【摘要】：目的:糖尿病發(fā)病率逐年增高,其中以二型糖尿病(T2DM)為主,預(yù)計(jì)到2030年全球罹患糖尿病人數(shù)將達(dá)到4.39億人。隨著生活水平的提高,肥胖患者逐漸增多,而肥胖會(huì)導(dǎo)致血漿中游離脂肪酸(Free Fatty Acids,FFAs)含量的升高,引發(fā)高脂血癥。已知高脂血癥會(huì)造成胰島β細(xì)胞損害,持續(xù)高濃度FFAs會(huì)誘導(dǎo)胰島β細(xì)胞凋亡,但其具體分子機(jī)制尚不清楚。最近的研究顯示胰島β細(xì)胞凋亡與由糖/脂毒性和胰島淀粉樣蛋白沉積所誘導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)。人胰島淀粉樣多肽(hIAPP)是和胰島素一起由胰島β細(xì)胞分泌的與糖代謝相關(guān)的多肽激素,其生理功能目前尚不清楚,但在T2DM患者尸檢的胰島中發(fā)現(xiàn)了這種多肽的淀粉樣沉積。多個(gè)研究認(rèn)為淀粉樣蛋白沉積的前提是該類蛋白早期出現(xiàn)了構(gòu)象變化,尤其是β折疊增多。β折疊增多,可能導(dǎo)致此類蛋白的降解異常,早期可能啟動(dòng)內(nèi)質(zhì)網(wǎng)UPR途徑以清除過量的淀粉樣蛋白,后期則可能導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激,激活內(nèi)質(zhì)網(wǎng)應(yīng)激途徑的細(xì)胞凋亡,導(dǎo)致胰島β細(xì)胞丟失。因此,明確FFAs與hIAPP之間的相互作用是如何導(dǎo)致β細(xì)胞損傷,可能有利于控制/延緩T2DM的發(fā)展。方法:1.化學(xué)實(shí)驗(yàn)hIAPP單獨(dú)或FFAs+hIAPP分別與硫黃素(ThT)37℃共同孵育,利用多功能酶標(biāo)儀連續(xù)檢測熒光強(qiáng)度,以反映hIAPP纖維化水平。hIAPP單獨(dú)或FFAs+hIAPP 37℃孵育7 h,利用透射電鏡觀察hIAPP纖維化形成。利用圓二色散方法檢測hIAPP構(gòu)象變化。制備POPC、POPS以及POPC/POPS組成的SUVs(肽脂摩爾比為1:10),分別與hIAPP,PA+hIAPP以及ThT于37℃孵育,利用多功能酶標(biāo)儀連續(xù)檢測熒光強(qiáng)度。2.細(xì)胞實(shí)驗(yàn)空質(zhì)粒和表達(dá)hIAPP的質(zhì)粒轉(zhuǎn)染INS-1細(xì)胞48h后,熒光顯微鏡下觀察轉(zhuǎn)染效率。INS-1細(xì)胞轉(zhuǎn)染空質(zhì)粒和hIAPP質(zhì)粒24h后,用不同濃度的PA處理細(xì)胞24h和48h,利用MTT方法檢測細(xì)胞存活率,real time RT-PCR檢測hIAPP m RNA、內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)基因GRP78、ATF6、CHOP m RNA相對(duì)表達(dá)量。INS-1細(xì)胞轉(zhuǎn)染空質(zhì)粒和hIAPP質(zhì)粒轉(zhuǎn)染24h后,用不同濃度的PA處理細(xì)胞48h,收集細(xì)胞,先用70%乙醇固定,PBS洗滌后,再用碘化丙啶(PI)染色,FCM檢測亞二倍體峰以反映細(xì)胞凋亡情況。結(jié)果:1.ThT熒光實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)200或400μM FFAs均能夠增加hIAPP纖維化形成。其中,200μM PA不但能夠促進(jìn)hIAPP纖維化的形成,同時(shí)也增加纖維化hIAPP的穩(wěn)定性。透射電鏡結(jié)果顯示PA明顯增加hIAPP纖維化的形成。CD實(shí)驗(yàn)結(jié)果顯示PA可以促進(jìn)hIAPP構(gòu)象由α螺旋狀態(tài)或無規(guī)卷曲轉(zhuǎn)變?yōu)棣抡郫B。在人工質(zhì)膜條件下,陰性脂質(zhì)POPS增加hIAPP的纖維化,POPS與PA協(xié)同對(duì)hIAPP的纖維化聚集有促進(jìn)作用。2.表達(dá)hIAPP基因的質(zhì)粒成功轉(zhuǎn)入了INS-1細(xì)胞,轉(zhuǎn)染效率為65%。轉(zhuǎn)染hIAPP的細(xì)胞其hIAPP m RNA水平顯著增加。轉(zhuǎn)染hIAPP質(zhì)粒的細(xì)胞加入PA處理后,與對(duì)照組(轉(zhuǎn)染空質(zhì)粒)比較,細(xì)胞存活率明顯降低,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)基因GRP78、ATF6、CHOP m RNA相對(duì)表達(dá)量明顯增加,細(xì)胞凋亡率明顯增加。