本文關(guān)鍵詞： LRP16 MIN6 胰島素 葡萄糖轉(zhuǎn)運子-2 胰十二指腸同源盒-1　出處：《中國人民解放軍軍醫(yī)進修學院》2009年碩士論文　論文類型：學位論文

【摘要】： LRP16基因是解放軍總醫(yī)院韓為東等在1999年首先克隆的一個人類基因。定位于染色體11q12.23,其表達產(chǎn)物是一個核蛋白。以往的研究發(fā)現(xiàn),LRP16基因為雌激素的靶基因,雌激素通過雌激素受體α(ERα)上調(diào)該基因,而LRP16同時又是ERα的共激活因子,能夠反饋增強ERα介導的轉(zhuǎn)錄活性。近年來大量研究發(fā)現(xiàn),雌激素具有保護胰島β細胞、促進胰島素合成與分泌的作用,該作用是否通過LRP16的介導值得關(guān)注。我們新近的研究發(fā)現(xiàn),人胰島β細胞瘤的免疫組化顯示LRP16蛋白表達量明顯高于正常對照。這提示LRP16基因可能在胰腺的生長、發(fā)育和胰島β細胞合成、分泌胰島素等方面具有重要作用。本研究通過在胰島β細胞系MIN6細胞中過表達LRP16基因,檢測葡萄糖刺激下胰島素的分泌功能(GSIS)和胰島素mRNAs的表達情況,并探討其可能機制。本研究共分為2部分: 一、LRP16基因?qū)IN6細胞的增殖和胰島素分泌功能的影響 目的:探討LRP16基因?qū)IN6細胞的增殖和胰島素分泌功能的影響及其可能機制。方法:1.用Western blot方法檢測MIN6細胞是否表達LRP16蛋白;2.用SuperFect脂質(zhì)體轉(zhuǎn)染法建立穩(wěn)定過表達LRP16基因的MIN6細胞株;3.用MTT法檢測細胞的增殖情況;4.分別用0 mmol/L、3 mmol/L和30 mmol/L葡萄糖刺激細胞,檢測胰島素分泌功能;5.用Western blot方法檢測葡萄糖轉(zhuǎn)運子-2(Glut-2)的蛋白表達情況。結(jié)果:1.MIN6細胞表達LRP16蛋白;2.過表達LRP16組的細胞增殖情況與對照組相比無顯著差別(p＞0.05);3.在0 mmol/L、3 mmol/L和30 mmol/L葡萄糖刺激下,過表達LRP16組的胰島素分泌量分別為對照組的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顯示過表達LRP16組的Glut-2蛋白量是對照組的1.76倍(p＜0.05)。結(jié)論:小鼠的胰島β細胞中表達LRP16蛋白;過表達LRP16基因不能促進MIN6細胞增殖,但是可以促進葡萄糖刺激的胰島素分泌(GSIS),該作用可能依賴于Glut-2的上調(diào),且不依賴葡萄糖濃度。 二、LRP16基因?qū)IN6細胞胰島素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)定過表達LRP16基因的MIN6細胞株;2.用Real-Time PCR法檢測胰島素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Pdx-1、MafA和NeuroD1蛋白的表達情況。結(jié)果:1.Real-Time PCR顯示過表達LRP16組的InsulinⅠmRNA和InsulinⅡmRNA的量分別是對照組的1.6倍和1.8倍(p＜0.05);2.過表達LRP16組的Pdx-1 mRNA的量是對照組的1.62倍(p＜0.05),而MafA和NeuroD1 mRNAs的量與對照組相比沒有顯著差別(p＞0.05);3.過表達LRP16組的Pdx-1蛋白量是對照組的2.04倍(p＜0.05),而MafA和NeuroD1的蛋白量與對照組相比沒有顯著差別(p＞0.05)。結(jié)論:過表達LRP16基因能夠促進胰島素mRNAs的合成,該作用可能是通過上調(diào)轉(zhuǎn)錄因子Pdx-1實現(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.

【學位授予單位】：中國人民解放軍軍醫(yī)進修學院
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R587.1;R346

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本文編號：1538194