β细胞内源性增殖新途径：α细胞转化为β细胞的机制及GLP-1的调控作用

发布时间：2018-05-30 10:50

本文选题：β细胞 + α细胞　；参考：《南方医科大学》2017年硕士论文

【摘要】：[背景]糖尿病患者数逐年增加,已经成为危害公共健康的重大难题,其发病的中心环节是胰岛β细胞数目减少和功能受损。目前糖尿病的主要治疗方式为药物治疗和胰腺移植。由于环境等因素的影响,移植后的外源性胰腺并不能很好地发挥调节血糖的作用,因此胰岛β细胞的内源性增殖成为人们关注的焦点。目前有研究发现胰腺外分泌细胞(胰腺导管细胞和胰腺腺泡细胞)以及胰腺外细胞(肝脏细胞)可以转化成为胰岛β细胞。在一些β细胞极端受损的糖尿病动物模型中,研究者观察到α细胞转化成为β细胞的现象,为糖尿病的治疗带来了新的思路。既往研究认为GLP-1有促进β细胞增生并抑制其凋亡的作用,但其具体机制尚不明确。有研究表明GLP-1作用于细胞表面的GLP-1受体,通过PI3K/Akt/FOX01信号通路增强PDX-1的反应性,PDX-1在维持β细胞形态功能中起到重要作用,并能促进非β细胞向β细胞转化。由此我们推测GLP-1通过细胞表面GLP-1受体激活细胞内PI3K/Akt/FOXO1信号通路,调节PDX-1、MafA、MafB等转录因子的表达,继而促进α细胞跨谱系转化为β细胞。[目的]本课题拟建立单次大剂量STZ诱导重度β细胞损伤的糖尿病大鼠模型,通过观察GLP-1干预后大鼠胰岛中内分泌细胞数目的变化,探讨GLP-1能否促进大鼠胰岛β细胞原位再生及其可能的机制。应用GLP-1受体抑制剂和PI3K酶抑制剂对糖尿病大鼠干预后,检测PI3K/Akt/FOXO1信号通路相关信号因子转录水平和蛋白表达水平,探讨GLP-1能否通过PI3K/Akt/FOX01信号通路促进α细胞转化成为β细胞。[内容]内容分为以下三大部分:第一部分GLP-1促进STZ诱导的糖尿病大鼠胰岛β细胞原位再生目的探讨GLP-1能否促进胰岛β细胞的原位再生。方法单次大剂量注射STZ建立糖尿病大鼠模型,将空腹血糖值大于28mmol/L的SD大鼠纳入研究,并将其分为五组:正常对照组、糖尿病模型组、GLP-150μg/kg干预组、GLP-1100μg/kg干预组、GLP-12000μg/kg干预组。每周监测各组大鼠血糖、体重变化;ELISA法检测C肽、胰岛素水平;大鼠胰腺切片insulin/gucagon双重免疫染色观察内分泌细胞数目变化。统计学方法为one-way ANOVA,P0.05差异具有统计学意义。结果与糖尿病模型组相比,GLP-1干预后糖尿病大鼠的血糖水平减低、体重下降;C肽、胰岛素水平上升;胰岛素分泌细胞数目呈GLP-1剂量依赖型增加,差异均具有统计学意义(P0.05)。结论GLP-1能够促进β细胞原位再生。第二部分GLP-1通过驱动胰岛内α细胞转分化为β细胞促进胰岛的原位再生及功能重建目的探讨GLP-1促进β细胞原位再生的可能机制。方法分组同第一部分,对大鼠胰腺冰冻切片行insulin/glucagon、insulin/amylase、insulin/Pan-CK双重免疫荧光染色,对大鼠胰腺石蜡切片行PCNA免疫组化染色。统计学方法为one-way ANOVA,P0.05差异具有统计学意义。结果GLP-1干预后,糖尿病大鼠胰岛中出现insulin/glucagon双阳性细胞,数目随GLP-1干预剂量增加而增加,差异具有统计学意义(P0.05)。大鼠胰腺切片中未观察到insulin/amylase、insulin/pan-ck双阳性细胞;PCNA免疫组化结果无明显差异(P0.05),且染色区域均位于α细胞分布区域。结论GLP-1促进β细胞的原位再生来自于α细胞的转化,无胰腺腺泡细胞或导管细胞的转化,也无α细胞的自我复制。第三部分GLP-1及其受体经PI3K/Akt/FOXO1通路上调PDX-1表达促进α细胞转分化为β细胞目的探讨GLP-1通过其受体经PI3K/Akt/FOXO1通路增强转录因子PDX-1、MafB的表达,下调MafA的表达,继而促进α细胞转分化为β细胞。方法建立糖尿病大鼠模型后,将SD大鼠分为7组:正常对照组、糖尿病模型组、GLP-1 50μμg/kg 干预组、GLP-1 100μg/kg 干预组、GLP-1 200μg/kg 干预组、GLP-1与GLP-1受体拮抗剂exendin(9-39)共同干预组、GLP-1与PI3K酶抑制剂 LY294002 共同干预组。RT-PCR 检测 GLP-1R、AKT、PDX-1、MafA、MafB、Foxol的mRNA表达结果;Western blot检测大鼠胰岛中GLP-1R、PDX-1、MafA、MafB、Akt的蛋白表达结果。结果PCR结果显示:糖尿病模型组的GLP-1R、Akt、PDX-1、FOXO1、MafA、MafB的表达较正常组明显下降。GLP-1干预后,GLP-1R、Akt、PDX-1、FOXOl MafA的mRNA表达量明显上升,MafBmRNA表达量下降,呈剂量依赖型。exendin(9-39)抑制 GLP-1 受体后,GLP-1R、Akt、PDX-1、Foxol、MafA 的mRNA表达量较GLP-1 100μg/kg干预组下降,而MafB表达量上升。LY294002抑制 PI3K 酶后,GLP-1R、Akt、PDX-1、Foxol、MafA 的 mRNA 表达量较GLP-1100μg/kg干预下降,而MafB表达量上升。Western blot结果与PCR结果相似,差异均具有统计学意义(P0.05)。结论GLP-1通过GLP-1受体及其下游的PI3K/Akt/FOXO1通路增强转录因子PDX-1、MafA的表达,下调MafB的表达,进而促进α细胞向β细胞转化。
[Abstract]:[background] the number of diabetic patients has increased year by year. It has become a major problem endangering public health. The central link of the disease is the decrease of the number of beta cells and impaired function. At present, the main treatment for diabetes is drug treatment and pancreas transplantation. The exogenous pancreas after transplantation is not very good because of the influence of environment and other factors. The endogenous proliferation of islet beta cells has become the focus of attention. Recent studies have shown that pancreatic exocrine cells (pancreatic duct cells and