本文選題：骨髓間充質(zhì)干細胞　切入點：異種移植　出處：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:體外分離培養(yǎng)鑒定大鼠骨髓間充質(zhì)干細胞(Rat bone marrow mesenchymal stem cells,Rat BM-MSCs)并初步探究其對小鼠實驗性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)的治療作用。方法:1.取SPF級大鼠骨髓,貼壁培養(yǎng)法獲得BM-MSCs,觀察細胞形態(tài)、對P3代細胞進行細胞表面抗原標志物CD29、CD90、CD106、CD45鑒定以及BM-MSCs成骨、成脂分化鑒定。2.取20只雌性C57BL/6小鼠,隨機分為三組:正常對照組(Control組)、PBS組和Rat BM-MSCs組。Rat BM-MSCs細胞移植后小鼠作為Rat BM-MSCs組,PBS移植后小鼠作為PBS組,未作處理小鼠作為正常對照組。MOG35-55聯(lián)合完全弗氏佐劑充分乳化后,背部皮下多點注射免疫,誘發(fā)小鼠EAE。3.小鼠免疫后第38d腹腔注射Rat BM-MSCs(1×106cells/200μl)進行治療,PBS組注射等體積PBS,10d后再次治療,每天進行神經(jīng)功能評分觀察各組小鼠神經(jīng)功能變化。4.二次移植治療12d后取各組小鼠脊髓:HE染色及Luxol Fast Blue染色觀察脊髓炎性細胞浸潤以及髓鞘脫失情況;5.Rat BM-MSCs細胞二次移植后第12d獲取各組小鼠脾臟制成單細胞懸液,脾細胞CFSE標記后10μg/ml ConA和MOG35-55刺激培養(yǎng)3d,收細胞,流式細胞術(shù)檢測脾細胞增殖情況;6.Rat BM-MSCs細胞二次移植后第12d收集小鼠外周血血清,Ellisa技術(shù)檢測正常對照組、PBS組和Rat BM-MSCs組外周血中細胞因子:干擾素γ(IFN-γ),白介素17(IL-17)的含量。結(jié)果:1.Rat BM-MSCs成功分離培養(yǎng),P3代細胞呈梭形或多角形,呈典型旋渦狀生長;流式細胞術(shù)顯示Rat BM-MSC P3代細胞表達抗原CD29、CD90、CD106,不表達CD45;BM-MSCs體外誘導(dǎo)能向成骨及成脂方向分化。2.C57BL/6小鼠發(fā)病后,EAE小鼠神經(jīng)功能癥狀加重,臨床評分逐漸升高,BM-MSCs移植后,Rat BM-MSCs組小鼠神經(jīng)功能缺損癥狀較PBS組減輕,評分降低,二次移植后臨床癥狀維持進一步好轉(zhuǎn)。3.HE染色結(jié)果顯示,Rat BM-MSCs組脊髓炎性細胞浸潤比同時間點PBS組減輕(P0.05);Luxol Fast Blue染色結(jié)果顯示,Rat BM-MSCs組脫髓鞘比同時間點PBS組減輕(P0.05);病理結(jié)果分析,BM-MSCs移植后小鼠脊髓炎癥浸潤和脫髓鞘情況得以改善。4.流式細胞術(shù)分析,ConA和MOG35-55刺激培養(yǎng)3d后,PBS組和Rat BM-MSCs組脾細胞增殖明顯增加,而Rat BM-MSCs組又較PBS組降低。5.ELISA結(jié)果分析,與正常對照組相比,PBS組小鼠外周血血漿中促炎細胞因子IFN-γ、IL-17含量升高;與PBS組相比,Rat BM-MSCs組小鼠外周血血漿促炎細胞因子IFN-γ、IL-17含量降低(P0.05)。結(jié)論:全骨髓貼壁培養(yǎng)法能有效分離純化BM-MSCs,大鼠BM-MSCs移植對小鼠EAE模型有治療作用,且其治療機制可能與抑制EAE小鼠體內(nèi)炎性T細胞增殖和調(diào)節(jié)細胞因子的分泌有關(guān)。
[Abstract]:Objective: to isolate and identify rat bone marrow mesenchymal stem cells (BM-MSCs) in vitro and to explore the therapeutic effect of BM-MSCs on experimental autoimmune autoimmune encephalomyelitis (EAE) in mice. BM-MSCswere obtained by adherent culture method. The morphology of P3 cells was observed. CD29, CD90, CD106, CD45, BM-MSCs osteogenesis, adipogenic differentiation, and 20 female C57BL/6 mice were identified. The mice were randomly divided into three groups: normal control group (control group) and Rat BM-MSCs group. The mice were treated as PBS group after Rat BM-MSCs transplantation, and the mice were emulsified with full Freund's adjuvant and without treatment as normal control group (.MOG35-55) and complete Freund's adjuvant (Freund's adjuvant). Rat BM-MSCs(1 脳 106cells/200 渭 l was injected intraperitoneally on the 38th day after immunization to induce mice to be immunized by subcutaneous multi-point injection on the back. The mice in the PBS group were treated again after 10 days of iso-volume PBSs injection. After 12 days of second transplantation, the spinal cord of each group was stained with Luxol Fast Blue and Luxol Fast HE staining to observe the infiltration of inflammatory cells in spinal cord and the demyelination of myelin sheath. On the 12th day after transplantation, the spleen of each group was obtained and made into single cell suspension. Splenocytes were cultured for 3 days with 10 渭 g/ml ConA and MOG35-55 after CFSE labeling. Detection of splenocyte proliferation by flow cytometry 6. The levels of cytokines (IFN- 緯, IL-17IL-17) in peripheral blood of mice in normal control group and Rat BM-MSCs group were detected by flow cytometry on the 12th day after secondary transplantation of BM-MSCs cells. Results: 1.Rat BM-MSCs successfully isolated and cultured pP3 cells in fusiform or polygonal shape. Flow cytometry showed that Rat BM-MSC P3 cells expressed antigen CD29, CD90, CD106, and CD45 BM-MSCs could induce osteogenesis and adipogenic differentiation in vitro. The clinical scores of BM-MSCs were gradually increased, and the neurological impairment symptoms in the BM-MSCs group were decreased compared with those in the PBS group, and the scores of the BM-MSCs were lower than those of the PBS group. The results of HE staining showed that the infiltration of inflammatory cells in spinal cord in BM-MSCs group was lighter than that in PBS group at the same time point. The results of Fast Blue staining showed that the demyelination of BM-MSCs group was lower than that of PBS group at the same time point, and the pathological changes of BM-MSCs group was lower than that of PBS group at the same time point. Results the inflammatory infiltration and demyelination of spinal cord were improved after transplantation of BM-MSCs. The proliferation of splenocytes was significantly increased in Rat BM-MSCs group and Rat BM-MSCs group after 3 days of culture stimulated by Cona and MOG35-55 by flow cytometry. Compared with the normal control group, the plasma pro-inflammatory cytokine IFN- 緯 IL-17 in the Rat BM-MSCs group was higher than that in the normal control group. Compared with PBS group, the plasma pro-inflammatory cytokine IFN- 緯 IL-17 in BM-MSCs group decreased P0.05.Conclusion: the whole bone marrow adherent culture method can effectively isolate and purify BM-MSCs. Rat BM-MSCs transplantation has therapeutic effect on mouse EAE model. The therapeutic mechanism may be related to the inhibition of inflammatory T cell proliferation and regulation of cytokine secretion in EAE mice.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R744.51

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