骨髓基质干细胞诱导分化为胰岛细胞的实验研究
发布时间：2018-08-31 11:14
【摘要】： 目的：胰岛细胞移植是治疗Ⅰ型糖尿病最有希望的方法,由于供体细胞不足和免疫排斥反应等问题,限制了胰岛细胞移植技术的应用。骨髓基质干细胞(BMSCs)具有获得容易,可体外复制和增殖,可以定向诱导分化为特定功能细胞的特征。因此,将BMSCs移植用于治疗糖尿病具有重要的临床应用价值。目前,尚未获得诱导干细胞向胰岛细胞分化的高效诱导方法；取得较好治疗效果的干细胞移植途径尚未确定。本实验主要探索体外诱导BMSCs分化为胰岛素分泌细胞的最佳条件；比较尾静脉、肾被膜和胰腺内注射三种干细胞移植途径对糖尿病模型动物的治疗作用,以筛选出干细胞治疗糖尿病的最佳移植途径。 方法：全骨髓贴壁法分离大鼠BMSCs.体外扩增后,部分细胞用于体外诱导实验,即向培养体系中添加胰腺提取液、尼克酰胺、Exendin-4及不同浓度的葡萄糖；观察分化细胞的形态变化；双硫腙染色法(DTZ)鉴定胰岛素分化细胞的生成；放射免疫法(RIA)检测上清液中胰岛素和C—肽水平。另一部分细胞进行BrdU标记。将标记的BMSCs,经尾静脉、肾被膜和胰腺内注射三种途径,移植入链脲佐菌素(STZ)制备糖尿病模型小鼠体内。于移植后3、7、14、28、42天测定血糖；免疫组织化学法检测移植细胞在胰腺中的迁移和分化；放射免疫学方法监测胰岛素及C-肽水平。结果：(1)在高糖或低糖培养基中加入诱导因子尼克酰胺、Exendin-4,细胞趋向形成细胞团,高糖培养条件下细胞成团趋势更明显。添加鼠胰腺提取液,细胞内颗粒增多、细胞脱落、死亡。(2)诱导组大部分细胞双硫腙染色呈棕红色,对照组细胞无阳性着色。(3)诱导组细胞经葡萄糖刺激后,能够分泌胰岛素和C-肽。(4)腹腔注射STZ3d后,小鼠血糖均≥16.7mmol/L,出现明显多饮、多尿等症状,确定糖尿病模型造模成功。(5)尾静脉、肾被膜及胰腺内移植组与糖尿病模型对照组相比,血糖均有所降低,胰岛素及C-肽均有所增加(P0.05),尤以胰腺移植组血糖下降、胰岛素和C-肽升高更为显著。(6)尾静脉和胰腺内移植组的胰腺内可见BrdU阳性标记细胞。肾被膜移植组的小鼠肾脏中有BrdU标记的细胞,胰腺未见BrdU阳性表达的细胞。 结论：(1)在尼克酰胺、Exendin-4联合诱导下,BMSCs可分化为胰岛样细胞,并且高糖培养基的效果要优于低糖培养基。(2)三种移植途径均可降低糖尿病模型小鼠的血糖,提高血液中胰岛素和C-肽的水平。其中胰腺内移植组的效果更明显。
[Abstract]:Objective: islet cell transplantation is the most promising method for the treatment of type 1 diabetes mellitus. The application of islet cell transplantation is limited due to donor cell insufficiency and immune rejection. Bone marrow stromal cells (BMSCs) (BMSCs) is easy to obtain, can be replicated and proliferated in vitro, and can be induced to differentiate into specific functional cells. Therefore, BMSCs transplantation has important clinical value in the treatment of diabetes. At present, no efficient induction method has been obtained to induce stem cells to differentiate into islet cells, and the way of stem cell transplantation to obtain better therapeutic effect has not been determined. The aim of this study was to investigate the optimal conditions for inducing BMSCs to differentiate into insulin-secreting cells in vitro, and to compare the therapeutic effects of three kinds of stem cell transplantation pathways: caudal vein, renal capsule and intrapancreatic injection on diabetic model animals. In order to screen the best transplantation of stem cells to treat diabetes. Methods: rat BMSCs. was isolated by whole bone marrow adherent method. After amplification in vitro, some cells were used in the induction experiment in vitro, that is, adding pancreatic extract, nicotinamide Exendin-4 and different concentrations of glucose to the culture system to observe the morphological changes of differentiated cells. Dithizone staining (DTZ) was used to identify insulin differentiation cells and radioimmunoassay (RIA) was used to detect the levels of insulin and C-peptide in supernatant. Another part of the cells were labeled with BrdU. Streptozotocin (STZ) was transplanted into streptozotocin (STZ) into diabetic mice by injecting labeled BMSCs, through tail vein, renal capsule and pancreas. The levels of insulin and C-peptide were detected by radioimmunoassay and the migration and differentiation of transplanted cells in pancreas were detected by immunohistochemistry. Results: (1) the cells tended to form cell clusters in high sugar or low sugar medium, and the tendency of cell cluster formation was more obvious under high glucose culture condition. (2) most of the cells in the induction group showed brown red dithizone staining, while no positive staining was found in the control group. (3) the cells in the induction group were stimulated by glucose. (4) after intraperitoneal injection of STZ3d, the blood glucose of mice was more than 16.7 mmol / L, with obvious symptoms of polyhydration and polyuria. (5) the model of diabetes was successfully established. (5) the tail vein, renal capsule and intrapancreatic transplantation group were compared with the diabetic model control group. Blood glucose was decreased, insulin and C-peptide were increased (P0.05), especially in pancreas transplantation group. (6) in caudal vein and intrapancreatic transplantation group, BrdU positive labeled cells were found in pancreas. There were BrdU labeled cells in kidney and no BrdU positive cells in pancreas. Conclusion: (1) BMSCs can differentiate into islet like cells induced by nicotinamide Exendin-4, and the effect of high glucose medium is better than that of low glucose medium. Increase the levels of insulin and C-peptide in the blood. The effect of intrapancreatic transplantation was more obvious.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329.28