pancreatic acinar cells) and pancreatic cells (liver cells) can be transformed into islet beta cells. In some diabetic animal models with extreme damage to beta cells The researchers have observed that the transformation of alpha cells into beta cells brings new ideas for the treatment of diabetes. Previous studies suggest that GLP-1 has the effect of promoting beta cell proliferation and inhibiting its apoptosis, but its specific mechanism is not clear. Studies have shown that GLP-1 acts on the GLP-1 receptor on the cell surface and increases through the PI3K/Akt/FOX01 signaling pathway. The reaction of strong PDX-1, PDX-1 plays an important role in maintaining the morphologic function of beta cells, and can promote the transformation of non beta cells to beta cells. Therefore, we speculate that GLP-1 regulates the transcription of PDX-1, MafA, MafB and other transcriptional factors through the activation of the intracellular PI3K/Akt/FOXO1 signaling pathway on the surface of the cell surface GLP-1 receptor, and then promotes the transformation of the trans lineage of alpha cells into beta cells. [Objective] [Objective] to establish a diabetic rat model with severe beta cell injury induced by a single large dose of STZ. By observing the changes in the number of endocrine cells in the islets of rats after GLP-1 intervention, it is discussed whether GLP-1 can promote the in situ regeneration of islet beta cells in rats and the possible mechanism. GLP-1 receptor inhibitors and PI3K enzyme inhibitors are used. On the prognosis of diabetic rats, the transcriptional level of PI3K/Akt/FOXO1 signaling pathway and protein expression level were detected, and whether GLP-1 could promote the transformation of alpha cells into beta cells through PI3K/Akt/FOX01 signaling pathway was divided into the following three parts: the first part GLP-1 promoted the pancreatic islet beta cell in STZ induced diabetic rats Objective to investigate whether GLP-1 could promote the in situ regeneration of islet beta cells. Method a single large dose of STZ was used to establish a diabetic rat model, and the SD rats with fasting blood glucose greater than 28mmol/L were included in the study, and they were divided into five groups: normal control group, diabetic model group, GLP-150 g/kg intervention group, GLP-1100 u g/kg intervention group, GLP-12000 micron g/. Kg intervention group. The blood glucose and weight change were monitored every week. The ELISA method was used to detect the C peptide and insulin level. The number of endocrine cells was observed by insulin/gucagon double immunostaining in the pancreas section of the rat. The statistical method was one-way ANOVA, and the difference of P0.05 was statistically significant. Compared with the diabetes model group, the result of GLP-1 was the prognosis of diabetes. The blood glucose level of rats decreased, body weight decreased, C peptide, insulin level increased, the number of insulin secreting cells increased in a GLP-1 dose dependent type, and the difference was