本文選題：人臍血間充質(zhì)干細(xì)胞 + 帕金森病　； 參考：《武漢大學(xué)》2017年博士論文

【摘要】：帕金森病是中老年人常見(jiàn)的慢性進(jìn)展性的神經(jīng)系統(tǒng)退行性疾病,主要病理變化為中腦黑質(zhì)的多巴胺能神經(jīng)元變性和壞死,致黑質(zhì)紋狀體的多巴胺能通路受損,最終導(dǎo)致體內(nèi)多巴胺含量降低,從而引起靜止性震顫、肌強(qiáng)直和姿勢(shì)步態(tài)異常等臨床表現(xiàn)。目前尚未徹底明確其病因及發(fā)病機(jī)制,可能與遺傳、環(huán)境、氧化應(yīng)激、免疫炎癥、細(xì)胞凋亡等多因素有關(guān)。目前臨床上尚無(wú)有效的治愈手段,藥物治療僅能改善臨床癥狀,并且需要維持治療,但長(zhǎng)期用藥后存在藥效衰減及并發(fā)癥增多等弊端。目前認(rèn)為細(xì)胞移植可作為治愈帕金森病的潛在方法。本研究首先研究人臍血間充質(zhì)干細(xì)胞(human umbilical cord-derived mesenchymal stem cells,hUC-MSCs)在體外向神經(jīng)細(xì)胞分化的可能性,再將其移植入PD模型大鼠腦內(nèi),應(yīng)用免疫組化及免疫熒光方法觀察移植的hUC-MSCs在腦中的分布,并探討hUC-MSCs移植治療帕金森病的機(jī)制與效果,為臨床應(yīng)用提供基礎(chǔ)。第一部分臍血間充質(zhì)干細(xì)胞的培養(yǎng)和鑒定目的:研究hUC-MSCs的生物學(xué)特性,探討在體外向神經(jīng)細(xì)胞誘導(dǎo)分化的可能性。方法:從臍血中提取hUC-MSCs進(jìn)行培養(yǎng)及誘導(dǎo)分化。培養(yǎng)基中添加表皮生長(zhǎng)因子EGF、堿性成纖維細(xì)胞生長(zhǎng)因子BFGF、膠質(zhì)細(xì)胞系源性神經(jīng)營(yíng)養(yǎng)因子GDNF、腦源性神經(jīng)營(yíng)養(yǎng)因子BDNF、全反式維甲酸RA,進(jìn)行誘導(dǎo)分化。用相差顯微鏡觀察hUC-MSCs的形態(tài)特征,流式細(xì)胞儀檢測(cè)hUC-MSCs表面標(biāo)志的表達(dá),免疫組織化學(xué)法檢測(cè)神經(jīng)細(xì)胞標(biāo)志的表達(dá)。結(jié)果:hUC-MSCs傳代培養(yǎng)后,細(xì)胞呈漩渦狀生長(zhǎng)。hUC-MSCs表面標(biāo)志用流式細(xì)胞儀檢測(cè)提示CD44、CD90、CD29、CD105的表達(dá)較強(qiáng),CD45、CD34及HLA-DR均不表達(dá),其中CD45代表白細(xì)胞表面標(biāo)志,后兩種代表造血前體細(xì)胞表面標(biāo)志。免疫組化染色Nestin、NSE、GFAP染色均為陽(yáng)性。TH染色為陰性,BrdU染色呈陽(yáng)性。結(jié)論:hUC-MSCs可向神經(jīng)細(xì)胞分化,具有神經(jīng)細(xì)胞表面標(biāo)志。第二部分晚期帕金森病大鼠模型的建立目的:制作穩(wěn)定的晚期帕金森病(PD)大鼠模型方法:利用腦立體定向儀,將6-羥基多巴胺注入大鼠內(nèi)側(cè)前腦束及右側(cè)中腦腹側(cè)被蓋區(qū)。用阿樸嗎啡誘導(dǎo)旋轉(zhuǎn)行為,取大鼠黑質(zhì)進(jìn)行免疫組織化學(xué)染色檢測(cè)多巴胺能神經(jīng)元TH表達(dá)。結(jié)果:PD大鼠黑質(zhì)進(jìn)行免疫組織化學(xué)染色,損毀側(cè)黑質(zhì)中的TH陽(yáng)性神經(jīng)元損失達(dá)對(duì)側(cè)90%以上。結(jié)論:通過(guò)6-OHDA注射可成功制作單側(cè)完全性損傷PD大鼠模型。第三部分hUC-MSCs移植治療晚期PD大鼠的實(shí)驗(yàn)研究目的:探討hUC-MSCs移植對(duì)PD大鼠的治療作用。方法:將大鼠隨機(jī)分為hUC-MSCs移植治療組(25只大鼠)和生理鹽水對(duì)照組(21只大鼠),觀察hUC-MSCs移植后PD大鼠的行為學(xué)改變。術(shù)后1周,制備大鼠腦冰凍切片,進(jìn)行免疫熒光化學(xué)染色檢測(cè)移植細(xì)胞hUC-MSCs的TH表達(dá)情況。移植術(shù)后2周和8周,分別對(duì)兩組大鼠紋狀體進(jìn)行免疫組織化學(xué)染色檢測(cè)TH陽(yáng)性細(xì)胞數(shù)存活率。移植術(shù)后8周對(duì)損毀側(cè)紋狀體進(jìn)行DA含量測(cè)定。結(jié)果:移植術(shù)后1周對(duì)移植的紋狀體區(qū)進(jìn)行免疫熒光化學(xué)染色,顯示BrdU標(biāo)記的TH陽(yáng)性細(xì)胞率為80%±34%。移植術(shù)后2周和8周,分別對(duì)兩組大鼠紋狀體進(jìn)行免疫組織化學(xué)染色,發(fā)現(xiàn)移植組TH陽(yáng)性細(xì)胞數(shù)明顯增多,移植術(shù)后8周對(duì)損毀側(cè)紋狀體進(jìn)行DA含量測(cè)定,hUC-MSCs移植組中DA含量為1754.12±14.79 pg/g,生理鹽水對(duì)照組為1687.94±14.82pg/g。結(jié)論:將hUC-MSCs移植到大鼠PD模型中,可改善PD大鼠旋轉(zhuǎn)行為,并且表達(dá)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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R742.5;R-332

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