本文编号：2214811
[Abstract]:Objective: islet cell transplantation is the most promising method for the treatment of type 1 diabetes mellitus. The application of islet cell transplantation is limited due to donor cell insufficiency and immune rejection. Bone marrow stromal cells (BMSCs) (BMSCs) is easy to obtain, can be replicated and proliferated in vitro, and can be induced to differentiate into specific functional cells. Therefore, BMSCs transplantation has important clinical value in the treatment of diabetes. At present, no efficient induction method has been obtained to induce stem cells to differentiate into islet cells, and the way of stem cell transplantation to obtain better therapeutic effect has not been determined. The aim of this study was to investigate the optimal conditions for inducing BMSCs to differentiate into insulin-secreting cells in vitro, and to compare the therapeutic effects of three kinds of stem cell transplantation pathways: caudal vein, renal capsule and intrapancreatic injection on diabetic model animals. In order to screen the best transplantation of stem cells to treat diabetes. Methods: rat BMSCs. was isolated by whole bone marrow adherent method. After amplification in vitro, some cells were used in the induction experiment in vitro, that is, adding pancreatic extract, nicotinamide Exendin-4 and different concentrations of glucose to the culture system to observe the morphological changes of differentiated cells. Dithizone staining (DTZ) was used to identify insulin differentiation cells and radioimmunoassay (RIA) was used to detect the levels of insulin and C-peptide in supernatant. Another part of the cells were labeled with BrdU. Streptozotocin (STZ) was transplanted into streptozotocin (STZ) into diabetic mice by injecting labeled BMSCs, through tail vein, renal capsule and pancreas. The levels of insulin and C-peptide were detected by radioimmunoassay and the migration and differentiation of transplanted cells in pancreas were detected by immunohistochemistry. Results: (1) the cells tended to form cell clusters in high sugar or low sugar medium, and the tendency of cell cluster formation was more obvious under high glucose culture condition. (2) most of the cells in the induction group showed brown red dithizone staining, while no positive staining was found in the control group. (3) the cells in the induction group were stimulated by glucose. (4) after intraperitoneal injection of STZ3d, the blood glucose of mice was more than 16.7 mmol / L, with obvious symptoms of polyhydration and polyuria. (5) the model of diabetes was successfully established. (5) the tail vein, renal capsule and intrapancreatic transplantation group were compared with the diabetic model control group. Blood glucose was decreased, insulin and C-peptide were increased (P0.05), especially in pancreas transplantation group. (6) in caudal vein and intrapancreatic transplantation group, BrdU positive labeled cells were found in pancreas. There were BrdU labeled cells in kidney and no BrdU positive cells in pancreas. Conclusion: (1) BMSCs can differentiate into islet like cells induced by nicotinamide Exendin-4, and the effect of high glucose medium is better than that of low glucose medium. Increase the levels of insulin and C-peptide in the blood. The effect of intrapancreatic transplantation was more obvious.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329.28
【引证文献】
相关硕士学位论文 前1条
1 李薇;携带Ubc9基因的病毒表达载体的构建及其在细胞中的表达[D];青岛大学;2011年
，本文编号：2214811
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