis in rats

Objective: To explore the effect of the human neural stem cells(hNSCs) transplantation on experimental autoimmune encephalomyelitis(EAE) in rats, and to explore and the status of transplanted hNSCs in the brain tissue of EAE rats. Methods: HNSCs were separated from brains of 9-15-week human embryo,cultured and induced to differentiate. EAE rat models were established and the hNSCs were transplanted into the rat intraventricularly 10,15,21 days after immunization. The neurological signs were taken in all the groups every day after immunization. The differentiation and migration of hNSCs in vivo and the number of the demyelinated foci was determined. Results: The neural stem cells were induced to differentiate into astrocytes,oligodendrocytes and neurons in vivo. Brdu,nestin,NSE,GFAP,CNP positive cells could be present in almost all tissue sections at every time point. Immunohistological results confirmed that the transplanted stem cells migrated to the demyelinated foci. The neural function in rats receiving transplantation was improved 30 days after immunization compared with control group (P < 0.05). The number of demyelinated foci decreased significantly 20 days after transplantation(P < 0.05). Conclusion: The hNSCs have multipotential differentiation properties in vivo. The transplanted cells differentiate into three main types of neural cells under the influcence of the EAE microenvironment signals.HNSCs transplantation intraventricularly could effectively improve the neurologic function of rats and reduce demyelinated foci.