胚胎干细胞源性神经前体细胞移植脑缺血大鼠局部细胞的免疫反应.doc

胚胎干细胞源性神经前体细胞移植脑缺血大鼠局部细胞的免疫反应梅爱农1，张苏明21福建医科大学省立临床医学院干部特诊一科，福建省临床老年病研究所，福建省立医院老年医学研究室，福建省福州市 350001；2华中科技大学同济医院神经内科，湖北省武汉市 430030Transplantation immunity phenomena after embryonic stem cell-derived neural progenitor cells are grafted into ischemic rat brainMei Ai-nong1, Zhang Su-ming21Department of Cadre, Provincial Clinical College, Fujian Medical University, Fujian Institute of Clinical Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fuzhou 350001, Fujian Province, China; 2Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China摘要背景：神经前体细胞的免疫原性各家研究结果不一，尤其是体内移植后的机体免疫反应模式需要进一步研究。目的：体外观察神经前体细胞组成型及诱导型主要组织相容性抗原表达情况；体内观察神经前体细胞移植入大鼠脑缺血组织后局部免疫细胞活化情况，探讨神经前体细胞的移植排斥可能性及模式。方法：自pCX-hrGFP ES-D3胚胎干细胞诱导分化神经前体细胞，流式细胞术体外检测主要组织相容性抗原，类分子表达及-干扰素诱导前后表达变化。实验分3组，磷酸盐缓冲液组、神经前体细胞组分别于大脑中动脉缺血大鼠模型造模后经侧脑室给予磷酸盐缓冲液注射及神经前体细胞移植，假手术组不造模。免疫组化法观察纹状区ED1+、CD4+、CD8+细胞浸润情况；淋巴细胞再刺激增殖实验观测神经前体细胞诱导移植大鼠颈部淋巴细胞的增殖指数。结果与结论：神经前体细胞组成型高表达主要组织相容性抗原类分子，几乎不表达主要组织相容性抗原类分子；经-干扰素诱导后，主要组织相容性抗原类分子进一步上调，主要组织相容性抗原类分子亦有轻度上调，提示神经前体细胞有可能引起机体免疫反应。移植实验表明，与假手术组相比，磷酸盐缓冲液组及神经前体细胞组均表现强烈的ED1+、CD4+、CD8+细胞浸润(P 0.05)，说明脑缺血损伤本身能导致局部免疫细胞活化；神经前体细胞组比磷酸盐缓冲液组有更强的ED1+、CD4+细胞浸润(P 0.05)，提示神经前体细胞移植可能导致局部免疫更进一步活化，且以CD4+T细胞反应为主。磷酸盐缓冲液组及神经前体细胞组神经前体细胞诱导下的增殖指数值均较假手术组升高(P 0.05)，提示脑组织局部炎症导致颈部淋巴细胞增殖性增加，而离体神经前体细胞不足以单独刺激致敏淋巴细胞增殖。中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：关键词：干细胞；移植；胚胎干细胞；神经前体细胞；免疫原性；脑缺血；主要组织相容性抗原；移植免疫； Abstract：BACKGROUND: Previous studies have showed inconsistent results in the immunogenicity of neural progenitor cells and further studies are required to confirm the model of immune response after transplantation in vivo.OBJECTIVE: To observe the constitutive and inducible expression of major histocompatibility complex (MHC) molecules on neural progenitor cells in vitro and the local immune cell activation in lesion tissue after neural progenitor cells grafted in ischemic rat brain, so as to explore the possibility and model of graft rejection of neural progenitor cells.METHODS: Neural progenitor cells were differentiated from pCX-eGFP ES-D3 embryonic stem cells and the constitutive and interferon-induced expression of MHC class I and II molecules on neural progenitor cells wasobserved in vitro by flow cytometry. Neural progenitor cells were then injected into the lateral ventricle of model animals after middle cerebral artery occlusion (MCAO) or sham surgery which were subdivided into the three groups as MCAO+neural progenitor cells injection group, MCAO+PBS injection group and sham+PBS injection group. A histological evaluation of ED1, CD8 and CD4 positive cells infiltration in the striatum and a proliferation assay of lymphocytes dissociated from cervical lymphnodes were performed after cell transplantation.RESULTS AND CONCLUSION: High constitutive expression of class I but no II MHC molecules was found on the surface of neural progenitor cells by flow cytometry. After interferon- induction, a further increase in class I molecules and a mild but detectable upregulation of class II molecules were found, suggesting the possibility of immune response initiated by neural progenitor cells because of the constitutive expression of class I and the inducible expression of MHC II molecules on cell surface. Rats in the MCAO+neural progenitor cells injection and MCAO+PBS injection group had a higher ED1+,CD4+ and CD8+ cells infiltration in the striatum compared with those in the sham group (P 0.05). Rats in the MCAO+neural progenitor cells group showed even higher ED1+, CD4+ cells infiltration compared with those in the MCAO+PBS injection group (P 0.05), suggesting that ischemic lesion itself was the main cause for the immunoreactions in brain, whereas the higher infiltration of CD4+ and ED1+ cells in the striatum of rats undergoing neural progenitor cell transplantation, indicating there is also a possible graft rejection directed to neural progenitor cells through CD4+-Th1 pathway. Compared with sham animals, rats in MCAO+neural progenitor cells injection group and MCAo+PBS injection group showed significantly higher lymphocytes proliferation (P 0.05). This suggests that neural progenitor cells in vitro alone cannot trigger a significant proliferation of lymphocytes.中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程全文链接：Key words：stem cells;brain ischemia;major histocompatibility complex;transplantation immunology;收稿日期:2014-02-07 中图分类号:R394.2通讯作者:张苏明，博士，主任医师，教授，博士生导师，华中科技大学同济医学院附属同济医院神经内科，湖北省武汉市 430030作者简介: 梅爱农，男，1971年生，湖北省秭归县人，汉族，2006年华中科技大学同济医学院毕业，博士，副主任医师，主要从事干细胞移植免疫方面的研究。引用本文:梅爱农,张苏明. 胚胎干细胞源性神经前体细胞移植脑缺血大鼠局部细胞的免疫反应J.中国组织工程研究, 2014,