LRP16基因对MIN6细胞功能的影响

发布时间：2018-02-26 13:24

本文关键词： LRP16 MIN6 胰岛素葡萄糖转运子-2 胰十二指肠同源盒-1　出处：《中国人民解放军军医进修学院》2009年硕士论文　论文类型：学位论文

【摘要】： LRP16基因是解放军总医院韩为东等在1999年首先克隆的一个人类基因。定位于染色体11q12.23,其表达产物是一个核蛋白。以往的研究发现,LRP16基因为雌激素的靶基因,雌激素通过雌激素受体α(ERα)上调该基因,而LRP16同时又是ERα的共激活因子,能够反馈增强ERα介导的转录活性。近年来大量研究发现,雌激素具有保护胰岛β细胞、促进胰岛素合成与分泌的作用,该作用是否通过LRP16的介导值得关注。我们新近的研究发现,人胰岛β细胞瘤的免疫组化显示LRP16蛋白表达量明显高于正常对照。这提示LRP16基因可能在胰腺的生长、发育和胰岛β细胞合成、分泌胰岛素等方面具有重要作用。本研究通过在胰岛β细胞系MIN6细胞中过表达LRP16基因,检测葡萄糖刺激下胰岛素的分泌功能(GSIS)和胰岛素mRNAs的表达情况,并探讨其可能机制。本研究共分为2部分: 一、LRP16基因对MIN6细胞的增殖和胰岛素分泌功能的影响目的:探讨LRP16基因对MIN6细胞的增殖和胰岛素分泌功能的影响及其可能机制。方法:1.用Western blot方法检测MIN6细胞是否表达LRP16蛋白;2.用SuperFect脂质体转染法建立稳定过表达LRP16基因的MIN6细胞株;3.用MTT法检测细胞的增殖情况;4.分别用0 mmol/L、3 mmol/L和30 mmol/L葡萄糖刺激细胞,检测胰岛素分泌功能;5.用Western blot方法检测葡萄糖转运子-2(Glut-2)的蛋白表达情况。结果:1.MIN6细胞表达LRP16蛋白;2.过表达LRP16组的细胞增殖情况与对照组相比无显著差别(p＞0.05);3.在0 mmol/L、3 mmol/L和30 mmol/L葡萄糖刺激下,过表达LRP16组的胰岛素分泌量分别为对照组的2.26倍(p＜0.05)、2.19倍(p＜0.05)和2.16倍(p＜0.05)(三者之间相比,p＞0.05);4.Westernblot显示过表达LRP16组的Glut-2蛋白量是对照组的1.76倍(p＜0.05)。结论:小鼠的胰岛β细胞中表达LRP16蛋白;过表达LRP16基因不能促进MIN6细胞增殖,但是可以促进葡萄糖刺激的胰岛素分泌(GSIS),该作用可能依赖于Glut-2的上调,且不依赖葡萄糖浓度。二、LRP16基因对MIN6细胞胰岛素mRNAs的合成及相关转录因子的影响目的:探讨LRP16基因对MIN6细胞胰岛素mRNAs合成的影响及可能相关的转录因子。方法:1.用superFect脂质体转染法建立稳定过表达LRP16基因的MIN6细胞株;2.用Real-Time PCR法检测胰岛素mRNAs的合成情况及转录因子胰十二指肠同源盒-1(Pdx-1)、MafA和NeuroD1 mRNAs的合成情况;3.用Western blot方法检测Pdx-1、MafA和NeuroD1蛋白的表达情况。结果:1.Real-Time PCR显示过表达LRP16组的InsulinⅠmRNA和InsulinⅡmRNA的量分别是对照组的1.6倍和1.8倍(p＜0.05);2.过表达LRP16组的Pdx-1 mRNA的量是对照组的1.62倍(p＜0.05),而MafA和NeuroD1 mRNAs的量与对照组相比没有显著差别(p＞0.05);3.过表达LRP16组的Pdx-1蛋白量是对照组的2.04倍(p＜0.05),而MafA和NeuroD1的蛋白量与对照组相比没有显著差别(p＞0.05)。结论:过表达LRP16基因能够促进胰岛素mRNAs的合成,该作用可能是通过上调转录因子Pdx-1实现的。
[Abstract]:LRP16 gene is a human gene of General Hospital of Chinese PLA Han Weidong in 1999. The first clone located on chromosome 11q12.23. Its expression is a nuclear protein. Previous studies have found that LRP16 gene as the target genes of estrogen, estrogen estrogen receptor alpha (ER alpha) by the gene, and at the same time is LRP16 coactivator ER alpha, feedback can enhance the transcriptional activity of ER alpha mediated. Recent studies have shown that estrogen can protect islet beta cells, promote insulin synthesis and secretion, the effect is mediated by LRP16 of concern. Our recent study found that human islet beta cell tumor immune group the LRP16 expression was significantly higher than that in normal control. This suggests that LRP16 gene may be in pancreatic islet beta cell growth, development and synthesis, which plays an important role in insulin secretion and so on. This research by The LRP16 gene was overexpressed in MIN6 cells of islet beta cell line, the secretion function of insulin (GSIS) and the expression of insulin mRNAs were detected, and the possible mechanism was explored. The study is divided into 2 parts.
The effect of LRP16 gene on the proliferation of MIN6 cells and the function of insulin secretion
