【摘要】：糖尿病是一種常見的嚴(yán)重代謝性疾病,而胰島移植是目前效果最好的治療方法,但是胰島來源匱乏嚴(yán)重制約了該療法的使用。通過定向誘導(dǎo)或基因工程的方法可以使非胰β細(xì)胞具有胰島素分泌功能,從而代替胰β細(xì)胞用于糖尿病的細(xì)胞治療,為糖尿病患者帶來了新希望。本研究以脂肪間充質(zhì)干細(xì)胞為研究對(duì)象,建立了誘導(dǎo)其形成胰島素分泌細(xì)胞的分化體系并研究了 miR-375過表達(dá)對(duì)分化的影響,為脂肪間充質(zhì)干細(xì)胞在糖尿病治療中的應(yīng)用奠定了基礎(chǔ)�；谂咛ジ杉�(xì)胞分化為胰島素分泌細(xì)胞(insulinproducing cells,IPCs)期間的關(guān)鍵步驟和規(guī)律,以及前期對(duì)人脂肪間充質(zhì)干細(xì)胞(human adipose tissue-derived mesenchymal stem,haMSCs)胰向分化的探索,確立五種含有不 同誘導(dǎo)因子的分化方案。通過使用這五種方案分別對(duì)脂肪間充質(zhì)干細(xì)胞進(jìn)行誘導(dǎo)分化以及對(duì)分化細(xì)胞進(jìn)行基因表達(dá)檢測(cè)和細(xì)胞形態(tài)學(xué)比較,最終確定了以第一階段添加小分子CYC、NOGGIN、B27和RA的基礎(chǔ)高糖培養(yǎng)液;第二階段添加Nic、GLP1、NOGGIN和B27的基礎(chǔ)高糖培養(yǎng)液;第三階段添加Nic、GLP1、IGF1和NOGGIN的IMDM培養(yǎng)液的三階段分化方案為最優(yōu)分化方案。我們通過使用該方案成功誘導(dǎo)haMSCs分化為IPCs,分化細(xì)胞表達(dá)胰腺相關(guān)基因Foxa2、Pdx1、Ngn3、Ins和Gcg,細(xì)胞胰島素免疫熒光染色為陽(yáng)性,分泌胰島素的ELISA檢測(cè)為陽(yáng)性。并發(fā)現(xiàn)BMP信號(hào)通路對(duì)胰島素分泌細(xì)胞的形成有調(diào)節(jié)作用,NOGGIN的加入有促進(jìn)Ins表達(dá)的作用。實(shí)驗(yàn)室所保存的人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells,hucMSC)可以作為 haMSC 分化過程中同期處理的對(duì)照細(xì)胞。miR-375在胰腺、胰島細(xì)胞發(fā)育和成熟起到重要的調(diào)控作用,控制其表達(dá)可能有助于干細(xì)胞向胰島素分泌細(xì)胞分化。為了探究基于前期建立的誘導(dǎo)分化方案過表達(dá)miR-375后對(duì)分化究竟有何影響,我們利用慢病毒載體,成功構(gòu)建穩(wěn)定過表達(dá)miR-375的haMSC細(xì)胞株,并將其誘導(dǎo)分化為IPCs;同時(shí),我們構(gòu)建了一種在分化第三階段瞬時(shí)過表達(dá)/抑制miR-375的脂質(zhì)體轉(zhuǎn)染體系,來探究過表達(dá)/抑制miR-375后對(duì)haMSC分化的影響。通過以上兩種方法,得出:miR-375 和 Ins表達(dá)呈正相關(guān);過表達(dá) miR-375 可以降低分化所得細(xì)胞中 Gcg 的表達(dá);在分化第三階段起始瞬時(shí)過表達(dá)miR-375可以上調(diào)Ngn3;miR-375過表達(dá)對(duì)Pdx1、Foxa2基因表達(dá)影響不大�？傊�,過表達(dá)miR-375可以促進(jìn)細(xì)胞向胰島素分泌細(xì)胞分化,并減少向胰高血糖素分泌細(xì)胞分化。
[Abstract]:Diabetes mellitus is a common severe metabolic disease, and islet transplantation is the most effective treatment at present, but the lack of islet source seriously restricts the use of the therapy. By means of directed induction or genetic engineering, non-pancreatic 尾 cells can be secreted by insulin instead of pancreatic 尾 cells, which brings new hope to diabetic patients. In this study, adipose mesenchymal stem cells (FMSCs) were used to induce the differentiation of insulin-secreting cells and the effect of overexpression of miR-375 on differentiation was studied. It lays a foundation for the application of adipose mesenchymal stem cells in the treatment of diabetes. Based on the key steps and rules during the differentiation of embryonic stem cells into insulinproducing cells, and the preliminary exploration of pancreatic differentiation of human adipose mesenchymal stem cells (human adipose tissue-derived mesenchymal stemma haMSCs), five differentiation schemes containing different inducible factors were established. By using these five schemes to induce the differentiation of adipose mesenchymal stem cells and to detect the gene expression of the differentiated cells and compare the morphology of the cells, the basic high glucose medium was determined by adding small molecule CYCnGINB27 and RA in the first stage. In the second stage, the basic high glucose medium of Nickel GLP1NOGGIN and B27 was added, and in the third stage, the three-stage differentiation scheme of IMDM medium supplemented with Nickel GLP1IGF1 and NOGGIN was the best. We successfully induced the differentiation of haMSCs into IPCs by using this protocol. The differentiated cells expressed the pancreatic associated genes Foxa2, Pdx1, Ngn3, ins and Gcg. The cells were positive for insulin immunofluorescence staining and ELISA positive for insulin secretion. It was also found that BMP signaling pathway could regulate the formation of insulin-secreting cells. The addition of NOGGIN could promote the expression of Ins. Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells hucMSC) can play an important role in the development and maturation of pancreatic islet cells. Controlling its expression may help stem cells differentiate into insulin-secreting cells. In order to investigate the effect of overexpression of miR-375 on differentiation based on the previously established induction protocol, we successfully constructed a stable haMSC cell line with overexpression of miR-375 by using lentivirus vector, and induced it to differentiate into IPCs. at the same time, We constructed a liposome transfection system for transient overexpression / inhibition of miR-375 in the third stage of differentiation to investigate the effect of overexpression / inhibition of miR-375 on the differentiation of haMSC. Through the above two methods, the positive correlation between the expression of miR-375 and Ins was found, and overexpression of miR-375 could decrease the expression of Gcg in differentiated cells. Transient overexpression of miR-375 in the third stage of differentiation could up-regulate the expression of Ngn3mmiR-375 and had little effect on the expression of Pdx1 + Foxa2 gene. In conclusion, overexpression of miR-375 can promote the differentiation of cells into insulin-secreting cells and reduce the differentiation to glucagon secreting cells.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1

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本文編號(hào)：2196637