人羊膜上皮细胞移植对亨廷顿病大鼠的治疗效应及其机制

发布时间：2019-03-30 16:40

【摘要】：目的：亨廷顿病(Huntington disease，HD)，是一种以中枢神经系统退行性病变为特征的常染色体显性遗传病，目前尚无有效治疗方法。人羊膜上皮细胞（human amniotic epithelial cells，hAECs）具有干细胞特性和免疫调节作用，为治疗神经退行性变疾病提供了可能。hAECs对HD动物模型病变是否具有修复作用尚不清楚。本实验旨在探讨hAECs移植治疗HD大鼠的疗效及其可能的生物学机制。方法：①采用机械-胰酶消化法分离hAECs，流式细胞术（Flow cytometry，FCM）和免疫组织化学染色检测表型。②SD大鼠右侧纹状体注射1μL喹啉酸（quinoline acid，QA）1.8×105μmol/L建立HD模型。成模动物随机分为模型组(n=16)、培养基对照组（n=16）和hAECs治疗组(n=16)。另设正常组（n=16）作为平行对照。hAECs组经纹状体QA损毁侧注射hAECs悬液10μl/只（含6×105个细胞），培养基组相同途径注射等体积无血清L-DMEM培养基。③hAECs移植后第4w和第8w皮下注射阿朴吗啡诱导旋转，观察各组大鼠的旋转行为；Morris水迷宫实验观察各组大鼠学习记忆能力。④HE染色观察脑组织病理改变。⑤CD68免疫组织化学染色观察纹状体区小胶质细胞的增生。⑥DARPP-32免疫荧光染色检测纹状体区GABA能神经元的损失情况。⑦人细胞核特异性抗原和神经元核蛋白（NeuN）免疫荧光双染色检测hAECs存活及分化。⑧FCM检测hAECs移植后第4w和第8w各组大鼠外周血单个核细胞中CD4+IFN-γ+（Th1）、CD4+IL-4+（Th2）、CD4+Foxp3+（Treg）、CD4+IL-17+（Th17）、CD4-CD8+IFN-γ+（CTL1）和CD4-CD8+IL-4+（CTL2）淋巴细胞亚群百分率的变化。结果：①用于治疗的第3代hAECs高表达CD29、CD44、CD49f、CD326、E-cad、CD73，CK19表达阳性，几乎不表达CD34、CD45、CD71、CD80、CD86和HLA-DR。②移植后第4w和第8w，hAECs移植组阿朴吗啡诱导旋转次数明显低于对照组和模型组(均P0.01)；Morris水迷宫实验显示，与模型组相比，hAECs移植后4w的第1d逃避潜伏期明显缩短(P0.05)。③hAECs组炎症反应较之模型组和培养基组明显减轻。④与模型组和培养基组比较，hAECs组小胶质细胞（CD68阳性）明显减少（均P0.01），，而GABA能神经元（DARPP-32阳性）显著增多（均P0.01）。⑤移植后第4w和第8w，病损纹状体区可见植入的细胞存活并表达NeuN。⑥hAECs移植后外周血Treg细胞百分比明显高于对照组及模型组（均P＜0.05）。结论：hAECs移植能明显改善HD大鼠运动功能和纹状体区病理损伤，其机制可能与其抑制炎症反应，抑制小胶质细胞增生，保护GABA能神经元有关。实验结果提示，hAECs可能成为临床HD细胞治疗有价值的供体细胞。
[Abstract]:Aim: Huntington's disease (Huntington disease,HD) is an autosomal dominant genetic disease characterized by degenerative lesions of the central nervous system. Human amniotic epithelial cells (human amniotic epithelial cells,hAECs) have the characteristics of stem cells and immunomodulatory effect. It is not clear whether hAECs can repair the pathological changes of HD animal model in the treatment of neurodegenerative diseases. The aim of this study was to investigate the therapeutic effect of hAECs transplantation on HD rats and its possible biological mechanism. Methods: (1) flow cytometry (Flow cytometry,FCM) of hAECs, was isolated by mechanical trypsin digestion method and phenotypes were detected by immunohistochemical staining. (2) HD model was established by injection of 1 渭 L quinolinic acid (quinoline acid,QA) into the right striatum of SD rats. The model animals were randomly divided into three groups: model group (n = 16), medium control group (n = 16) and hAECs group (n = 16). Another normal group (n = 16) was injected with 10 渭 l hAECs suspension (containing 6 脳 10 ~ 5 cells) via striatum QA lesion side in parallel control hAECs group. The rotation was induced by subcutaneous injection of apomorphine at the 4th and 8th week after transplantation, and the rotational behavior of the rats in each group was observed. In the culture medium group, the same volume of serum-free L-DMEM medium was injected in the same way. Morris water maze test was used to observe the learning and memory ability of rats in each group. 4HE staining was used to observe the pathological changes of brain tissue. 5CD68 immunohistochemical staining was used to observe the proliferation of microglia in striatum. 6 DARPP-32 immunofluorescence staining was used to detect the proliferation of microglial cells in striatum. Loss of GABA neurons in striatum. Survival and differentiation of hAECs detected by immunofluorescence double staining of 7 human nuclear specific antigen and nuclear protein (NeuN). Peripheral blood samples of rats at 4th and 8th week after hAECs transplantation were detected by flow cytometry (FCM). CD4 IFN- 纬 (Th1) in blood mononuclear cells, The percentage changes of lymphocyte subsets of CD4 IL-4 (Th2), CD4 Foxp3 (Treg), CD4 IL-17 (Th17), CD4-CD8 IFN- 纬 (CTL1) and CD4-CD8 IL-4 (CTL2) were observed. Results: 1High expression of CD29,CD44,CD49f,CD326,E-cad,CD73,CK19 was found in the third generation of hAECs, but almost no expression of CD34,CD45,CD71,CD80,CD86 and HLA-DR.2 was found at the 4th and 8th week after transplantation. The number of rotation induced by apomorphine in hAECs transplantation group was significantly lower than that in control group and model group (P0.01). The Morris water maze test showed that the escape latency on the 1st day after transplantation of hAECs was significantly shorter than that in the model group (P0.05), and the inflammatory response in the 3hAECs group was significantly less than that in the model group and the culture medium group. 4 compared with the model group and the culture medium group, the inflammatory response of the 3hAECs group was significantly reduced. In hAECs group, the number of microglial cells (CD68 positive) decreased significantly (P0.01), while the number of GABA positive neurons (DARPP-32 positive) increased significantly (P0.01). (5) at the 4th and 8th week after transplantation, the number of microglial cells (DARPP-32 positive) was significantly increased. The percentage of Treg cells in peripheral blood after NeuN.6hAECs transplantation was significantly higher than that in control group and model group (P < 0.05), and the survival rate of implanted cells in striatum was significantly higher than that in control group and model group (P < 0.05). Conclusion: hAECs transplantation can significantly improve motor function and striatum pathological injury in HD rats, and its mechanism may be related to its inhibition of inflammatory response, inhibition of proliferation of microglia and protection of GABA neurons. The results suggest that hAECs may be a valuable donor cell for the treatment of clinical HD cells.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.2

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