【摘要】：第一部分：脂毒性誘導(dǎo)β細(xì)胞去分化 目的：在離體模型中觀察脂毒性對(duì)小鼠胰島功能狀態(tài)的影響和在體模型中探討高脂飲食對(duì)胰島p細(xì)胞功能及其分化狀態(tài)的影響。 方法：分離C57BL/6J小鼠胰島,加棕櫚酸培養(yǎng)24h、48h、72h, MTT檢測(cè)細(xì)胞活性,ELISA測(cè)細(xì)胞凋亡,定量PCR和免疫印記分別測(cè)定PDX1mRNA(?)口蛋白表達(dá)情況；高脂飲食喂養(yǎng)C57BL/6J小鼠8周,4周和8周時(shí)行腹腔葡萄糖耐量試驗(yàn)(IPGTT)與胰島素釋放試驗(yàn)(IPITT)觀察糖代謝情況以及ELISA法檢測(cè)血漿胰島素水平,共聚焦結(jié)合免疫熒光觀察胰島結(jié)構(gòu)及各種細(xì)胞標(biāo)記物(p細(xì)胞：FoxO1、胰島素和PDX1; α細(xì)胞：胰高血糖素；干細(xì)胞：OCT4、 Nanog)的表達(dá),并檢測(cè)Ki67和Caspase-3分別觀察胰島細(xì)胞增殖和凋亡。 結(jié)果：0.5mmol/L的棕櫚酸培養(yǎng)24小時(shí),48小時(shí)或72小時(shí)均降低胰島細(xì)胞活力和增加細(xì)胞凋亡,并呈現(xiàn)時(shí)間依賴性(p0.05)。48小時(shí)的棕櫚酸暴露顯著降低胰島細(xì)胞PDX1mRNA和蛋白水平(p0.05),但對(duì)胰島素mRNA表達(dá)無影響(p0.05)。高脂飲食增加胰島素的分泌(p0.01)；計(jì)算曲線下面積(AUC)顯示高脂飲食組中AUCIPGTT和AUCIPITT增加,8周相對(duì)于4周時(shí)更明顯(p0.05)；高脂飲食喂養(yǎng)增加HOMA-IR指數(shù)(p0.05)。高脂飲食降低β細(xì)胞表達(dá)FoxO1、胰島素和PDX1,增加干細(xì)胞標(biāo)記物OCT4和Nanog的表達(dá)。高脂飲食促進(jìn)胰島細(xì)胞Caspase-3表達(dá)；高脂飲食增加胰島細(xì)胞表達(dá)Ki67,但所示增殖的胰島細(xì)胞總體數(shù)量仍極少。 結(jié)論：1.離體和在體模型中脂毒性誘導(dǎo)胰島功能損傷和胰島素抵抗,呈現(xiàn)時(shí)間依賴性； 2.高脂飲食促進(jìn)胰島細(xì)胞增殖,誘導(dǎo)細(xì)胞凋亡； 3.高脂飲食損傷β細(xì)胞,促使胰島細(xì)胞去分化成為骨髓干細(xì)胞。 第二部分：前α細(xì)胞形成與胰島內(nèi)源性GLP-1系統(tǒng)的激活及機(jī)制 目的：在離體和在體脂毒性模型,通過觀察胰島內(nèi)GLP-1和GLP-1形成關(guān)鍵酶PC1/3表達(dá)變化,探討胰島內(nèi)GLP-1系統(tǒng)激活水平及氧化應(yīng)激在其中扮演的作用。 方法：分離正常C57BL/6J小鼠胰島,棕櫚酸培養(yǎng)胰島24-72h,ELISA法觀察培養(yǎng)液和細(xì)胞裂解液中GLP-1水平,定量PCR和免疫印記觀察PC1/3mRNA和蛋白水平變化情況,藉此獲得最佳的胰島內(nèi)GLP-1系統(tǒng)激活的刺激條件。高脂飲食喂養(yǎng)C57BL/6J小鼠8周,ELISA法測(cè)空腹?fàn)顟B(tài)下血清中GLP-1濃度,Q-PCR胰腺組織PC1/3mRNA表達(dá),免疫熒光觀察前a細(xì)胞形成情況。并采用DCFH-DA法檢測(cè)棕櫚酸誘導(dǎo)生成活性氧(ROS)水平,添加抗氧化劑N-乙酰半胱氨酸(NAC)進(jìn)一步觀察細(xì)胞裂解液中PCl/3mRNA和GLP-1水平變化。 結(jié)果：0.5mmol/L的棕櫚酸培養(yǎng)離體胰島24小時(shí),48小時(shí)或72小時(shí)分別增加細(xì)胞培養(yǎng)中的GLP-1水平3.15-,6.55-和5.62倍(p0.05)。在胰島細(xì)胞裂解中PC1/3mRNA和蛋白水平及GLP-1的濃度與細(xì)胞培養(yǎng)液中結(jié)果呈現(xiàn)相似的變化(p0.05)。相對(duì)于棕櫚酸處理離體胰島48小時(shí),72小時(shí)后GLP-1和PC1/3表達(dá)水平呈現(xiàn)下降趨勢(shì)。8周高脂飲食喂養(yǎng)顯著促進(jìn)前a細(xì)胞形成,以及增加小鼠胰島細(xì)胞PC1/3的表達(dá)及血漿GLP-1濃度(p0.05)。相對(duì)于對(duì)照組,0.5mmol/L棕櫚酸培養(yǎng)胰島24小時(shí),48小時(shí)或72小時(shí)分別增加ROS水平3.01-,5.12-和6.45倍。在棕櫚酸培養(yǎng)胰島之前添加0.5mmol/LNAC使胰島細(xì)胞活性從59.56%增加至84.94%,而凋亡降低64.84%。NAC也顯著降低細(xì)胞裂解物中PC1/3mRNA水平和GLP-1的濃度,但不能完全恢復(fù)正常(p0.05)。 結(jié)論：1.無論長(zhǎng)期暴露于棕櫚酸還是高脂飲食均上調(diào)胰島GLP-1及其關(guān)鍵酶PC1/3表達(dá),即激活胰島內(nèi)GLP-1系統(tǒng)； 2.棕櫚酸增加活性氧生成,拮抗氧化應(yīng)激減輕胰島細(xì)胞損傷和部分逆轉(zhuǎn)胰島內(nèi)GLP-1系統(tǒng)活化,表明氧化應(yīng)激部分介導(dǎo)脂毒性激活胰島內(nèi)GLP-1系統(tǒng)。 第三部分：胰島內(nèi)源性的GLP-1的保護(hù)作用及機(jī)制 目的：在脂毒性模型中,改變GLP-1受體的活性觀察胰島內(nèi)源性的GLP-1對(duì)胰島細(xì)胞的保護(hù)作用,以及通過檢測(cè)氧化應(yīng)激和炎癥反應(yīng)探討GLP-1的作用機(jī)制。 方法：分離正常C57BL/6J小鼠胰島并棕櫚酸培養(yǎng),通過添加GLP-1R受體拮抗劑Exendin9-39或激動(dòng)劑利納魯肽,觀察胰島細(xì)胞活性、凋亡水平及PDX1表達(dá)的變化,檢測(cè)ROS水平、NA(D)PH氧化酶成分(NOX4, p22phox和gp91phox)和抗氧化基因(SOD2和Gpx-1)的mRNA表達(dá)水平以及炎癥因子(TNF-a, IL-1β和IL-6)表達(dá)水平。