胰腺微环境下间充质干细胞分化为胰岛样细胞的机制初探

发布时间：2018-01-04 22:12

本文关键词：胰腺微环境下间充质干细胞分化为胰岛样细胞的机制初探　出处：《暨南大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的骨髓间充质干细胞(BMSCs)植入胰腺微环境下,观察对糖尿病模型鼠的影响,植入后细胞的再分布,初步探讨植入的BMSCs分化为胰岛样细胞的机制。方法从同种异体SD大鼠骨髓中获取BMSCs,纯化后用逆转录病毒(RV)转染绿色荧光蛋白(EGFP)标记,以多点注射的方法移植入糖尿病大鼠胰腺包膜下。移植后动态监测血糖、胰岛素和C-肽变化;通过EGFP示踪观察植入的BMSCs的分布;移植后1、2、3、5、8及12周,取EGFP标记的胰腺组织,Realtime-PCR检测PDX-1、ngn 3、nestin、Nkx 2.2、pax 4和EGFP的表达;移植后8周取EGFP标记的胰腺组织,免疫荧光染色和荧光原位杂交检测检测EGFP和胰岛素、EGFP和PDX-1的共表达,流式细胞术分析EGFP标记的胰腺组织细胞DNA倍性。结果移植6d后糖尿病鼠血糖开始降低,24d后接近于正常鼠水平(150±42.0mg/dL);移植14d后胰岛素、C肽水平开始升高,到56d稳定在较高水平(1.0±0.2μg/L,1.2±0.3 nmol/L)。移植8周,植入细胞稳定表达EGFP,植入细胞在胰腺组织分布于胰岛(12.46%)、血管(4.4%)、导管(3.21%)、腺泡(9.24%)和组织间隙(70.69%)。免疫荧光染色发现有EGFP和胰岛素共表达细胞(5.1696),FISH发现有EGFP和PDX-1 mRNA共表达细胞(0.96%);移植到胰腺微环境中的BMSCs,1周时检测到PDX-1、ngn 3、nestin、Nkx 2.2和pax 4的表达,2到3周时各基因表达水平持续上调,5周左右达到最大值,以后逐渐下调,12周时nestin、ngn 3、pax 4不表达。流式细胞术测定EGFP标记的胰腺组织中细胞核型为整二倍体或四倍体,未见多倍体或非整倍体细胞。结论BMSCs胰腺包膜下移植后,糖尿病大鼠血糖降低、胰岛素和C肽水平升高:BMSCs植入后在胰腺中发生再分布;胰腺微环境下,BMSCs能跨胚层分化为胰岛样细胞,基因表达符合β细胞分化的时序性变化,没有与胰腺组织细胞发生融合。
[Abstract]:Objective to observe the effect of bone marrow mesenchymal stem cells (BMSCs) implanted into pancreatic microenvironment on diabetic model rats and the redistribution of cells after implantation. To explore the mechanism of the differentiation of implanted BMSCs into islet-like cells. Methods BMSCs were obtained from allogeneic SD rat bone marrow and then transfected with green fluorescent protein (EGFP) labeled with retrovirus RV. The pancreatic capsule was transplanted into diabetic rats by multi-point injection. The changes of blood glucose, insulin and C-peptide were monitored dynamically after transplantation. The distribution of implanted BMSCs was observed by EGFP tracer. At 8 and 12 weeks after transplantation, PDX-1ngn 3 ngn 3 nestin was detected in pancreatic tissue labeled with EGFP. The expression of Nkx 2.2 pPAX4 and EGFP; The pancreatic tissues labeled with EGFP were taken 8 weeks after transplantation. The co-expression of EGFP, insulin and PDX-1 were detected by immunofluorescence staining and fluorescence in situ hybridization. DNA ploidy of pancreatic tissue cells labeled with EGFP was analyzed by flow cytometry. Results after 6 days of transplantation, the blood glucose level of diabetic rats began to decrease after 24 days and was close to that of normal mice at 150 卤42.0 mg / d L ~ (-1). After 14 days of transplantation, the level of insulin C peptide began to increase, and at 56 days it was stable at a higher level of 1.0 卤0.2 渭 g / L, 1.2 卤0.3 nmol / L 路L ~ (-1) 路L ~ (-1), and was transplanted for 8 weeks. EGFP was expressed stably in the implanted cells. The implanted cells were distributed in the pancreatic islets (12.46%), and the vascular vessels were 4.4%, and the ducts were 3.21% (P < 0.05). EGFP and insulin co-expression cells were found by immunofluorescence staining. EGFP and PDX-1 mRNA co-expressed cells were found in FISH. The expression of Nkx2.2 and pax _ 4 was detected at 1 week after transplantation into pancreatic microenvironment. After 2 to 3 weeks, the expression level of each gene continued to up-regulate and reached the maximum at about 5 weeks, and then gradually down-regulated nestinine ngn 3 at 12 weeks. Pax _ 4 was not expressed. The nuclear type of pancreatic tissue labeled with EGFP was determined by flow cytometry to be diploid or tetraploid, but no polyploid or aneuploidy cells were found. Conclusion after subcapsular transplantation of BMSCs, blood glucose in diabetic rats decreased, insulin and C-peptide levels increased and redistributed in pancreas after implantation. BMSCs could differentiate into islet like cells in pancreatic microenvironment. The gene expression was consistent with the temporal changes of 尾 -cell differentiation and did not fuse with pancreatic tissue cells.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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