【摘要】：目的：亨廷頓病(Huntington disease，HD)，是一種以中樞神經(jīng)系統(tǒng)退行性病變?yōu)樘卣鞯某Ｈ旧w顯性遺傳病，目前尚無有效治療方法。人羊膜上皮細胞（human amniotic epithelial cells，hAECs）具有干細胞特性和免疫調(diào)節(jié)作用，為治療神經(jīng)退行性變疾病提供了可能。hAECs對HD動物模型病變是否具有修復作用尚不清楚。本實驗旨在探討hAECs移植治療HD大鼠的療效及其可能的生物學機制。 方法：①采用機械-胰酶消化法分離hAECs，流式細胞術（Flow cytometry，F(xiàn)CM）和免疫組織化學染色檢測表型。②SD大鼠右側(cè)紋狀體注射1μL喹啉酸（quinoline acid，QA）1.8×105μmol/L建立HD模型。成模動物隨機分為模型組(n=16)、培養(yǎng)基對照組（n=16）和hAECs治療組(n=16)。另設正常組（n=16）作為平行對照。hAECs組經(jīng)紋狀體QA損毀側(cè)注射hAECs懸液10μl/只（含6×105個細胞），培養(yǎng)基組相同途徑注射等體積無血清L-DMEM培養(yǎng)基。③hAECs移植后第4w和第8w皮下注射阿樸嗎啡誘導旋轉(zhuǎn)，觀察各組大鼠的旋轉(zhuǎn)行為；Morris水迷宮實驗觀察各組大鼠學習記憶能力。④HE染色觀察腦組織病理改變。⑤CD68免疫組織化學染色觀察紋狀體區(qū)小膠質(zhì)細胞的增生。⑥D(zhuǎn)ARPP-32免疫熒光染色檢測紋狀體區(qū)GABA能神經(jīng)元的損失情況。⑦人細胞核特異性抗原和神經(jīng)元核蛋白（NeuN）免疫熒光雙染色檢測hAECs存活及分化。⑧FCM檢測hAECs移植后第4w和第8w各組大鼠外周血單個核細胞中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）淋巴細胞亞群百分率的變化。 結(jié)果：①用于治療的第3代hAECs高表達CD29、CD44、CD49f、CD326、E-cad、CD73，CK19表達陽性，幾乎不表達CD34、CD45、CD71、CD80、CD86和HLA-DR。②移植后第4w和第8w，hAECs移植組阿樸嗎啡誘導旋轉(zhuǎn)次數(shù)明顯低于對照組和模型組(均P0.01)；Morris水迷宮實驗顯示，與模型組相比，hAECs移植后4w的第1d逃避潛伏期明顯縮短(P0.05)。③hAECs組炎癥反應較之模型組和培養(yǎng)基組明顯減輕。④與模型組和培養(yǎng)基組比較，hAECs組小膠質(zhì)細胞（CD68陽性）明顯減少（均P0.01），，而GABA能神經(jīng)元（DARPP-32陽性）顯著增多（均P0.01）。⑤移植后第4w和第8w，病損紋狀體區(qū)可見植入的細胞存活并表達NeuN。⑥hAECs移植后外周血Treg細胞百分比明顯高于對照組及模型組（均P＜0.05）。 結(jié)論：hAECs移植能明顯改善HD大鼠運動功能和紋狀體區(qū)病理損傷，其機制可能與其抑制炎癥反應，抑制小膠質(zhì)細胞增生，保護GABA能神經(jīng)元有關。實驗結(jié)果提示，hAECs可能成為臨床HD細胞治療有價值的供體細胞。
[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- 緯 (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- 緯 (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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R742.2

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