高脂喂養(yǎng)C57BL/6J小鼠8周,于第4周開始皮下注射利納魯肽4周,免疫熒光觀察胰島細(xì)胞結(jié)構(gòu)改變及炎癥信號(hào)通路NF-κB的改變。結(jié)果：聯(lián)合棕櫚酸和Exendin9-39培養(yǎng)48小時(shí),胰島細(xì)胞活性從57.82%降至40.28%,細(xì)胞凋亡從0.48至0.72(p0.05)。聯(lián)合棕櫚酸和利納魯肽培養(yǎng)離體胰島,顯著增加胰島的生存能力和降低胰島細(xì)胞凋亡,并均接近對(duì)照組水平(p0.05)；也顯著地上調(diào)PDX1mRNA表達(dá)7.70倍,均能被Exendin9-39所逆轉(zhuǎn)(p0.05)。Exendin9-39阻斷GLP-1受體信號(hào)增加ROS生成,而利納魯肽抑制NA(D)PH氧化酶成分和上調(diào)抗氧化基因(SOD2和Gpx-1) mRNA的表達(dá),進(jìn)而降低ROS生成(p0.05)。利納魯肽還抑制棕櫚酸誘導(dǎo)的炎癥因子(TNF-a,IL-1β和IL-6)表達(dá)(p0.05)。利拉魯肽顯著增加β細(xì)胞標(biāo)記物PDX1, NKX6.1和GLUT2的mRNA的表達(dá)(p0.05)。低脂喂養(yǎng)組中,胰島結(jié)構(gòu)呈現(xiàn)α細(xì)胞外周和β細(xì)胞核心的特性,而高脂喂養(yǎng)使得α細(xì)胞分散且比例增高,增加中小胰島的面積。利納魯肽恢復(fù)胰島的正常結(jié)構(gòu)和胰島素的表達(dá),降低α細(xì)胞/β細(xì)胞比例和α重量的變化。利納魯肽還可抑制胰島內(nèi)p65的表達(dá)。 結(jié)論：1.拮抗GLP-1受體活性,加重細(xì)胞損傷,惡化β細(xì)胞功能； 2.胰島內(nèi)源性的GLP-1通過維持胰島內(nèi)氧化應(yīng)激的平衡和抑制炎癥信號(hào)通路保護(hù)p細(xì)胞和維持正常胰島結(jié)構(gòu)。
[Abstract]:Part I: lipotoxicity induced dedifferentiation of beta cells
AIM: To observe the effect of lipid toxicity on the function and differentiation of mouse pancreatic islets in vitro and in vivo.
Methods: Islets of C57BL/6J mice were isolated and cultured with palmitic acid for 24 hours, 48 hours, 72 hours, MTT assay for cell activity, ELISA for cell apoptosis, quantitative PCR and immunoblotting for the expression of PDX1 mRNA (?) oral protein, and high fat diet for 8 weeks, 4 weeks and 8 weeks for intraperitoneal glucose tolerance test (IPGTT) and insulin release test (IPITT). Glucose metabolism and plasma insulin levels were measured by ELISA. Confocal and immunofluorescence were used to observe the structure of islets and the expression of various cell markers (p cells: FoxO1, insulin and PDX1; alpha cells: glucagon; stem cells: OCT4, Nanog), and the proliferation and apoptosis of islets were observed by Ki67 and Caspase-3, respectively.
Results: 0.5mmol/L palmitic acid culture for 24 hours, 48 hours or 72 hours decreased the activity of islet cells and increased cell apoptosis, and showed a time-dependent (p0.05). 48 hours palmitic acid exposure significantly decreased the levels of PDX1 mRNA and protein in islet cells (p0.05), but had no effect on insulin mRNA expression (p0.05). High-fat diet increased insulin expression. Secretion (p0.01); calculated area under curve (AUC) showed that AUCIPGTT and AUCIPITT increased in the high-fat diet group, especially at 8 weeks compared with 4 weeks (p0.05); high-fat diet increased HOMA-IR index (p0.05). High-fat diet decreased the expression of FoxO1, insulin and PDX1 in beta cells, and increased the expression of stem cell markers OCT4 and Nanog. High-fat diet promoted pancreas. Caspase-3 expression in islet cells and Ki67 expression in islet cells were increased by high-fat diet, but the total number of proliferated islet cells was still very small.
