發(fā)布時(shí)間：2018-04-02 11:39

  本文選題：骨髓間質(zhì)干細(xì)胞　切入點(diǎn)：胰島　出處：《鄭州大學(xué)》2006年碩士論文

【摘要】：目的：1型糖尿病是針對(duì)胰島β細(xì)胞的特異性自身免疫性疾病，需終身依賴胰島素治療。但是胰島素治療并不能從根本上治愈該病，不僅給患者帶來(lái)很大的痛苦，對(duì)社會(huì)和家庭也是沉重的負(fù)擔(dān)。新的治療策略就是尋找合適的細(xì)胞以替代β細(xì)胞發(fā)揮生理功能。然而，供體來(lái)源的匱乏以及移植免疫排斥反應(yīng)極大地阻礙了該方法的推廣和應(yīng)用。骨髓間質(zhì)干細(xì)胞(MSC)是一種具有自我更新和多向分化潛能，存在于骨髓的非造血成體干細(xì)胞，這類細(xì)胞呈現(xiàn)穩(wěn)定的表型，易于分離、培養(yǎng)，，自我復(fù)制，有高度增殖潛力，而且在一定條件下具有跨系分化潛力。本研究采用于細(xì)胞培養(yǎng)及誘導(dǎo)分化技術(shù)，分離培養(yǎng)小鼠骨髓間質(zhì)干細(xì)胞，使其轉(zhuǎn)分化為分泌胰島素細(xì)胞。 材料與方法：實(shí)驗(yàn)用雄性成年昆明小鼠(4周～6周)，用5ml注射器沖出股骨、脛骨中的骨髓，分別培養(yǎng)于完全低糖DMEM培養(yǎng)基中1小時(shí)，含1％二甲基亞砜的無(wú)血清DMEM中3天和完全高糖DMEM中7天。取培養(yǎng)第1天、第3天、第10天的細(xì)胞置于倒置顯微鏡下觀察細(xì)胞的生長(zhǎng)情況及形態(tài)變化；培養(yǎng)第10天時(shí)細(xì)胞免疫化學(xué)染色檢測(cè)細(xì)胞內(nèi)胰島素、胰高血糖素抗體；以RT-PCR法檢測(cè)第1天和10天時(shí)細(xì)胞內(nèi)Insulin和Glucagon基因的表達(dá)，用western blot檢測(cè)第10天時(shí)細(xì)胞內(nèi)胰島素蛋白的表達(dá)，用雙硫腙(DTZ)染色試驗(yàn)檢測(cè)胰島樣細(xì)胞團(tuán)內(nèi)是否有Insulin合成，以及葡萄糖刺激的胰島素釋放試驗(yàn)檢驗(yàn)胰島樣細(xì)胞是否能夠初步模擬體內(nèi)β細(xì)胞的生理功能。 結(jié)果： 1．小鼠骨髓間質(zhì)干細(xì)胞的原代培養(yǎng) 接種后可見絕大部分細(xì)胞成圓球形，單個(gè)存在，偶可見未解離的3-5個(gè)細(xì)胞
[Abstract]:Objective Type 1 diabetes mellitus is a specific autoimmune disease for islet 尾 cells, which needs to be treated with insulin for life. But insulin therapy can not cure the disease fundamentally, and it not only brings great pain to the patients. It's also a heavy burden for society and families. The new treatment strategy is to find the right cells to replace beta cells. However, The lack of donor sources and allograft rejection have greatly hindered the development and application of this method. Bone marrow mesenchymal stem cells (MSCs) are non-hematopoietic adult stem cells with self-renewal and multi-differentiation potential. These cells exhibit stable phenotypes, easy to isolate, culture, self-replicate, have high proliferative potential, and have cross-line differentiation potential under certain conditions. Mouse bone marrow mesenchymal stem cells were isolated and cultured to differentiate into insulin-secreting cells. Materials and methods: male adult Kunming mice were used to culture bone marrow of femur and tibia with 5ml syringe for 1 hour, respectively. In serum free DMEM containing 1% dimethyl sulfoxide for 3 days and 7 days for complete high glucose DMEM, the cells were cultured on day 1, day 3 and day 10 to observe the growth and morphological changes of the cells under inverted microscope. The intracellular insulin and glucagon antibody were detected by immunocytochemical staining on the 10th day of culture, the expression of Insulin and Glucagon genes were detected by RT-PCR method on the 1st and 10th day, and the expression of insulin protein was detected by western blot on the 10th day. Dithizone dithizone (DTZ) staining was used to detect whether there was Insulin synthesis in islet like cells and glucose stimulated insulin release test was used to test whether islet like cells could mimic the physiological function of 尾 cells in vivo. Results:. 1. Primary culture of mouse bone marrow mesenchymal stem cells. After inoculation, most of the cells were spherical, single, and occasionally 3-5 cells were not dissociated.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2

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本文編號(hào)：1700268