胚胎干细胞治疗脊髓损伤的实验研究

Experimental study on transplantation of embryonic stem cells in treating spinal cord injury

目的探讨未经诱导的胚胎干细胞（Embryonic stem cells,ESCs）移植对SD大鼠脊髓损伤（Spinal cord injury,SCI）神经功能恢复的影响。方法取受孕昆明系小鼠的囊胚进行胚胎干细胞的提取、建系,以流式细胞仪测试阶段特异性胚胎干细胞表面抗原（stage-specific embryonic antigens 1,SSEA-1）,进行干性鉴定并进行5-溴脱氧尿嘧啶核苷（5-bromodeoxyuridine,5-Brd U）染色示踪。将30只SD大鼠制备为T10脊髓损伤模型,将存活的26只大鼠随机分为对照组（A组,8只）,只行脊髓损伤手术而不行特殊治疗;生理盐水治疗组（B组,9只）,于损伤区域局部注射20μL生理盐水;细胞治疗组（C组,9只）,于损伤区域局部注射20μL标记好的ESCs,总细胞数为2×106个。术后1、2、3、4、5、6周采用BBB后肢运动功能评分观察大鼠神经功能恢复情况,并在第6周取损伤脊髓行病理组织HE染色和免疫组织化学染色观察。结果 ESCs经体外培养,呈典型的＂鸟巢＂状生长,碱性磷酸酶（alkaline-phosphatase,AKP）染色阳性,SSEA-1表达阳性,并成功标记上5-Brd U。大鼠实验,BBB运动功能评分显示脊髓损伤开始0-1周内,三组大鼠运动功能恢复情况差别无统计学意义（P〉0.05）。第2-6周,C组与A、B组比较差异有统计学意义（P〈0.05）。HE染色观察：三组皆有损伤区域内空洞形成及局部细胞坏死,但C组明显较A、B两组小。免疫组化染色观察C组在损伤区域表达双染的神经巢蛋白（Neural nest protein,Nestin）、神经元特异性蛋白（Neuron-specific nuclear protein,Neu N）及神经胶质纤维酸性蛋白（Glial fibrillaryacidic protein,GFAP）。结论将未分化的ESCs局部移植入SCI大鼠体内后,能够分化成神经元及神经胶质细胞,对肢体功能的改善有一定的促进作用。

Objective To explore the effect of transplantation of embryonic stem cells（ESCs）on neurological functionalrecovery of injured spinal cord in SD mouse. Methods The blastocysts from mouse of Kunming species were collected,cultured on the embryo fibroblast cells feeder layers（MEFs）and tested stage specific embryonic antigens 1 by flow cytometry,then indentified and marked with 5-bromodeoxyuridine（5-Brd U）. T10 spinal cord impact damage mode was made with 30 SDrats; the 26 survived rats were randomly divided into three groups： Control group（group A,n=8）operated without specialtreatment; Physiological saline treatment group（group B,n=9）injected with 20 μL sodium chloride; Cell therapy group（groupC,n=9）marked with ESCs injections,the total number of cells were 2 × 106. BBB function test was performed to study thefunction improvement operated after 1,2,3,4,5,6 weeks respectively,and the cell survival and differentiation in the spinalcord were observed by HE staining and immunohistochemistry staining of pathological tissues. Results ESCs cultured invitro displayed nest-like clonal cluster. AKP and SSEA-1 were positive. the ESCs were successfully marked with 5-Brd U.Statistical siginificance was noticed in BBB scoring of group C compared with groups A and B a week after operation（P〉0.05）.Statistical siginificance in BBB scoring was also noticed between group C and group A,B two weeks after operation（P〈0.05）.The HE staining revealed there were dead tissue in SCI area,but the area of group C was smaller than other 2 groups.Immunohistochemical results showed that ESCs can express Nestin,Neu N and GFAP in the SCI area induced by the microenvironment. Conclusion ESCs could differentiate into neurons and glial cells and promote the recovery of neural function ofrats after transplanted into SCI rats.