人脐血间充质干细胞治疗晚期帕金森病的模型鼠实验研究

发布时间：2018-06-02 12:53

本文选题：人脐血间充质干细胞 + 帕金森病　；参考：《武汉大学》2017年博士论文

【摘要】：帕金森病是中老年人常见的慢性进展性的神经系统退行性疾病,主要病理变化为中脑黑质的多巴胺能神经元变性和坏死,致黑质纹状体的多巴胺能通路受损,最终导致体内多巴胺含量降低,从而引起静止性震颤、肌强直和姿势步态异常等临床表现。目前尚未彻底明确其病因及发病机制,可能与遗传、环境、氧化应激、免疫炎症、细胞凋亡等多因素有关。目前临床上尚无有效的治愈手段,药物治疗仅能改善临床症状,并且需要维持治疗,但长期用药后存在药效衰减及并发症增多等弊端。目前认为细胞移植可作为治愈帕金森病的潜在方法。本研究首先研究人脐血间充质干细胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)在体外向神经细胞分化的可能性,再将其移植入PD模型大鼠脑内,应用免疫组化及免疫荧光方法观察移植的hUC-MSCs在脑中的分布,并探讨hUC-MSCs移植治疗帕金森病的机制与效果,为临床应用提供基础。第一部分脐血间充质干细胞的培养和鉴定目的:研究hUC-MSCs的生物学特性,探讨在体外向神经细胞诱导分化的可能性。方法:从脐血中提取hUC-MSCs进行培养及诱导分化。培养基中添加表皮生长因子EGF、碱性成纤维细胞生长因子BFGF、胶质细胞系源性神经营养因子GDNF、脑源性神经营养因子BDNF、全反式维甲酸RA,进行诱导分化。用相差显微镜观察hUC-MSCs的形态特征,流式细胞仪检测hUC-MSCs表面标志的表达,免疫组织化学法检测神经细胞标志的表达。结果:hUC-MSCs传代培养后,细胞呈漩涡状生长。hUC-MSCs表面标志用流式细胞仪检测提示CD44、CD90、CD29、CD105的表达较强,CD45、CD34及HLA-DR均不表达,其中CD45代表白细胞表面标志,后两种代表造血前体细胞表面标志。免疫组化染色Nestin、NSE、GFAP染色均为阳性。TH染色为阴性,BrdU染色呈阳性。结论:hUC-MSCs可向神经细胞分化,具有神经细胞表面标志。第二部分晚期帕金森病大鼠模型的建立目的:制作稳定的晚期帕金森病(PD)大鼠模型方法:利用脑立体定向仪,将6-羟基多巴胺注入大鼠内侧前脑束及右侧中脑腹侧被盖区。用阿朴吗啡诱导旋转行为,取大鼠黑质进行免疫组织化学染色检测多巴胺能神经元TH表达。结果:PD大鼠黑质进行免疫组织化学染色,损毁侧黑质中的TH阳性神经元损失达对侧90%以上。结论:通过6-OHDA注射可成功制作单侧完全性损伤PD大鼠模型。第三部分hUC-MSCs移植治疗晚期PD大鼠的实验研究目的:探讨hUC-MSCs移植对PD大鼠的治疗作用。方法:将大鼠随机分为hUC-MSCs移植治疗组(25只大鼠)和生理盐水对照组(21只大鼠),观察hUC-MSCs移植后PD大鼠的行为学改变。术后1周,制备大鼠脑冰冻切片,进行免疫荧光化学染色检测移植细胞hUC-MSCs的TH表达情况。移植术后2周和8周,分别对两组大鼠纹状体进行免疫组织化学染色检测TH阳性细胞数存活率。移植术后8周对损毁侧纹状体进行DA含量测定。结果:移植术后1周对移植的纹状体区进行免疫荧光化学染色,显示BrdU标记的TH阳性细胞率为80%±34%。移植术后2周和8周,分别对两组大鼠纹状体进行免疫组织化学染色,发现移植组TH阳性细胞数明显增多,移植术后8周对损毁侧纹状体进行DA含量测定,hUC-MSCs移植组中DA含量为1754.12±14.79 pg/g,生理盐水对照组为1687.94±14.82pg/g。结论:将hUC-MSCs移植到大鼠PD模型中,可改善PD大鼠旋转行为,并且表达TH蛋白。
[Abstract]:Parkinson's disease is a common and chronic progressive neurodegenerative disease of the middle and old people. The main pathological change is the degeneration and necrosis of dopaminergic neurons in the substantia nigra of the middle brain. The dopamine pathway in the substantia nigra is impaired, which eventually leads to the decrease of dopamine content in the body, resulting in static tremor, myotonic and postural gait. At present, the etiology and pathogenesis have not been thoroughly clarified, which may be related to many factors such as heredity, environment, oxidative stress, immune inflammation, cell apoptosis and so on. There is no effective cure at present. Drug therapy can only improve clinical symptoms and need maintenance therapy, but there is a drug effect attenuation and complications after long-term medication. It is believed that cell transplantation can be used as a potential cure for Parkinson's disease. This study first studies the possibility of differentiation of human umbilical cord blood mesenchymal stem cells (human umbilical cord-derived mesenchymal stem cells, hUC-MSCs) into neural cells in vitro, and then transplants them into the brain of PD model rats and applies immunohistochemistry and immunity. The distribution of hUC-MSCs in the brain was observed by the immunofluorescence method, and the mechanism and effect of hUC-MSCs transplantation in the treatment of Parkinson's disease were discussed, and the basis for clinical application was provided. The first part of the cultivation and identification of umbilical cord blood mesenchymal stem cells: study the biological characteristics of hUC-MSCs and explore the possibility of inducing differentiation to neural cells in vitro. Methods: hUC-MSCs was extracted from umbilical cord blood and cultured and induced to differentiate. The culture medium added epidermal growth