FFAs通过影响hIAPP构象和内质网UPR加重胰岛β细胞损伤的初步研究
发布时间：2019-06-14 20:13
【摘要】：目的:糖尿病发病率逐年增高,其中以二型糖尿病(T2DM)为主,预计到2030年全球罹患糖尿病人数将达到4.39亿人。随着生活水平的提高,肥胖患者逐渐增多,而肥胖会导致血浆中游离脂肪酸(Free Fatty Acids,FFAs)含量的升高,引发高脂血症。已知高脂血症会造成胰岛β细胞损害,持续高浓度FFAs会诱导胰岛β细胞凋亡,但其具体分子机制尚不清楚。最近的研究显示胰岛β细胞凋亡与由糖/脂毒性和胰岛淀粉样蛋白沉积所诱导的内质网应激相关。人胰岛淀粉样多肽(hIAPP)是和胰岛素一起由胰岛β细胞分泌的与糖代谢相关的多肽激素,其生理功能目前尚不清楚,但在T2DM患者尸检的胰岛中发现了这种多肽的淀粉样沉积。多个研究认为淀粉样蛋白沉积的前提是该类蛋白早期出现了构象变化,尤其是β折叠增多。β折叠增多,可能导致此类蛋白的降解异常,早期可能启动内质网UPR途径以清除过量的淀粉样蛋白,后期则可能导致内质网应激,激活内质网应激途径的细胞凋亡,导致胰岛β细胞丢失。因此,明确FFAs与hIAPP之间的相互作用是如何导致β细胞损伤,可能有利于控制/延缓T2DM的发展。方法:1.化学实验hIAPP单独或FFAs+hIAPP分别与硫黄素(ThT)37℃共同孵育,利用多功能酶标仪连续检测荧光强度,以反映hIAPP纤维化水平。hIAPP单独或FFAs+hIAPP 37℃孵育7 h,利用透射电镜观察hIAPP纤维化形成。利用圆二色散方法检测hIAPP构象变化。制备POPC、POPS以及POPC/POPS组成的SUVs(肽脂摩尔比为1:10),分别与hIAPP,PA+hIAPP以及ThT于37℃孵育,利用多功能酶标仪连续检测荧光强度。2.细胞实验空质粒和表达hIAPP的质粒转染INS-1细胞48h后,荧光显微镜下观察转染效率。INS-1细胞转染空质粒和hIAPP质粒24h后,用不同浓度的PA处理细胞24h和48h,利用MTT方法检测细胞存活率,real time RT-PCR检测hIAPP m RNA、内质网应激相关基因GRP78、ATF6、CHOP m RNA相对表达量。INS-1细胞转染空质粒和hIAPP质粒转染24h后,用不同浓度的PA处理细胞48h,收集细胞,先用70%乙醇固定,PBS洗涤后,再用碘化丙啶(PI)染色,FCM检测亚二倍体峰以反映细胞凋亡情况。结果:1.ThT荧光实验结果发现200或400μM FFAs均能够增加hIAPP纤维化形成。其中,200μM PA不但能够促进hIAPP纤维化的形成,同时也增加纤维化hIAPP的稳定性。透射电镜结果显示PA明显增加hIAPP纤维化的形成。CD实验结果显示PA可以促进hIAPP构象由α螺旋状态或无规卷曲转变为β折叠。在人工质膜条件下,阴性脂质POPS增加hIAPP的纤维化,POPS与PA协同对hIAPP的纤维化聚集有促进作用。2.表达hIAPP基因的质粒成功转入了INS-1细胞,转染效率为65%。转染hIAPP的细胞其hIAPP m RNA水平显著增加。转染hIAPP质粒的细胞加入PA处理后,与对照组(转染空质粒)比较,细胞存活率明显降低,内质网应激相关基因GRP78、ATF6、CHOP m RNA相对表达量明显增加,细胞凋亡率明显增加。在转染hIAPP质粒的细胞中,PA与hIAPP二者共同作用较hIAPP单独作用,细胞存活率明显降低,内质网应激和细胞凋亡率均明显增加。结论:1.FFAs尤其是PA可以改变hIAPP的构象,促使其转变为β折叠,并促进hIAPP纤维化形成。2.在人工质膜条件下,阴性脂质POPS增加hIAPP的纤维化,POPS与PA协同对hIAPP的纤维化聚集有促进作用。3.PA促进hIAPP构象向β折叠转变,促进hIAPP的纤维化,诱导了内质网应激,引起细胞凋亡,加重胰岛β细胞损伤。
[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，
本文编号：2499655
[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，
本文编号：2499655
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