本文關(guān)鍵詞： LRP16 MIN6 胰島素 葡萄糖轉(zhuǎn)運(yùn)子-2 胰十二指腸同源盒-1　出處：《中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： LRP16基因是解放軍總醫(yī)院韓為東等在1999年首先克隆的一個(gè)人類(lèi)基因。定位于染色體11q12.23,其表達(dá)產(chǎn)物是一個(gè)核蛋白。以往的研究發(fā)現(xiàn),LRP16基因?yàn)榇萍に氐陌谢?雌激素通過(guò)雌激素受體α(ERα)上調(diào)該基因,而LRP16同時(shí)又是ERα的共激活因子,能夠反饋增強(qiáng)ERα介導(dǎo)的轉(zhuǎn)錄活性。近年來(lái)大量研究發(fā)現(xiàn),雌激素具有保護(hù)胰島β細(xì)胞、促進(jìn)胰島素合成與分泌的作用,該作用是否通過(guò)LRP16的介導(dǎo)值得關(guān)注。我們新近的研究發(fā)現(xiàn),人胰島β細(xì)胞瘤的免疫組化顯示LRP16蛋白表達(dá)量明顯高于正常對(duì)照。這提示LRP16基因可能在胰腺的生長(zhǎng)、發(fā)育和胰島β細(xì)胞合成、分泌胰島素等方面具有重要作用。本研究通過(guò)在胰島β細(xì)胞系MIN6細(xì)胞中過(guò)表達(dá)LRP16基因,檢測(cè)葡萄糖刺激下胰島素的分泌功能(GSIS)和胰島素mRNAs的表達(dá)情況,并探討其可能機(jī)制。本研究共分為2部分: 一、LRP16基因?qū)IN6細(xì)胞的增殖和胰島素分泌功能的影響 目的:探討LRP16基因?qū)IN6細(xì)胞的增殖和胰島素分泌功能的影響及其可能機(jī)制。方法:1.用Western blot方法檢測(cè)MIN6細(xì)胞是否表達(dá)LRP16蛋白;2.用SuperFect脂質(zhì)體轉(zhuǎn)染法建立穩(wěn)定過(guò)表達(dá)LRP16基因的MIN6細(xì)胞株;3.用MTT法檢測(cè)細(xì)胞的增殖情況;4.分別用0 mmol/L、3 mmol/L和30 mmol/L葡萄糖刺激細(xì)胞,檢測(cè)胰島素分泌功能;5.用Western blot方法檢測(cè)葡萄糖轉(zhuǎn)運(yùn)子-2(Glut-2)的蛋白表達(dá)情況。結(jié)果:1.MIN6細(xì)胞表達(dá)LRP16蛋白;2.過(guò)表達(dá)LRP16組的細(xì)胞增殖情況與對(duì)照組相比無(wú)顯著差別(p＞0.05);3.在0 mmol/L、3 mmol/L和30 mmol/L葡萄糖刺激下,過(guò)表達(dá)LRP16組的胰島素分泌量分別為對(duì)照組的2.26倍(p＜0.05)、2.19倍(p＜0.05)和2.16倍(p＜0.05)(三者之間相比,p＞0.05);4.Westernblot顯示過(guò)表達(dá)LRP16組的Glut-2蛋白量是對(duì)照組的1.76倍(p＜0.05)。結(jié)論:小鼠的胰島β細(xì)胞中表達(dá)LRP16蛋白;過(guò)表達(dá)LRP16基因不能促進(jìn)MIN6細(xì)胞增殖,但是可以促進(jìn)葡萄糖刺激的胰島素分泌(GSIS),該作用可能依賴(lài)于Glut-2的上調(diào),且不依賴(lài)葡萄糖濃度。 二、LRP16基因?qū)IN6細(xì)胞胰島素mRNAs的合成及相關(guān)轉(zhuǎn)錄因子的影響 目的:探討LRP16基因?qū)IN6細(xì)胞胰島素mRNAs合成的影響及可能相關(guān)的轉(zhuǎn)錄因子。方法:1.用superFect脂質(zhì)體轉(zhuǎn)染法建立穩(wěn)定過(guò)表達(dá)LRP16基因的MIN6細(xì)胞株;2.用Real-Time PCR法檢測(cè)胰島素mRNAs的合成情況及轉(zhuǎn)錄因子胰十二指腸同源盒-1(Pdx-1)、MafA和NeuroD1 mRNAs的合成情況;3.用Western blot方法檢測(cè)Pdx-1、MafA和NeuroD1蛋白的表達(dá)情況。結(jié)果:1.Real-Time PCR顯示過(guò)表達(dá)LRP16組的InsulinⅠmRNA和InsulinⅡmRNA的量分別是對(duì)照組的1.6倍和1.8倍(p＜0.05);2.過(guò)表達(dá)LRP16組的Pdx-1 mRNA的量是對(duì)照組的1.62倍(p＜0.05),而MafA和NeuroD1 mRNAs的量與對(duì)照組相比沒(méi)有顯著差別(p＞0.05);3.過(guò)表達(dá)LRP16組的Pdx-1蛋白量是對(duì)照組的2.04倍(p＜0.05),而MafA和NeuroD1的蛋白量與對(duì)照組相比沒(méi)有顯著差別(p＞0.05)。結(jié)論:過(guò)表達(dá)LRP16基因能夠促進(jìn)胰島素mRNAs的合成,該作用可能是通過(guò)上調(diào)轉(zhuǎn)錄因子Pdx-1實(shí)現(xiàn)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.

【學(xué)位授予單位】：中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R587.1;R346

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本文編號(hào)：1538194