GDF11对2型糖尿病小鼠胰岛β细胞的保护作用及机制研究
发布时间：2018-11-13 14:51
【摘要】：研究背景2型糖尿病是一种年龄相关性疾病,随着病程进展,胰岛β细胞功能及含量逐渐衰减。研究发现生长分化因子]1(GDF11)可以改善多种器官衰老表型及正向调控胰岛β细胞的分化成熟。但是,尚无关于GDF11对成年胰岛β细胞作用的文献报道。研究目的本研究旨在明确GDF11对糖尿病小鼠胰岛β细胞功能及含量的影响及可能机制。方法购买6周龄雄性SPF级C57BL/6小鼠,用高脂饮食联合链脲佐菌素(STZ)建立诱发性2型糖尿病小鼠模型(即HFD/STZ小鼠)。购买6周龄雄性SPF级db/db小鼠作为自发性2型糖尿病模型。本研究选择了诱发性和自发性2型糖尿病动物模型作为研究对象,通过外源性补充或中和循环中的GDF11水平,观察GDF11对小鼠胰岛β细胞的作用及机制。每周定期测定各组小鼠的摄食量、体重及空腹血糖。干预完成后,各组小鼠行腹腔葡萄糖耐量实验(IPGTT),并测定基线及糖负荷30min的血浆胰岛素水平。实验结束时,ELISA检测血清生理生化水平;ELISA检测血清及胰腺内胰岛素、胰高血糖素水平;采用RT-PCR分析胰岛PDX-1、MafA、NKX6.1、Insulin2基因转录水平。取胰腺组织行HE染色及免疫荧光定量分析胰岛形态及细胞含量变化。Western blot检测胰岛相关蛋白表达水平。结果研究结果显示,增加循环内GDF11水平对正常小鼠糖脂代谢无明显影响。但是,补充GDF11可显著降低两种糖尿病模型小鼠的空腹血糖、HbAlc及血脂水平,降低胰高血糖素浓度,改善糖耐量,同时明显增加空腹及糖负荷30min后的胰岛素水平,上调胰岛PDX-1、MafA、NKX6.1、Insulin2基因表达。另外,rGDFl1和AAV-GDF11干预后可维持β细胞含量,降低β细胞凋亡率,但是对α细胞含量、β细胞大小及增殖率无明显影响。而且,补充GDF11可以增加胰腺内胰岛素含量,但对胰高血糖素含量无明显作用。另外,中和循环中GDF11对正常小鼠无明显作用,但可进一步加重糖尿病小鼠高血糖,损害糖耐量,抑制胰岛素分泌功能及升高β细胞凋亡率。机制方面,补充GDF11可显著增加P-Smad2表达水平,但对P-Smad3水平无明显作用。同时,补充GDF11可升高P-AKT及P-Fox01表达水平,但对P-S6K1水平无明显影响。结论综上所述,本研究首次证实GDF11可能通过激活TGF-β/Smad2及PI3K-AKT-FoxOl信号通路改善糖尿病小鼠糖脂代谢,抑制胰高血糖素的释放,保护胰岛β细胞功能,减少β细胞凋亡。然而,使用抗体中和血液中GDF11可诱导相反作用。GDF11可能在胰岛β细胞功能及含量中扮演重要角色,对探讨糖尿病的防治方法提供了新的思路。
[Abstract]:Background Type 2 diabetes mellitus (T2DM) is an age-related disease. With the progression of the disease, the function and content of islet 尾 cells decrease gradually. It was found that growth differentiation factor] 1 (GDF11) could improve the senescence phenotype of many organs and positively regulate the differentiation and maturation of islet 尾 cells. However, there is no literature about the effect of GDF11 on adult islet 尾-cells. Objective to investigate the effect of GDF11 on the function and content of islet 尾 cells in diabetic mice and its possible mechanism. Methods six week-old male SPF grade C57BL/6 mice were purchased and induced type 2 diabetic mice (HFD/STZ mice) were established by high fat diet combined with streptozotocin (STZ). Six week old male SPF db/db mice were purchased as a model of spontaneous type 2 diabetes mellitus. In this study, the animal models of induced and spontaneous type 2 diabetes mellitus were selected to observe the effect and mechanism of GDF11 on islet 尾 cells in mice by exogenous supplementation or neutralization of GDF11 levels in circulation. The food intake, body weight and fasting blood glucose of each group were measured regularly every week. After the intervention, the plasma insulin levels of baseline and glucose loaded 30min were measured by intraperitoneal glucose tolerance test (IPGTT),) in each group. At the end of the experiment, serum physiological and biochemical levels were detected by ELISA, insulin and glucagon levels in serum and pancreas were detected by ELISA, and PDX-1,MafA,NKX6.1,Insulin2 gene transcription level of islet was analyzed by RT-PCR. The changes of pancreatic morphology and cell content in pancreatic tissue were determined by HE staining and immunofluorescence. The expression of islet associated protein was detected by. Western blot. Results the results showed that increasing the level of GDF11 in circulation had no significant effect on the metabolism of glucose and lipid in normal mice. However, supplementation of GDF11 could significantly decrease the levels of fasting blood glucose, HbAlc and blood lipid, decrease the concentration of glucagon, improve glucose tolerance, and increase the insulin level after 30min. Upregulation of islet PDX-1,MafA,NKX6.1,Insulin2 gene expression. In addition, rGDFl1 and AAV-GDF11 could maintain 尾 cell content and decrease 尾 cell apoptosis rate, but had no significant effect on 伪 cell content, 尾 cell size and proliferation rate. In addition, supplementation of GDF11 could increase insulin content in pancreas, but had no effect on glucagon content. In addition, GDF11 had no obvious effect on normal mice in neutralization cycle, but it could further aggravate hyperglycemia, damage glucose tolerance, inhibit insulin secretion and increase the rate of 尾 cell apoptosis in diabetic mice. In terms of mechanism, supplementation of GDF11 could significantly increase the level of P-Smad2 expression, but had no effect on P-Smad3 level. At the same time, supplementation of GDF11 could increase the expression of P-AKT and P-Fox01, but had no significant effect on the level of P-S6K1. Conclusion in conclusion, GDF11 may improve glucose and lipid metabolism, inhibit glucagon release, protect islet 尾 cell function and decrease 尾 cell apoptosis by activating TGF- 尾 / Smad2 and PI3K-AKT-FoxOl signaling pathway in diabetic mice for the first time. GDF11 may play an important role in the function and content of islet 尾 cells, which provides a new idea for the prevention and treatment of diabetes mellitus.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