factor EGF, basic fibroblast growth factor BFGF, glial derived neurotrophic factor GDNF, brain derived neurotrophic factor BDNF, all trans retinoic acid RA, to induce differentiation. The morphological characteristics of hUC-MSCs were observed by phase contrast microscope. The expression of hUC-MSCs surface markers was detected by flow cytometry, and the expression of nerve cell markers was detected by immunohistochemistry. Results: after hUC-MSCs generation, the cells showed whirlpool like growth.HUC-MSCs surface markers to detect CD44, CD90, CD29, CD105, and CD45, CD34 and HLA-DR were not expressed, and CD45 represented white. Surface markers of cell surface, the latter two represent the surface markers of hematopoietic progenitor cells. Immunohistochemical staining Nestin, NSE, GFAP staining were negative for positive.TH staining, BrdU staining was positive. Conclusion: hUC-MSCs can differentiate into neural cells and have neural cell surface markers. The purpose of the establishment of the second part of the late stage Parkinson's disease rat model is to make stable The method of advanced Parkinson's disease (PD) rat model: injection of 6- hydroxy dopamine into the medial forebrain and the ventral tegmental area of the right middle brain by using brain stereotactic apparatus. The rotation behavior was induced by apomorphine, and the rat substantia nigra was examined by immunohistochemical staining to detect the expression of dopaminergic TH. Results: the substantia nigra of PD rats was immunized. Chemical staining, the loss of TH positive neurons in the damaged lateral substantia nigra was more than 90% of the contralateral side. Conclusion: the unilateral complete injury PD rat model can be successfully made by 6-OHDA injection. The purpose of the experimental study on the treatment of late PD rats by third part of hUC-MSCs transplantation is to explore the therapeutic effect of hUC-MSCs transplantation on PD rats. Methods: the rats were randomly divided into hUC- MSCs transplantation group (25 rats) and saline control group (21 rats) were used to observe the behavioral changes of PD rats after hUC-MSCs transplantation. 1 weeks after operation, the frozen section of rat brain was prepared, and the TH expression of hUC-MSCs in the transplanted cells was detected by immunofluorescence chemical staining. 2 and 8 weeks after transplantation, the striatum was immunized in two groups of rats respectively. The survival rate of TH positive cells was detected by histochemical staining. The DA content of the damaged striatum was measured at 8 weeks after transplantation. Results: immunofluorescence staining was performed on the striatum at 1 weeks after transplantation, indicating that the TH positive cell rate of the BrdU markers was 2 and 8 weeks after the 80% + 34%. transplantation, and the striatum of the two groups of rats was exempting, respectively. The number of TH positive cells in the transplanted group was significantly increased. The content of DA in the damaged lateral striatum was measured at 8 weeks after transplantation. The DA content in the hUC-MSCs transplantation group was 1754.12 + 14.79 pg/g, and the normal saline control group was 1687.94 + 14.82pg/g. conclusion: hUC-MSCs transplantation to the rat PD model could improve the rotation behavior of the PD rats. And the expression of TH protein.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.5;R-332

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