TXNIP在衰老导致的胰岛β细胞功能障碍中的作用及其机制研究

发布时间：2018-03-09 06:19

本文选题：衰老　切入点：胰岛功能障碍　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：第一部分高脂饲养促进衰老导致的胰岛功能障碍及TXNIP表达目的:探讨衰老在胰腺功能障碍中的作用及衰老与TXNIP表达的关系。方法:收集不同年龄段(青年、中年、老年)正常人体胰腺组织标本,用免疫组化方法检测TXNIP蛋白表达水平。48只4周龄C57BL/6J小鼠随机均分为普食组和高脂组,两组再按照4个时间段(3个月、6个月、9个月、12个月)饲养至所需月龄。观察小鼠一般生长情况,检测各年龄段各组小鼠GTT、ITT及基础代谢指标,检测TXNIP、TRX及衰老相关蛋白p16的表达水平。结果:随着年龄的增长,正常人体胰岛中TXNIP表达水平增加(p0.01)。随着年龄的增长,小鼠空腹血糖逐渐增高(p0.05),小鼠胰岛素分泌水平逐渐下降(p0.05),胰岛素抵抗逐渐增加(p0.05);与同月龄普食组小鼠相比,6、9、12月龄高脂组小鼠空腹血糖较高(p0.01);各月龄普食组小鼠胰岛素分泌功能均较好,胰岛素抵抗均较轻。随着年龄增加,高脂组小鼠血中TC、TG、LDL含量逐渐增加(p0.01)。随着年龄增加,胰腺组织中TXNIP、P16蛋白表达逐渐增加,TRX蛋白表达逐渐降低(均p0.05)。同年龄段小鼠胰腺组织,与普食组相比,9、12月龄高脂组的TXNIP表达水平明显升高(p0.05);3、6、12月龄高脂组的p16表达水平明显升高(p0.05),而TRX表达水平较低(p0.05)。结论:高脂饲养促进衰老导致的小鼠胰岛素分泌功能降低和胰岛素抵抗增加,促进小鼠代谢紊乱,同时促进衰老导致的胰腺组织TXNIP表达增加。第二部分TXNIP促进衰老导致的胰岛功能障碍目的:探讨TXNIP在衰老导致的胰岛功能障碍中的作用和机制方法:24只4周龄C57BL/6J小鼠(WT)和TXNIP基因敲出鼠(TXNIP-/-),按照4个时间段(3个月、6个月、9个月、12个月)普食饲养至所需月龄。观察小鼠一般生长情况,检测各年龄段各组小鼠GTT、ITT及基础代谢指标,检测TXNIP、TRX及衰老相关蛋白p16的表达水平。结果:与同年龄段WT小鼠相比,TXNIP-/-组小鼠空腹血糖较低(p0.05),小鼠胰岛素分泌功能较好(p0.05),胰岛素抵抗较弱(p0.05)。与同年龄段WT小鼠相比,各月龄TXNIP-/-组的体重均较低((p0.05);6、9、12月龄TXNIP-/-组的TG较低(p0.05);9、12月龄LDL较低(p0.05)。同年龄段小鼠胰腺组织,与WT小鼠相比较,9、12月龄TXNIP-/-组的TRX表达水平明显较高(p0.01);6、9、12月龄TXNIP-/-组的P16表达水平明显较低(p0.05)。结论:TXNIP促进衰老导致的胰岛素分泌功能减弱和胰岛素抵抗,促进衰老导致的小鼠代谢紊乱和衰老相关蛋白p16的表达。第三部分TXNIP促进胰岛β细胞衰老的作用及相关机制目的:研究TXNIP和EZH2对MIN6细胞衰老的影响及其相关机制及其相互作用关系。方法:将处于对数生长期的MIN6细胞,进行EZH2、TXNIP过表达及TXNIP沉默慢病毒转染,用荧光显微镜和Westen-blot方法检测转染效能。将MIN6细胞分为空载组(LV-GFP)组、TXNIP过表达(LV-TXNIP)组和TXNIP沉默组(LV-TXNIPsi RNA)组。利用流式测定细胞周期;利用CCK-8评估细胞增值活力;采用Annexin V/PI双染色法检测细胞凋亡;用DHE法测定ROS表达;用Westen-blot法检测TXNIP、TRX、p16、活化的Caspase蛋白的表达水平及p38 MAPK通路激活水平。结果:感染慢病毒72小时后,病毒转染成功。与LV-GFP组相比较,LV-GFP-TXNIP组的增殖出现G1-S期阻滞,细胞生存率明显降低,而其凋亡率和ROS水平明显增加,p16、活化的Caspase3蛋白表达及p38 MAPK磷酸化水平明显增高(均P0.01)。LV-GFP-TXNIPsi RNA组和LV-EZH2组的细胞生存率明显升高,而其凋亡率和ROS水平明显降低(均P0.01),P16、活化的Caspase3蛋白表达及p38 MAPK磷酸化水平明显降低(均P0.05)。LV-EZH2组的p16、TXNIP蛋白表达明显增高(均P0.01),且TRX蛋白表达明显降低(P0.01)。其次,p38MAPK特异性抑制剂作用后,LV-GFP-TXNIP组的p16及活化的Caspase3蛋白表达均显著减少(均P0.01)。结论:TXNIP表达上调能够抑制MIN6细胞增殖,促进细胞的衰老和凋亡,且p38 MAPK信号通路可能参与其中,同时,过表达EZH2能增加细胞生存率,减少细胞的凋亡和过氧化物的产生,其可能机制为EZH2可通过抑制TXNIP的表达,从而抑制p16的表达,延缓细胞衰老。
[Abstract]:The first part of high fat feeding promoting islet dysfunction and aging caused by TXNIP expression objective: To explore the relationship between expression of senescence in pancreatic dysfunction and its role in aging and TXNIP. Methods: We collected in different age groups (young, middle-aged, elderly) of normal human pancreatic tissue specimens by immunohistochemical method to detect the TXNIP protein expression level of.48 is only 4 week old C57BL/6J mice were randomly divided into normal diet group and high-fat group, the two groups in 4 time periods (3 months, 6 months, 9 months, 12 months) raised to the required month old mice were observed. The general growth situation, the detection of all ages of mice GTT, detection of TXNIP ITT and the basic metabolism, the expression level of TRX and senescence associated protein p16. Results: with the increase of age, increase the level of TXNIP expression in normal human pancreatic islets (P0.01). With the increase of age, fasting blood glucose of mice increased gradually (P0.05), insulin secretion in mice Decreased (P0.05), insulin resistance (P0.05) gradually increased; compared with the same age normal diet group, high fat group were higher fasting blood glucose levels 6,9,12 months (P0.01 months); the normal diet group were insulin secretion and insulin resistance are good, are lighter. With the increase of age, high fat mice serum TC, TG, LDL content increased gradually (P0.01). With the increase of age, TXNIP in pancreatic tissues, P16 protein expression increased, TRX protein expression decreased gradually (P0.05). With the age of pancreatic tissue of mice, compared with normal diet group, high fat group 9,12 the expression level of TXNIP months increased significantly (P0.05); 3,6,12 high fat group p16 months old expression level increased significantly (P0.05), and the expression level of TRX is lower (P0.05). Conclusion: high fat diet to promote insulin secretion in mice Aging leads to decreased and increased insulin resistance, promote mouse metabolism, and promote decline The old in pancreatic tissue increased expression of TXNIP. The second part of the TXNIP to promote aging caused by islet dysfunction Objective: To explore the effect and mechanism of TXNIP in aging caused by islet dysfunction in 24 C57BL/6J mice aged 4 weeks (WT) and TXNIP gene knockout mice (TXNIP-/-), according to the 4 time periods (3 months. 