牛磺酸对体外培养大鼠胰腺干细胞增殖及分化影响的研究

发布时间：2018-02-05 00:49

  本文关键词： 胰腺干细胞 牛磺酸 增殖 分化　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：目前糖尿病严重影响患者的生活质量,并且可引发严重的糖尿病并发症。传统的治疗手段主要通过药物刺激体内胰岛素分泌,或者体外注射胰岛素达到平稳血糖的目的,却不能彻底修复糖尿病患者的胰岛病变。近年来,有研究发现胰腺内存在胰腺干细胞,并且可在体外诱导形成具有功能性的胰岛β细胞,这为治疗糖尿病提供了新的思路。临床和动物试验均证明,补充牛磺酸能够降低糖尿病患者的血糖水平,改善临床症状,并防止并发症的发生。课题组前期试验证明,应用牛磺酸能够促进STZ诱导糖尿病大鼠胰岛干细胞的增殖,上调胰腺干细胞向胰岛β细胞分化标志因子的表达,提示牛磺酸可促进糖尿病状态下胰岛干细胞的增殖分化,但其作用机制尚不清楚。因此本研究通过体外试验分离培养大鼠胰腺干细胞,探讨牛磺酸胰腺干细胞增殖及分化作用机理,为应用牛磺酸防治糖尿病提供理论依据。本试验选取180-220gSD大鼠采用机械消化法分离培养出原代大鼠胰腺干细胞,经胰腺干细胞标志物PDX1、Nestin和CK19免疫组化鉴定后,培养液中添加不同浓度(5mmol/L、10mmol/L、15mmol/L和20mmol/L)的牛磺酸对4-6代并处于对数生长期的胰腺干细胞进行孵育,处理24h、48h、72h、96h、120h、144h后MTS法检测胰腺干细胞的增殖活性,并确定牛磺酸处理的适宜浓度;在适宜浓度牛磺酸下连续处理原代胰腺干细胞至第4代,提取胰腺干细胞总蛋白,western-blot法检测胰腺干细胞发育过程中调控因子(PDX1、Ptfla、Ngn3、Nestin、CK19、SOX9)蛋白表达水平的变化。试验结果显示:免疫组化鉴定胰腺干细胞标志物PDX1、Nestin和CK19均表达呈阳性,说明分离得到的细胞为胰腺干细胞;10mmol/L和15mmol/L牛磺酸浓度组细胞增殖活性高于其余各组;胰腺干细胞调控因子蛋白表达量变化表明:1Ommol/L牛磺酸组与对照组相比PDX1和Ptfla表达量无明显差异,但CK19表达量表达量升高且差异显著(P0.05);15mmol/L牛磺酸组与对照组比较,PDX1、CK19、Ptfla表达量上调差异极显著(P0.01);与对照组相比牛磺酸组SOX9和Ngn3表达量均上.调差异极显著(P0.01),但Nestin表达量无差异(P0.05)。结果证明:1.牛磺酸可明显促进体外培养胰腺干细胞的增殖活性,且适宜添加浓度为10mmol/L和15mmol/L。2.牛磺酸可显著促进体外培养胰腺干细胞PDX1、Ptfla、Ngn3、CK19、SOX9的表达,而对Nestin无影响,表明牛磺酸可促进胰腺干细胞分化。3.经过进一步分析,牛磺酸促胰腺干细胞分化机制可能与对Wnt通路、Notch通路以及FGF10通路的调控有关。
[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.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
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本文编号：1491637