体外诱导大鼠骨髓干细胞分化为胰岛素分泌细胞
发布时间：2018-11-15 09:09
【摘要】： 目的:糖尿病是一种严重危害人们健康的慢性疾病。胰岛细胞移植可能是不依赖胰岛素治愈糖尿病的最佳方法。但由于移植材料匮乏,这种治疗方法的推广受到了限制。目前研究表明多种来源干细胞可以通过体外诱导分化为胰岛素分泌细胞,这为胰岛细胞移植提供了新的来源。因骨髓干细胞具有多种分化潜能、取材容易、可以避免免疫排斥反应等优点,使其成为诱导分化胰岛细胞的最佳候选干细胞。有研究显示骨髓干细胞在体外可以诱导分化为胰岛素分泌细胞。各种诱导方法虽然不尽相同,但总体上都比较繁琐,而且大多都应用细胞因子。这大大地限制了骨髓干细胞作为胰岛细胞移植的来源在临床上的广泛应用。本实验尝试在体外通过简单的非细胞因子方法诱导大鼠骨髓干细胞,使其分化为胰岛素分泌细胞,并应用一系列方法对诱导的细胞进行检测,目的在于探索一种不应用任何细胞因子,简单易行的诱导方法,从而为骨髓干细胞作为胰岛细胞移植的来源在临床上实际应用提供进一步的理论依据及新思路。 方法:1、制备鼠尾胶原及铺鼠尾胶原的玻片及六孔板。2、分离大鼠骨髓细胞。3、将分离的骨髓细胞分别设为DMSO及高糖环境培养的诱导组和低糖环境培养的对照组。4、通过倒置显微镜观察诱导组和对照组细胞形态的改变。5、通过双硫腙染色的方法检测诱导组及对照组细胞胰岛素的合成。6、通过免疫组化的方法检测诱导组及对照组细胞在蛋白质水平上胰岛素的表达。7、通过RT-PCR的方法检测诱导组及对照组细胞在mRNA水平上胰岛素的表达。8、通过ELISA的方法检测诱导组及对照组细胞分泌到细胞外的胰岛素含量。 结果:1、诱导组形成的细胞团在形态上与胰岛十分相似,与对照组形成的细胞团明显不同。2、用双硫腙对诱导组和对照组的细胞团进行染色,诱导组的细胞团呈现猩红色,表明细胞中有锌离子,提示有胰岛素的合成,而对照组不显色。3、免疫组化染色结果显示诱导组除团样细胞胞浆中有胰岛素外,圆形、椭圆形和部分梭形的分散的单个细胞中也含有胰岛素。对照组的细胞中无胰岛素存在。4、RT-PCR结果显示在mRNA水平上诱导组细胞表达Ins1和Ins2,而对照组不表达。5、ELISA方法检测诱导组细胞分泌到培养液上清中的胰岛素的量平均为(2.01±0.77)μg/L,而对照组为(0.63±0.43)μg/L。两者比较有显著性差异(P0.05)。 结论:用二甲基亚砜和高糖环境的方法诱导骨髓干细胞,能够形成在形态上与胰岛十分相似的细胞团。诱导组细胞不仅在mRNA水平及在蛋白水平表达胰岛素,而且能将其分泌到细胞外,提示二甲基亚砜和高糖环境能诱导骨髓干细胞分化为胰岛素分泌细胞。
[Abstract]:Objective: diabetes mellitus is a chronic disease that seriously endangers people's health. Islet cell transplantation may be the best way to cure diabetes without insulin dependence. However, due to the lack of transplantation materials, the promotion of this treatment is limited. Recent studies have shown that many stem cells can differentiate into insulin-secreting cells through in vitro induction, which provides a new source for islet cell transplantation. Bone marrow stem cells are the best candidate stem cells for inducing pancreatic islet cells because they have many potential of differentiation and can avoid immune rejection. Studies have shown that bone marrow stem cells can be induced to differentiate into insulin-secreting cells in vitro. Although all kinds of induction methods are not the same, they are more complicated in general, and most of them use cytokines. This greatly limits the clinical application of bone marrow stem cells as a source of islet cell transplantation. In this study, we tried to induce rat bone marrow stem cells to differentiate into insulin secreting cells by a simple non-cytokine method in vitro, and a series of methods were used to detect the induced cells. The aim of this study was to explore a simple and easy induction method without any cytokines, so as to provide further theoretical basis and new ideas for the clinical application of bone marrow stem cells as the source of islet cell transplantation. Methods: 1. The vitreous plates and six-hole plates of rat tail collagen and rat tail collagen were prepared. 2Rat bone marrow cells were isolated. 3. The isolated bone marrow cells were divided into DMSO and high glucose culture induction group and low glucose environment culture control group respectively. The morphologic changes of cells in induction group and control group were observed by inverted microscope, and insulin synthesis in induced group and control group was detected by dithizone staining. The expression of insulin at the protein level was detected by immunohistochemical method, and the expression of insulin on the mRNA level was detected by RT-PCR method in the cells of the induced group and the control group, and the expression of insulin in the cells of the induced group and the control group at the level of mRNA was detected by the method of RT-PCR. The levels of extracellular insulin secreted into the cells of the induction group and the control group were detected by ELISA. Results: 1. The cell clusters formed in the induced group were very similar to the pancreatic islets in morphology, and were obviously different from those in the control group. 2. Dithizone was used to stain the cell clusters of the induced group and the control group, and the cells in the induced group were scarlet red. The results showed that there was zinc ion in the cells, indicating the synthesis of insulin, but not in the control group. The results of immunohistochemical staining showed that the cells in the induction group were round except for insulin in the cytoplasm of clusterlike cells. The elliptical and partially fusiform scattered individual cells also contain insulin. The results of RT-PCR showed that the expression of Ins1 and Ins2, was induced at the mRNA level in the control group, but not in the control group. The average amount of insulin secreted into the supernatant by ELISA was (2.01 卤0.77) 渭 g / L in the induced group and (0.63 卤0.43) 渭 g / L in the control group. There was significant difference between the two groups (P0.05). Conclusion: bone marrow stem cells induced by dimethyl sulfoxide and high glucose environment can form cell clusters similar to pancreatic islets in morphology. The induction group not only expressed insulin at the level of mRNA and protein, but also secreted it into extracellular, suggesting that dimethyl sulfoxide and high glucose environment could induce bone marrow stem cells to differentiate into insulin secreting cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2