在轉(zhuǎn)染hIAPP質(zhì)粒的細(xì)胞中,PA與hIAPP二者共同作用較hIAPP單獨(dú)作用,細(xì)胞存活率明顯降低,內(nèi)質(zhì)網(wǎng)應(yīng)激和細(xì)胞凋亡率均明顯增加。結(jié)論:1.FFAs尤其是PA可以改變hIAPP的構(gòu)象,促使其轉(zhuǎn)變?yōu)棣抡郫B,并促進(jìn)hIAPP纖維化形成。2.在人工質(zhì)膜條件下,陰性脂質(zhì)POPS增加hIAPP的纖維化,POPS與PA協(xié)同對(duì)hIAPP的纖維化聚集有促進(jìn)作用。3.PA促進(jìn)hIAPP構(gòu)象向β折疊轉(zhuǎn)變,促進(jìn)hIAPP的纖維化,誘導(dǎo)了內(nèi)質(zhì)網(wǎng)應(yīng)激,引起細(xì)胞凋亡,加重胰島β細(xì)胞損傷。
[Abstract]:Objective: The incidence of diabetes is increasing year by year, including type 2 diabetes mellitus (T2DM), and it is expected that the number of diabetes in the world will reach a total of 4.39 billion people by 2030. With the improvement of living standard, the obesity patients gradually increase, and the obesity can lead to the increase of free fatty acids (FFAs) in the plasma, which causes the hyperlipidemia. It is known that hyperlipoidemia can cause islet cell damage, and continuous high concentration of FFAs can induce apoptosis of pancreatic islet cells, but its specific molecular mechanism is not clear. Recent studies have shown that pancreatic islet cell apoptosis is associated with endoplasmic reticulum stress induced by sugar/ lipotoxicity and islet amyloid deposition. The human islet amyloid polypeptide (hIAPP) is a polypeptide hormone related to glucose metabolism, which is secreted by islet-derived cells, together with insulin, and its physiological function is not yet clear, but the amyloid deposition of such a polypeptide is found in the pancreatic islet of an autopsy in a patient with T2DM. In many studies, the premise of amyloid deposition is that the early appearance of these proteins is a conformational change, especially in the case of fold increase. In the late stage, the endoplasmic reticulum UPR pathway may be initiated to remove excess amyloid protein, which may lead to the endoplasmic reticulum stress, the activation of the endoplasmic reticulum stress pathway, and the loss of pancreatic islet cells. Therefore, it is clear how the interaction between FFAs and hIAPP is responsible for controlling/ delaying the development of T2DM. Method:1. The chemical experiment hIAPP alone or FFAs + hIAPP was incubated with Tht 37 鈩，

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