[Abstract]:Background Type 2 diabetes mellitus (T2DM) is an age-related disease. With the progression of the disease, the function and content of islet 尾 cells decrease gradually. It was found that growth differentiation factor] 1 (GDF11) could improve the senescence phenotype of many organs and positively regulate the differentiation and maturation of islet 尾 cells. However, there is no literature about the effect of GDF11 on adult islet 尾-cells. Objective to investigate the effect of GDF11 on the function and content of islet 尾 cells in diabetic mice and its possible mechanism. Methods six week-old male SPF grade C57BL/6 mice were purchased and induced type 2 diabetic mice (HFD/STZ mice) were established by high fat diet combined with streptozotocin (STZ). Six week old male SPF db/db mice were purchased as a model of spontaneous type 2 diabetes mellitus. In this study, the animal models of induced and spontaneous type 2 diabetes mellitus were selected to observe the effect and mechanism of GDF11 on islet 尾 cells in mice by exogenous supplementation or neutralization of GDF11 levels in circulation. The food intake, body weight and fasting blood glucose of each group were measured regularly every week. After the intervention, the plasma insulin levels of baseline and glucose loaded 30min were measured by intraperitoneal glucose tolerance test (IPGTT),) in each group. At the end of the experiment, serum physiological and biochemical levels were detected by ELISA, insulin and glucagon levels in serum and pancreas were detected by ELISA, and PDX-1,MafA,NKX6.1,Insulin2 gene transcription level of islet was analyzed by RT-PCR. The changes of pancreatic morphology and cell content in pancreatic tissue were determined by HE staining and immunofluorescence. The expression of islet associated protein was detected by. Western blot. Results the results showed that increasing the level of GDF11 in circulation had no significant effect on the metabolism of glucose and lipid in normal mice. However, supplementation of GDF11 could significantly decrease the levels of fasting blood glucose, HbAlc and blood lipid, decrease the concentration of glucagon, improve glucose tolerance, and increase the insulin level after 30min. Upregulation of islet PDX-1,MafA,NKX6.1,Insulin2 gene expression. In addition, rGDFl1 and AAV-GDF11 could maintain 尾 cell content and decrease 尾 cell apoptosis rate, but had no significant effect on 伪 cell content, 尾 cell size and proliferation rate. In addition, supplementation of GDF11 could increase insulin content in pancreas, but had no effect on glucagon content. In addition, GDF11 had no obvious effect on normal mice in neutralization cycle, but it could further aggravate hyperglycemia, damage glucose tolerance, inhibit insulin secretion and increase the rate of 尾 cell apoptosis in diabetic mice. In terms of mechanism, supplementation of GDF11 could significantly increase the level of P-Smad2 expression, but had no effect on P-Smad3 level. At the same time, supplementation of GDF11 could increase the expression of P-AKT and P-Fox01, but had no significant effect on the level of P-S6K1. Conclusion in conclusion, GDF11 may improve glucose and lipid metabolism, inhibit glucagon release, protect islet 尾 cell function and decrease 尾 cell apoptosis by activating TGF- 尾 / Smad2 and PI3K-AKT-FoxOl signaling pathway in diabetic mice for the first time. GDF11 may play an important role in the function and content of islet 尾 cells, which provides a new idea for the prevention and treatment of diabetes mellitus.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
【参考文献】
相关期刊论文 前3条
1 李倩;郝志明;;生长分化因子11:TGF-β超家族的新成员[J];生物化学与生物物理进展;2015年07期
2 闫柄文;刘毅;裴海峰;王Y，
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