Conclusion: 1. In vitro and in vivo, lipotoxicity induces islet dysfunction and insulin resistance in a time-dependent manner.
2. high fat diet promotes islet cell proliferation and induces apoptosis.
3. high fat diet damages beta cells and promotes islet cells to dedifferentiate into bone marrow stem cells.
The second part: the formation of pre alpha cells and the activation and mechanism of islet endogenous GLP-1 system.
AIM: To investigate the expression of PC1/3, the key enzyme of GLP-1 and GLP-1 formation, and the role of oxidative stress in the activation of GLP-1 system.
Methods: Islets of normal C57BL/6J mice were isolated and cultured with palmitic acid for 24-72 hours. The levels of GLP-1 in culture medium and cell lysate were observed by ELISA. The changes of PC1/3 mRNA and protein levels were observed by quantitative PCR and immunoblotting. The optimal stimulating conditions for activation of GLP-1 system in islets were obtained. The concentration of GLP-1 in serum, the expression of PC1/3 mRNA in pancreatic tissue by Q-PCR and the formation of pre-A cells were detected by immunofluorescence. The levels of reactive oxygen species (ROS) induced by palmitic acid were detected by DCFH-DA method, and the levels of PCl/3 mRNA and GLP-1 in cell lysate were further observed by adding antioxidant N-acetylcysteine (NAC).
Results: The levels of GLP-1 in islets cultured with 0.5mmol/L palmitic acid for 24 hours, 48 hours or 72 hours increased by 3.15-, 6.55-and 5.62 times respectively (p0.05). The levels of PC1/3 mRNA and protein and the concentration of GLP-1 in islets lysis were similar to those in cell culture medium (p0.05) compared with those treated with palmitic acid in vitro. The expression of GLP-1 and PC1/3 in islets decreased after 48 hours and 72 hours. High-fat diet significantly promoted the formation of pre-A cells and increased the expression of PC1/3 in islet cells and plasma GLP-1 concentration (p0.05). Compared with the control group, the ROS level of islets cultured with 0.5mmol/L palmitic acid for 24 hours, 48 hours or 72 hours increased by 3.01-, 5.05-, respectively. The addition of 0.5mmol/LNAC before palmitic acid culture increased the activity of islet cells from 59.56% to 84.94% and decreased apoptosis by 64.84%. NAC also significantly decreased the levels of PC1/3 mRNA and GLP-1 in the lysates, but could not completely recover to normal (p0.05).
Conclusion: 1. Both long-term exposure to palmitic acid and high-fat diet up-regulate the expression of GLP-1 and its key enzyme PC1/3 in islets, that is, activate the GLP-1 system in islets.
