本文選題：AWRK6 + Min6細(xì)胞��； 參考：《遼寧大學(xué)》2017年碩士論文

【摘要】：內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)在二型糖尿病發(fā)病中發(fā)揮著重要的作用,ERS能夠促進(jìn)胰島β細(xì)胞的凋亡,所以研究ERS與胰島β細(xì)胞之間的機(jī)制對(duì)預(yù)防二型糖尿病具有重要的理論意義。AWRK6(SWVGKHGKKFGLKKHKKH)是本實(shí)驗(yàn)室從林蛙表皮提取出的一種新型抗菌肽并加以改造,抗菌肽AWRK6即可以促進(jìn)Min6細(xì)胞增殖還能促進(jìn)胰島素分泌,而且抗菌肽在體內(nèi)不容易被降解,AWRK6的這些特性均在實(shí)驗(yàn)室前期研究中得到驗(yàn)證。前期實(shí)驗(yàn)結(jié)果表明,抗菌肽AWRK6對(duì)ERS導(dǎo)致的胰島細(xì)胞凋亡有著的抑制效果。為了探討抗菌肽抑制凋亡的作用機(jī)制,用MTT比色法、免疫印跡法(Western Blot)等方法,分析AWRK6對(duì)ERS導(dǎo)致的凋亡的抑制機(jī)制。利用MTT比色法檢測(cè)MIN6存活率,使用衣霉素作為誘導(dǎo)ERS藥物。實(shí)驗(yàn)結(jié)果表明,抗菌肽AWRK6在100nmol/L下MIN6細(xì)胞存活率與衣霉素TM組相比有著差異性(P0.01)。Western blot結(jié)果表明,已誘導(dǎo)產(chǎn)生內(nèi)質(zhì)網(wǎng)應(yīng)激的胰島細(xì)胞在抗菌肽處理后,抗凋亡分子Epac2表達(dá)量增加,內(nèi)質(zhì)網(wǎng)應(yīng)激分子Chop和Bip表達(dá)量降低,促凋亡分子Bax表達(dá)量降低,并且在加入GLP-1受體抑制劑后,抗菌肽AWRK6緩解內(nèi)質(zhì)網(wǎng)應(yīng)激抑制凋亡的功能受到一定程度上的抑制。為了確定AWRK6在體內(nèi)的降血糖作用和對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激產(chǎn)生凋亡的影響,使用鏈脲佐菌素來(lái)構(gòu)建糖尿病的動(dòng)物模型。其中根據(jù)糖耐量實(shí)驗(yàn)(OGTT)我們發(fā)現(xiàn),用AWRK6處理過(guò)的二型糖尿病大鼠,其糖耐受能力得到顯著地提高。提取胰島組織蛋白做Western blot,還發(fā)現(xiàn)經(jīng)抗菌肽處理后能夠使抗凋亡分子Epac2蛋白量增加,內(nèi)質(zhì)網(wǎng)應(yīng)激分子Bip和Chop蛋白量減少,促凋亡因子Bax的蛋白量降低。本研究結(jié)果闡明了抗菌肽AWRK6通過(guò)改善內(nèi)質(zhì)網(wǎng)應(yīng)激,抑制胰島細(xì)胞凋亡的分子機(jī)制,為進(jìn)一步將AWRK6研發(fā)成為二型糖尿病治療藥物奠定了理論和實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.1

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