本文關(guān)鍵詞： 胰腺干細(xì)胞 �；撬� 增殖 分化　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目前糖尿病嚴(yán)重影響患者的生活質(zhì)量,并且可引發(fā)嚴(yán)重的糖尿病并發(fā)癥。傳統(tǒng)的治療手段主要通過藥物刺激體內(nèi)胰島素分泌,或者體外注射胰島素達到平穩(wěn)血糖的目的,卻不能徹底修復(fù)糖尿病患者的胰島病變。近年來,有研究發(fā)現(xiàn)胰腺內(nèi)存在胰腺干細(xì)胞,并且可在體外誘導(dǎo)形成具有功能性的胰島β細(xì)胞,這為治療糖尿病提供了新的思路。臨床和動物試驗均證明,補充�；撬崮軌蚪档吞悄虿』颊叩难撬�,改善臨床癥狀,并防止并發(fā)癥的發(fā)生。課題組前期試驗證明,應(yīng)用�；撬崮軌虼龠MSTZ誘導(dǎo)糖尿病大鼠胰島干細(xì)胞的增殖,上調(diào)胰腺干細(xì)胞向胰島β細(xì)胞分化標(biāo)志因子的表達,提示�；撬峥纱龠M糖尿病狀態(tài)下胰島干細(xì)胞的增殖分化,但其作用機制尚不清楚。因此本研究通過體外試驗分離培養(yǎng)大鼠胰腺干細(xì)胞,探討�；撬嵋认俑杉�(xì)胞增殖及分化作用機理,為應(yīng)用�；撬岱乐翁悄虿√峁├碚撘罁�(jù)。本試驗選取180-220gSD大鼠采用機械消化法分離培養(yǎng)出原代大鼠胰腺干細(xì)胞,經(jīng)胰腺干細(xì)胞標(biāo)志物PDX1、Nestin和CK19免疫組化鑒定后,培養(yǎng)液中添加不同濃度(5mmol/L、10mmol/L、15mmol/L和20mmol/L)的�；撬釋�4-6代并處于對數(shù)生長期的胰腺干細(xì)胞進行孵育,處理24h、48h、72h、96h、120h、144h后MTS法檢測胰腺干細(xì)胞的增殖活性,并確定�；撬崽幚淼倪m宜濃度;在適宜濃度�；撬嵯逻B續(xù)處理原代胰腺干細(xì)胞至第4代,提取胰腺干細(xì)胞總蛋白,western-blot法檢測胰腺干細(xì)胞發(fā)育過程中調(diào)控因子(PDX1、Ptfla、Ngn3、Nestin、CK19、SOX9)蛋白表達水平的變化。試驗結(jié)果顯示:免疫組化鑒定胰腺干細(xì)胞標(biāo)志物PDX1、Nestin和CK19均表達呈陽性,說明分離得到的細(xì)胞為胰腺干細(xì)胞;10mmol/L和15mmol/L牛磺酸濃度組細(xì)胞增殖活性高于其余各組;胰腺干細(xì)胞調(diào)控因子蛋白表達量變化表明:1Ommol/L�；撬峤M與對照組相比PDX1和Ptfla表達量無明顯差異,但CK19表達量表達量升高且差異顯著(P0.05);15mmol/L�；撬峤M與對照組比較,PDX1、CK19、Ptfla表達量上調(diào)差異極顯著(P0.01);與對照組相比�；撬峤MSOX9和Ngn3表達量均上.調(diào)差異極顯著(P0.01),但Nestin表達量無差異(P0.05)。結(jié)果證明:1.�；撬峥擅黠@促進體外培養(yǎng)胰腺干細(xì)胞的增殖活性,且適宜添加濃度為10mmol/L和15mmol/L。2.牛磺酸可顯著促進體外培養(yǎng)胰腺干細(xì)胞PDX1、Ptfla、Ngn3、CK19、SOX9的表達,而對Nestin無影響,表明�；撬峥纱龠M胰腺干細(xì)胞分化。3.經(jīng)過進一步分析,�；撬岽僖认俑杉�(xì)胞分化機制可能與對Wnt通路、Notch通路以及FGF10通路的調(diào)控有關(guān)。
[Abstract]:At present, diabetes seriously affecting the quality of life of patients and can cause serious complications of diabetes. The conventional therapy drugs to stimulate insulin secretion by insulin in vitro, or reach the purpose of steady blood glucose, but can not completely repair diabetes islet lesions. In recent years, studies have found that pancreatic and pancreatic stem cells in memory. It can induce pancreatic beta cell function of in vitro, which provides a new way for the treatment of diabetes mellitus. Clinical and animal experiments showed that taurine can reduce blood glucose levels in diabetic patients, improve clinical symptoms, and prevent complications. Our preliminary tests show that the application of taurine can promote cells induced by STZ the proliferation of pancreatic islet of diabetic rats, increase the pancreatic stem cells into islet beta cell differentiation marker gene expression in taurine Can promote cell proliferation and differentiation of islet stem under diabetic state, but its mechanism is still unclear. Therefore this study by in vitro isolation and culture of rat pancreatic stem cells, to investigate the effect of pancreatic stem cells proliferation and differentiation mechanism, provide theoretical basis for prevention and treatment of diabetes mellitus. Application of taurine in this experiment 180-220gSD rats were isolated and cultured from primary rat pancreatic stem cells by mechanical digestion, the pancreatic stem cell markers PDX1, Nestin and CK19 immunohistochemical staining after cultured with different concentrations (5mmol/L, 10mmol/L, 15mmol/L and 20mmol/L) of the 4-6 generation of taurine and the logarithmic phase of growth of pancreatic stem cells were incubated with 24h. 48h, 72h, 96h, 120h, cell proliferation activity was detected by MTS 144H after pancreatic stem, and the optimal concentration of taurine; continuous primary pancreatic stem cells in the treatment of taurine under suitable concentration To the fourth generation of pancreatic stem cells, extracted total protein, Western-blot method for detection of pancreatic stem cell regulatory factors in the development process (PDX1, Ptfla, Ngn3, Nestin, CK19, SOX9) expression. Test results showed that: immunohistochemical identification of pancreatic stem cell markers PDX1, Nestin and CK19 were positive. That the cells isolated pancreatic stem cells; 10mmol/L and 15mmol/L taurine concentration group cell proliferation activity is higher than those of other groups; pancreatic stem cell regulatory factor protein expression changes showed no difference between 1Ommol/L PDX1 and taurine group compared with the control group Ptfla expression, but CK19 expression significantly increased (P0.05); 15mmol/L group, taurine group and control PDX1, CK19, Ptfla expression increased significantly (P0.01); compared with the control group, taurine group SOX9 and Ngn3 expression were adjusted. Significant differences (P0.01), but Ne There was no difference in the expression of stin (P0.05). The results showed that: 1. taurine can significantly promote pancreatic stem cells proliferation in vitro, and the suitable concentration of 10mmol/L and 15mmol/L.2. of taurine can significantly promote pancreatic stem cells cultured in vitro PDX1, Ptfla, Ngn3, CK19, SOX9 expression, but had no effect on Nestin, suggests that taurine can to promote the differentiation of pancreatic stem cells.3. after further analysis, to promote pancreatic stem cell differentiation mechanism of taurine may be related to the Wnt pathway, Notch pathway and FGF10 pathway regulation.

【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1
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本文編號：1491637