2. Palmitic acid increases ROS production, antagonizes antioxidant stress to alleviate islet cell injury and partially reverses the activation of GLP-1 system in islets, suggesting that oxidative stress partially mediates lipid toxicity to activate GLP-1 system in islets.
The third part: the protective effect and mechanism of endogenous GLP-1 in islets.
AIM: To observe the protective effect of endogenous GLP-1 on islet cells by altering the activity of GLP-1 receptor in a lipotoxic model, and to explore the mechanism of GLP-1 by detecting oxidative stress and inflammation.
Methods: Islets of normal C57BL/6J mice were isolated and cultured with palmitic acid. The activity, apoptosis and PDX1 expression of islet cells were observed by adding GLP-1R receptor antagonist Exendin 9-39 or agonist Linalopeptide. The levels of ROS, NA (D) PH oxidase (NOX4, p22phox and gp91phox) and the mRNA expression of antioxidant genes (SOD2 and Gpx-1) were detected. Levels of inflammatory factors (TNF-a, IL-1beta and IL-6) were measured. High-fat-fed C57BL/6J mice were subcutaneously injected with linalopeptide for 8 weeks and 4 weeks. Immunofluorescence was used to observe the changes of islet cell structure and inflammation signal pathway NF-kappa B. Results: After 48 hours of culture with palmitic acid and Exendin 9-39, the activity of islet cells decreased from 57.82%. By 40.28%, the apoptosis was from 0.48 to 0.72 (p0.05). The combination of palmitic acid and linalopeptide significantly increased the viability of islets and decreased the apoptosis of islets, which were close to the level of control group (p0.05). The expression of PDX1 mRNA was also significantly up-regulated by 7.70 times and could be reversed by Exendin 9-39 (p0.05). Exendin 9-39 blocked the GLP-1 receptor signaling. Liraglutide also inhibited the expression of inflammatory factors (TNF-a, IL-1beta and IL-6) induced by palmitic acid (p0.05). Liraglutide significantly increased the mRNAs of beta cell markers PDX1, NKX6.1 and GLUT2. In low-fat diet group, the structure of pancreatic islets showed the characteristics of periphery of alpha cells and core of beta cells, while high-fat diet made alpha cells disperse and increase the proportion of small and medium-sized islets, increased the area of small and medium-sized islets. It can also inhibit the expression of p65 in islets.
Conclusion: 1. antagonizing the activity of GLP-1 receptor, aggravating cell damage and worsening the function of beta cell.
2. Endogenous GLP-1 protects P cells and maintains normal islet structure by maintaining the balance of oxidative stress and inhibiting inflammation signaling in islets.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.1