[Abstract]:Objective: diabetes mellitus is a chronic disease that seriously endangers people's health. Islet cell transplantation may be the best way to cure diabetes without insulin dependence. However, due to the lack of transplantation materials, the promotion of this treatment is limited. Recent studies have shown that many stem cells can differentiate into insulin-secreting cells through in vitro induction, which provides a new source for islet cell transplantation. Bone marrow stem cells are the best candidate stem cells for inducing pancreatic islet cells because they have many potential of differentiation and can avoid immune rejection. Studies have shown that bone marrow stem cells can be induced to differentiate into insulin-secreting cells in vitro. Although all kinds of induction methods are not the same, they are more complicated in general, and most of them use cytokines. This greatly limits the clinical application of bone marrow stem cells as a source of islet cell transplantation. In this study, we tried to induce rat bone marrow stem cells to differentiate into insulin secreting cells by a simple non-cytokine method in vitro, and a series of methods were used to detect the induced cells. The aim of this study was to explore a simple and easy induction method without any cytokines, so as to provide further theoretical basis and new ideas for the clinical application of bone marrow stem cells as the source of islet cell transplantation. Methods: 1. The vitreous plates and six-hole plates of rat tail collagen and rat tail collagen were prepared. 2Rat bone marrow cells were isolated. 3. The isolated bone marrow cells were divided into DMSO and high glucose culture induction group and low glucose environment culture control group respectively. The morphologic changes of cells in induction group and control group were observed by inverted microscope, and insulin synthesis in induced group and control group was detected by dithizone staining. The expression of insulin at the protein level was detected by immunohistochemical method, and the expression of insulin on the mRNA level was detected by RT-PCR method in the cells of the induced group and the control group, and the expression of insulin in the cells of the induced group and the control group at the level of mRNA was detected by the method of RT-PCR. The levels of extracellular insulin secreted into the cells of the induction group and the control group were detected by ELISA. Results: 1. The cell clusters formed in the induced group were very similar to the pancreatic islets in morphology, and were obviously different from those in the control group. 2. Dithizone was used to stain the cell clusters of the induced group and the control group, and the cells in the induced group were scarlet red. The results showed that there was zinc ion in the cells, indicating the synthesis of insulin, but not in the control group. The results of immunohistochemical staining showed that the cells in the induction group were round except for insulin in the cytoplasm of clusterlike cells. The elliptical and partially fusiform scattered individual cells also contain insulin. The results of RT-PCR showed that the expression of Ins1 and Ins2, was induced at the mRNA level in the control group, but not in the control group. The average amount of insulin secreted into the supernatant by ELISA was (2.01 卤0.77) 渭 g / L in the induced group and (0.63 卤0.43) 渭 g / L in the control group. There was significant difference between the two groups (P0.05). Conclusion: bone marrow stem cells induced by dimethyl sulfoxide and high glucose environment can form cell clusters similar to pancreatic islets in morphology. The induction group not only expressed insulin at the level of mRNA and protein, but also secreted it into extracellular, suggesting that dimethyl sulfoxide and high glucose environment could induce bone marrow stem cells to differentiate into insulin secreting cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.2
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