本文選題：β細胞 + α細胞��； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：[背景]糖尿病患者數逐年增加,已經成為危害公共健康的重大難題,其發(fā)病的中心環(huán)節(jié)是胰島β細胞數目減少和功能受損。目前糖尿病的主要治療方式為藥物治療和胰腺移植。由于環(huán)境等因素的影響,移植后的外源性胰腺并不能很好地發(fā)揮調節(jié)血糖的作用,因此胰島β細胞的內源性增殖成為人們關注的焦點。目前有研究發(fā)現胰腺外分泌細胞(胰腺導管細胞和胰腺腺泡細胞)以及胰腺外細胞(肝臟細胞)可以轉化成為胰島β細胞。在一些β細胞極端受損的糖尿病動物模型中,研究者觀察到α細胞轉化成為β細胞的現象,為糖尿病的治療帶來了新的思路。既往研究認為GLP-1有促進β細胞增生并抑制其凋亡的作用,但其具體機制尚不明確。有研究表明GLP-1作用于細胞表面的GLP-1受體,通過PI3K/Akt/FOX01信號通路增強PDX-1的反應性,PDX-1在維持β細胞形態(tài)功能中起到重要作用,并能促進非β細胞向β細胞轉化。由此我們推測GLP-1通過細胞表面GLP-1受體激活細胞內PI3K/Akt/FOXO1信號通路,調節(jié)PDX-1、MafA、MafB等轉錄因子的表達,繼而促進α細胞跨譜系轉化為β細胞。[目的]本課題擬建立單次大劑量STZ誘導重度β細胞損傷的糖尿病大鼠模型,通過觀察GLP-1干預后大鼠胰島中內分泌細胞數目的變化,探討GLP-1能否促進大鼠胰島β細胞原位再生及其可能的機制。應用GLP-1受體抑制劑和PI3K酶抑制劑對糖尿病大鼠干預后,檢測PI3K/Akt/FOXO1信號通路相關信號因子轉錄水平和蛋白表達水平,探討GLP-1能否通過PI3K/Akt/FOX01信號通路促進α細胞轉化成為β細胞。[內容]內容分為以下三大部分:第一部分GLP-1促進STZ誘導的糖尿病大鼠胰島β細胞原位再生目的探討GLP-1能否促進胰島β細胞的原位再生。方法單次大劑量注射STZ建立糖尿病大鼠模型,將空腹血糖值大于28mmol/L的SD大鼠納入研究,并將其分為五組:正常對照組、糖尿病模型組、GLP-150μg/kg干預組、GLP-1100μg/kg干預組、GLP-12000μg/kg干預組。每周監(jiān)測各組大鼠血糖、體重變化;ELISA法檢測C肽、胰島素水平;大鼠胰腺切片insulin/gucagon雙重免疫染色觀察內分泌細胞數目變化。統(tǒng)計學方法為one-way ANOVA,P0.05差異具有統(tǒng)計學意義。結果與糖尿病模型組相比,GLP-1干預后糖尿病大鼠的血糖水平減低、體重下降;C肽、胰島素水平上升;胰島素分泌細胞數目呈GLP-1劑量依賴型增加,差異均具有統(tǒng)計學意義(P0.05)。結論GLP-1能夠促進β細胞原位再生。第二部分GLP-1通過驅動胰島內α細胞轉分化為β細胞促進胰島的原位再生及功能重建目的探討GLP-1促進β細胞原位再生的可能機制。方法分組同第一部分,對大鼠胰腺冰凍切片行insulin/glucagon、insulin/amylase、insulin/Pan-CK雙重免疫熒光染色,對大鼠胰腺石蠟切片行PCNA免疫組化染色。統(tǒng)計學方法為one-way ANOVA,P0.05差異具有統(tǒng)計學意義。結果GLP-1干預后,糖尿病大鼠胰島中出現insulin/glucagon雙陽性細胞,數目隨GLP-1干預劑量增加而增加,差異具有統(tǒng)計學意義(P0.05)。大鼠胰腺切片中未觀察到insulin/amylase、insulin/pan-ck雙陽性細胞;PCNA免疫組化結果無明顯差異(P0.05),且染色區(qū)域均位于α細胞分布區(qū)域。結論GLP-1促進β細胞的原位再生來自于α細胞的轉化,無胰腺腺泡細胞或導管細胞的轉化,也無α細胞的自我復制。第三部分GLP-1及其受體經PI3K/Akt/FOXO1通路上調PDX-1表達促進α細胞轉分化為β細胞目的探討GLP-1通過其受體經PI3K/Akt/FOXO1通路增強轉錄因子PDX-1、MafB的表達,下調MafA的表達,繼而促進α細胞轉分化為β細胞。方法建立糖尿病大鼠模型后,將SD大鼠分為7組:正常對照組、糖尿病模型組、GLP-1 50μμg/kg 干預組、GLP-1 100μg/kg 干預組、GLP-1 200μg/kg 干預組、GLP-1與GLP-1受體拮抗劑exendin(9-39)共同干預組、GLP-1與PI3K酶抑制劑 LY294002 共同干預組。RT-PCR 檢測 GLP-1R、AKT、PDX-1、MafA、MafB、Foxol的mRNA表達結果;Western blot檢測大鼠胰島中GLP-1R、PDX-1、MafA、MafB、Akt的蛋白表達結果。結果PCR結果顯示:糖尿病模型組的GLP-1R、Akt、PDX-1、FOXO1、MafA、MafB的表達較正常組明顯下降。GLP-1干預后,GLP-1R、Akt、PDX-1、FOXOl MafA的mRNA表達量明顯上升,MafBmRNA表達量下降,呈劑量依賴型。exendin(9-39)抑制 GLP-1 受體后,GLP-1R、Akt、PDX-1、Foxol、MafA 的mRNA表達量較GLP-1 100μg/kg干預組下降,而MafB表達量上升。LY294002抑制 PI3K 酶后,GLP-1R、Akt、PDX-1、Foxol、MafA 的 mRNA 表達量較GLP-1100μg/kg干預下降,而MafB表達量上升。Western blot結果與PCR結果相似,差異均具有統(tǒng)計學意義(P0.05)。結論GLP-1通過GLP-1受體及其下游的PI3K/Akt/FOXO1通路增強轉錄因子PDX-1、MafA的表達,下調MafB的表達,進而促進α細胞向β細胞轉化。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.1

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本文編號：1955032