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6 楊剛毅;;GLP-1與脂肪肝——從實(shí)驗(yàn)室研究到臨床應(yīng)用[A];中華醫(yī)學(xué)會(huì)第十二次全國(guó)內(nèi)分泌學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2013年

7 楊新波;;治療糖尿病新靶點(diǎn)GLP-1及其相關(guān)新藥簡(jiǎn)介[A];中國(guó)成人醫(yī)藥教育論壇（4）[C];2011年

8 王敏楠;韓玉冰;李強(qiáng);郭琳;楊玉梅;李鵬杰;王薇;張金超;;GLP-1對(duì)棕櫚酸誘導(dǎo)的INS-1細(xì)胞損傷的保護(hù)作用及機(jī)制初探[A];中華醫(yī)學(xué)會(huì)第十二次全國(guó)內(nèi)分泌學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2013年

9 尹嘉晶;李艷波;王洋;;GLP-1對(duì)胰島素抵抗環(huán)境下胰島B細(xì)胞自噬的影響[A];中華醫(yī)學(xué)會(huì)第十次全國(guó)內(nèi)分泌學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2011年

10 李圣堅(jiān);薛耀明;李佳;朱波;張巧玲;陳毅光;;GLP-1通過抑制NF-κB信號(hào)通路減少IL-1β對(duì)INS-1細(xì)胞的損傷作用[A];2010中國(guó)醫(yī)師協(xié)會(huì)內(nèi)分泌代謝科醫(yī)師分會(huì)年會(huì)論文匯編[C];2010年

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3 劉芳芳;1、等熱量不同成分的食物對(duì)2型糖尿病患者血漿GLP-1 的即時(shí)影響2、亞臨床甲狀腺功能減退與血漿同型半胱氨酸水平之間的關(guān)系:一項(xiàng)薈萃分析[D];山東大學(xué);2014年

4 李文娟;軟脂酸對(duì)腸道GLP-1分泌細(xì)胞活性及脂質(zhì)代謝酶的影響[D];華中科技大學(xué);2013年

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6 孫歡歡;功能性消化不良患者血清GLP-1水平變化及意義[D];泰山醫(yī)學(xué)院;2013年

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9 邱惠瓊;參芪復(fù)方辯證加味對(duì)T2DM大血管病變患者GLP-1和Gg表達(dá)影響的臨床研究[D];成都中醫(yī)藥大學(xué);2014年

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