Objective: To evaluate the effect of LRP16 gene on proliferation and insulin secretion of MIN6 cells and its possible mechanism. Methods: 1. test whether MIN6 cells express LRP16 protein by Western blot method; 2. SuperFect using liposome transfection method to establish a stable MIN6 cell line expressing LRP16 gene; proliferation of 3. cells were detected by MTT method; 4. with 0 mmol/L, 3 mmol/L and 30 mmol/L glucose stimulated insulin secretion test cells; 5. using the Western blot method for detection of glucose transporter -2 (Glut-2) protein expression. Results: the expression of LRP16 protein in 1.MIN6 cells; 2. expression showed no difference between cell proliferation and the control group LRP16 group (P > 0.05); 3. in 0 mmol/L, 3 mmol/L and 30 mmol/L glucose stimulation, overexpression of LRP16 group insulin secretion were 2.26 times higher than that of control group (P < 0.05), 2.19 times (P < 0.05) and 2.16 times (P < 0.05 (three) Compared between, P > 0.05); 4.Westernblot showed that over expression of Glut-2 protein in LRP16 group was 1.76 times higher than the control group (P < 0.05). Conclusion: the expression of LRP16 protein in pancreatic beta cells in mice; overexpression of LRP16 gene can promote the proliferation of MIN6 cells, but can promote glucose stimulated insulin secretion (GSIS), the effect may be dependent on the upregulation of Glut-2, and does not depend on the concentration of glucose.
Two, the effect of LRP16 gene on the synthesis of insulin mRNAs and related transcription factors in MIN6 cells
Objective: To investigate the effect of LRP16 gene on the synthesis of mRNAs and MIN6 cells insulin related transcription factors. Methods: 1. superFect using liposome transfection method to establish a stable MIN6 cell line expressing LRP16 gene; synthesis of 2. using Real-Time PCR method to detect insulin mRNAs and transcription factors in pancreatic and duodenal homeobox -1 (Pdx-1). The synthesis of MafA and NeuroD1 mRNAs; 3. Pdx-1 detected by Western blot method, the expression of MafA and NeuroD1 protein. Results: 1.Real-Time PCR showed that over expression of LRP16 group Insulin 1 mRNA and Insulin II mRNA volume control group respectively 1.6 times and 1.8 times (P < 0.05); 2. overexpression of LRP16 group the Pdx-1 mRNA is 1.62 times higher than that of control group (P < 0.05), while MafA and NeuroD1 the amount of mRNAs compared with the control group had no significant difference (P > 0.05); 3. overexpression of Pdx-1 protein in LRP16 group was 2.04 times higher than the control group (P < 0.05), and M The protein content of afA and NeuroD1 was not significantly different from that of the control group (P > 0.05). Conclusion: over expression of LRP16 gene can promote the synthesis of insulin mRNAs, which may be achieved by upregulated transcription factor Pdx-1.

【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R587.1;R346

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