statistically significant (P0.05). Conclusion GLP-1 can promote in situ regeneration of beta cells. Second part GLP-1 promotes the islet's in situ redifferentiation through the transformation of alpha cells in the pancreatic island to beta cells. Objective to explore the possible mechanism of GLP-1 to promote the in situ regeneration of beta cells. Methods the first part was grouped with the first part of the rat pancreas frozen section with insulin/glucagon, insulin/amylase, insulin/Pan-CK double immunofluorescence staining. The PCNA immunohistochemical staining was performed on the paraffin section of the pancreas in rats. The statistical method was one-way ANOVA, P0.05. Results the difference was statistically significant. Results after GLP-1, insulin/glucagon double positive cells appeared in the islets of diabetic rats, the number increased with the increase of GLP-1 intervention dose, and the difference was statistically significant (P0.05). The insulin/amylase, insulin/pan-ck double positive cells were not observed in the pancreas slices of the rats, and the results of PCNA immunohistochemistry were not obvious. The difference (P0.05) and the staining area are located in the region of alpha cells. Conclusion GLP-1 promotes the in situ regeneration of beta cells from the transformation of alpha cells, no transformation of pancreatic acinar cells or ductal cells, and no self replication of alpha cells. The third part of GLP-1 and its receptors are up regulated by PDX-1 expression through the PI3K/Akt/ FOXO1 pathway to promote the transformation of alpha cells to beta cells. The purpose of the study was to explore the expression of PDX-1, MafB, and down regulation of MafA expression through its receptor through PI3K/Akt/FOXO1 pathway, and then to promote the conversion of MafA to beta cells. After establishing the diabetic rat model, the SD rats were divided into 7 groups: normal control group, diabetic model group, GLP-1 50 mu g/kg intervention group, GLP-1 100 u g/kg. Intervention group, GLP-1 200 g/kg intervention group, GLP-1 and GLP-1 receptor antagonist exendin (9-39) Co intervention group, GLP-1 and PI3K enzyme inhibitor LY294002 common intervention group.RT-PCR detection GLP-1R, AKT, PDX-1, concerned protein expression results. The results showed that the expression of GLP-1R, Akt, PDX-1, FOXO1, MafA, MafB in the diabetic model group was significantly lower than that in the normal group, and the expression of GLP-1R, Akt, PDX-1, FOXOl MafA was obviously increased, and the expression decreased. P-1 100 mu g/kg intervention group decreased, while MafB expression increased.LY294002 inhibition of PI3K enzyme, GLP-1R, Akt, PDX-1, Foxol, MafA mRNA expression was lower than that of GLP-1100. The K/Akt/FOXO1 pathway enhances the expression of transcription factor PDX-1 and MafA, down regulating the expression of MafB, thereby promoting the transformation of alpha cells into beta cells.
【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

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