6 months, 9 months, 12 months) of general feeding to the required months. Observe the growth of mice, the detection of all ages of mice GTT, detection of TXNIP ITT and basic metabolism, and the expression level of TRX and senescence associated protein p16. Results: compared with the same age WT mice. TXNIP-/- group were lower fasting blood glucose (P0.05), mouse insulin secretion function better (P0.05), insulin resistance is weak (P0.05). Compared with the same age WT mice at different ages, the weight of group TXNIP-/- was lower ((P0.05); 6,9,12 month old TXNIP-/- group TG was lower (P0.05); 9,12 months of age Low LDL (P0.05). With pancreatic tissue age mice compared with WT mice, 9,12 month old TXNIP-/- group TRX expression levels were significantly higher (P0.01); the expression level of 6,9,12 month old TXNIP-/- group P16 was significantly lower (P0.05). Conclusion: TXNIP promotes senescence induced insulin secretion and decreased insulin resistance, promote the expression of senescence caused by metabolic disorders and senescence associated protein p16. The third part of TXNIP to promote the aging of islet beta cell function and related mechanisms: To study the effect of TXNIP and EZH2 on MIN6 cell senescence and its mechanism and their interaction relationship. Methods: MIN6 cells in the exponential growth phase, EZH2 over expression of TXNIP and TXNIP, silent lentivirus transfection, fluorescence microscope and Westen-blot method to detect the transfection efficiency. MIN6 cells were divided into no-load group (LV-GFP) group, over expression of TXNIP (LV-TXNIP) group and TXNIP group (LV- silencing TXNIPsi RNA) group. The cell cycle was determined by flow cytometry; using CCK-8 to assess cell proliferation activity; using Annexin V/PI double staining method to detect cell apoptosis; the expression of ROS was determined by DHE method; Westen-blot method was used to detect TXNIP, TRX, p16, activated the expression level of Caspase protein and p38 MAPK activation level. Results: slow infection virus 72 hours after transfection. Compared with LV-GFP group, LV-GFP-TXNIP group, the proliferation of G1-S phase arrest, cell survival rate decreased significantly, and the apoptosis rate and ROS level increased significantly, the level of p16, Caspase3 protein expression and p38 MAPK phosphorylation increased significantly (P0.01) and.LV-GFP-TXNIPsi RNA group group LV-EZH2 the cell survival rate was significantly increased, and the apoptosis rate and ROS levels were significantly lower (P0.01), P16, Caspase3 protein expression and p38 phosphorylation level of MAPK activation was significantly lower in group.LV-EZH2 (P0.05) p16, TXNIP Protein expression increased significantly (P0.01), and the expression of TRX protein was significantly decreased (P0.01). Secondly, a specific inhibitor of p38MAPK function, the p16 group the expression of LV-GFP-TXNIP and activation of Caspase3 protein were significantly decreased (P0.01). Conclusion: the upregulation of TXNIP expression could inhibit the proliferation of MIN6 cells, promote cell senescence and apoptosis. P38 and MAPK signaling pathway may be involved, at the same time, over expression of EZH2 could increase the cell survival rate, apoptosis and cell superoxide production, the possible mechanism is that EZH2 can inhibit the expression of TXNIP, and inhibited p16 protein expression, cell senescence.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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