抗菌肽AWRK6通过缓解内质网应激抑制MIN6细胞凋亡的分子机制

发布时间：2018-05-17 00:03

本文选题：AWRK6 + Min6细胞　；参考：《辽宁大学》2017年硕士论文

【摘要】：内质网应激(ERS)在二型糖尿病发病中发挥着重要的作用,ERS能够促进胰岛β细胞的凋亡,所以研究ERS与胰岛β细胞之间的机制对预防二型糖尿病具有重要的理论意义。AWRK6(SWVGKHGKKFGLKKHKKH)是本实验室从林蛙表皮提取出的一种新型抗菌肽并加以改造,抗菌肽AWRK6即可以促进Min6细胞增殖还能促进胰岛素分泌,而且抗菌肽在体内不容易被降解,AWRK6的这些特性均在实验室前期研究中得到验证。前期实验结果表明,抗菌肽AWRK6对ERS导致的胰岛细胞凋亡有着的抑制效果。为了探讨抗菌肽抑制凋亡的作用机制,用MTT比色法、免疫印迹法(Western Blot)等方法,分析AWRK6对ERS导致的凋亡的抑制机制。利用MTT比色法检测MIN6存活率,使用衣霉素作为诱导ERS药物。实验结果表明,抗菌肽AWRK6在100nmol/L下MIN6细胞存活率与衣霉素TM组相比有着差异性(P0.01)。Western blot结果表明,已诱导产生内质网应激的胰岛细胞在抗菌肽处理后,抗凋亡分子Epac2表达量增加,内质网应激分子Chop和Bip表达量降低,促凋亡分子Bax表达量降低,并且在加入GLP-1受体抑制剂后,抗菌肽AWRK6缓解内质网应激抑制凋亡的功能受到一定程度上的抑制。为了确定AWRK6在体内的降血糖作用和对内质网应激产生凋亡的影响,使用链脲佐菌素来构建糖尿病的动物模型。其中根据糖耐量实验(OGTT)我们发现,用AWRK6处理过的二型糖尿病大鼠,其糖耐受能力得到显著地提高。提取胰岛组织蛋白做Western blot,还发现经抗菌肽处理后能够使抗凋亡分子Epac2蛋白量增加,内质网应激分子Bip和Chop蛋白量减少,促凋亡因子Bax的蛋白量降低。本研究结果阐明了抗菌肽AWRK6通过改善内质网应激,抑制胰岛细胞凋亡的分子机制,为进一步将AWRK6研发成为二型糖尿病治疗药物奠定了理论和实验依据。
[Abstract]:Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis of type 2 diabetes mellitus. ERS can promote the apoptosis of islet 尾 cells. Therefore, the study of the mechanism between ERS and islet 尾 cells has important theoretical significance in preventing type 2 diabetes. AWRK6 SWVGKHGKFGLKKHKKH) is a new antimicrobial peptide extracted from the epidermis of Rana chensinensis in our laboratory and modified. Antimicrobial peptide AWRK6 can not only promote the proliferation of Min6 cells, but also promote insulin secretion, and these characteristics of antimicrobial peptides which are not easily degraded in vivo have been verified in laboratory studies. The previous results showed that the antimicrobial peptide AWRK6 could inhibit the apoptosis of islet cells induced by ERS. In order to investigate the mechanism of antibacterial peptides inhibiting apoptosis, the inhibition mechanism of AWRK6 on apoptosis induced by ERS was analyzed by means of MTT colorimetry and Western blotting. The survival rate of MIN6 was detected by MTT colorimetry, and the drug of ERS induced by chlortetracycline was used. The results showed that the survival rate of MIN6 cells under 100nmol/L was significantly different from that of 100nmol/L TM group. Western blot results showed that the expression of anti-apoptotic molecule Epac2 in islet cells which had induced endoplasmic reticulum stress was increased after treatment with antimicrobial peptides. The expression of endoplasmic reticulum stress molecules (Chop and Bip) and apoptosis-promoting molecules (Bax) were decreased, and the inhibition of endoplasmic reticulum stress (ER) induced by antibacterial peptide AWRK6 was inhibited to some extent after the addition of GLP-1 receptor inhibitor. In order to determine the hypoglycemic effect of AWRK6 in vivo and the effect of endoplasmic reticulum stress on apoptosis, streptozotocin was used to construct an animal model of diabetes mellitus. According to the glucose tolerance test (OGTT) we found that the glucose tolerance of type 2 diabetic rats treated with AWRK6 was significantly improved. The islet tissue proteins were extracted as Western blots. it was also found that the amount of anti-apoptotic Epac2 protein increased, the endoplasmic reticulum stress molecule Bip and Chop protein decreased, and the pro-apoptotic factor Bax protein decreased after treatment with antimicrobial peptides. The results of this study demonstrated the molecular mechanism of antimicrobial peptide AWRK6 to inhibit the apoptosis of islet cells by improving endoplasmic reticulum stress, which laid a theoretical and experimental basis for the further development of AWRK6 as a therapeutic drug for type 2 diabetes.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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