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

Transplantation immunity phenomena after embryonic stem cell-derived neural progenitor cells are grafted into ischemic rat brain

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Ⅰ,Ⅱ类分子表达及γ-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Ⅰ类分子,几乎不表达主要组织相容性抗原Ⅱ类分子;经γ-干扰素诱导后,主要组织相容性抗原Ⅰ类分子进一步上调,主要组织相容性抗原Ⅱ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<0.01),但前两组增殖指数值比较差异无显著性意义(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。

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 neura progenitor cells. METHODS： Neural progenitor cells were differentiated from pCX-eGFP ES-D3 embryonic stem cells and the constitutive and interferon-y-induced expression of MHC class I and II molecules on neural progenitor cells was observed 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-y 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＋neuraJ progenitor cells injection and MCAO＋PBS injection group had a higher EDI＊,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 EDI＊, 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 EDI＊ 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＊-Thl pathway. Compared with sham animals, rats in MCAO＋neural progenitor cells injection group and MCAo＋PBS injection group showed significantly higher lymphocytes proliferation （P 〈 0.01 ）, but there was no difference in lymphocytes proliferation between MCAO＋neural progenitor cells and MCAO＋PBS groups （P 〉 0.05）. This suggests that neural progenitor cells in vitro alone cannot trigger a significant proliferation of lymphocytes.