新型重组VPAC2激动剂RD的制备及促进胰岛素功能的分子机制

doi:10.13523/j.cb.20141109

中国生物工程杂志

2014, Vol. 34

Issue (11): 60-66 DOI: 10.13523/j.cb.20141109

研究报告

新型重组VPAC2激动剂RD的制备及促进胰岛素功能的分子机制

马义, 罗天杰, 洪岸

暨南大学细胞生物学系暨南大学生物医药研究院广东省生物工程药物重点实验室基因工程药物国家工程研究中心广州 510632

Preparation of the Novel Recombinant VPAC2 Receptor Agonist RD and Its Molecular Mechanism of Promoteing Insulin Fuction

MA Yi, LUO Tian-jie, HONG An

Department of Cell Biology of Jinan University, Institute of Biological Medicine of Jinan University, National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China

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摘要：

利用DNA重组、原核表达、Chitin-Beads柱和HPLC纯化、质谱鉴定等技术,制备了一种新型具有抗2型糖尿病功能的VPAC2受体激动剂RD,并初步研究和揭示了其在Ⅱ型糖尿病治疗中有效促进胰岛素信号传导的分子机制。实验结果表明:利用基因重组技术制备的VPAC2受体激动剂RD的分子量为3 785.0 Da,纯度为96%;将重组肽作用于正常或胰岛素抵抗的3T3-L1 脂肪细胞(IR模型细胞),1和5μmol/L 重组肽RD可促进正常3T3-L1脂肪细胞IRS-1 蛋白的表达(分别增加36%和42%),而促进IR模型细胞IRS-1 蛋白的表达增加更为明显(分别增加55%和63%)。IR模型细胞经1,5和10μmol/L重组肽RD处理后,pIRS1(ser307)的表达水平分别比降低了5.9%,10.7%和32.7%。在IR模型细胞中,5和10μmol/L RD处理组,IRS-2蛋白的表达水平分别降低12.8%和40.6%;而1,5和10μmol/L RD各处理组pIRS2蛋白的表达水平分别降低35.1%,40.8%和48.5%。5 and 10μmol/L RD处理的IR模型细胞中Akt蛋白的表达显著增强,表达量分别增加74%和77%。1,5 和10μmol/L的重组肽RD处理的IR模型细胞中,Akt Ser473磷酸化水平分别降低33.9%,64.0%和71.1%;Akt Thr308磷酸化水平分别升高13.5%,78.6%和83.3%。建立了重组VPAC2受体激动剂RD的制备技术,并在体外细胞水平检测了其效果(显著促进正常3T3-L1脂肪细胞及IR模型细胞IRS-1 蛋白的表达;降低IR模型细胞pIRS1(ser307),IRS-2,pIRS2蛋白的表达;促进IR模型细胞Akt蛋白的表达及Akt Thr308磷酸化水平等),为阐明其在2型糖尿病治疗中的分子作用机制及药用研发提供了实验基础。

关键词： 基因重组; VPAC2受体激动剂; 糖尿病; 胰岛素

Abstract:

Using genetic recombinant, prokaryotic expression, purification by Chitin-Beads column and HPLC, and identification by mass spectrometry technologies, a new VPAC2-specific agonist RD with the function of anti-type 2 diabetes was prepared, and its molecular mechanism of the effectively promoting insulin signal transduction in treating diabetes was studied. The results show that the molecular weight of the prepared RD is 3.785kDa. and its purity is 96%. Treating normal and insulin resistant 3T3-L1 adipocytes (IR model cells) with recombinant RD,the 1μmol/L and 5μmol/L RD treated normal 3T3-L1 adipocytes groups can significantly promote the expression of the IRS-1 protein, with an expression increase of 36% and 42% respectively. After the IR model cells were treated with the 1μmol/L and 5μmol/L RD, the IRS-1 expressions were increased by 55% and 63% respectively. After the IR model cells were treated with 1,5 and 10μmol/L RD, the pIRS-1(Ser307) expressions were decreased by 5.9%, 10.7% and 32.7% respectively. 5μmol/L and 10μmol/L RD treated IR model cells groups can decrease the expression of the IRS-2 protein by 12.8% and 40.6% respectively, and 1,5 and 10μmol/L MHDBAY treated IR model cells groups, the expressions of the pIRS-2 protein were decreased by 35.1%, 40.8% and 48.5% respectively. The expressions of the Akt protein in the IR model cells in 5μM and 10μM MHDBAY treated group were significantly increased, with an expression increase of 74% and 77% respectively. In IR model cells treated with 1, 5, 10μM RD, the phosphorylation levels of Akt Ser473 were decreased by,33.9%,64.0% and 71.1% respectively, and the phosporylation levels of Akt Thr308 were increased by 13.5%,78.6% and 83.3% respectively. This study established the technical preparation of the recombinant VPAC2 receptor agonist RD, and its biological effects in cell level were detected (significantly promote IRS-1 expression in normal 3T3-L1 adipocytes and IR model cell, decrease pIRS1 (Ser307), IRS-2, pIRS2 expression in IR cell model, and promote Akt expression and Akt Thr308 phosphorylation level, etc.). That may provide the experimental basis for illuminating molecular mechanism of RD in treating diabetes and its pharmaceutical research and development.

Key words: Genetic recombinant VPAC2-specific agonist Diabetes Insulin

收稿日期: 2014-08-13 出版日期: 2014-11-25

ZTFLH:

Q78

基金资助:

国家自然科学基金面上项目(81373314),广东省自然科学基金(S2012010008756),高等学校博士学科点专项科研基金(20124401120012),广东省高等学校科技创新(2012KJCX0015),广东省教育部产学研结合(2010B090400544),广州市科技计划(2011J4300112),中央高校基本科研业务费专项资金(21612408),科技部科技型中小企业技术创新基金(11C26214413218)资助项目

通讯作者: 马义,tmayi@jnu.edu.cn. E-mail: tmayi@jnu.edu.cn

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引用本文:

马义, 罗天杰, 洪岸. 新型重组VPAC2激动剂RD的制备及促进胰岛素功能的分子机制[J]. 中国生物工程杂志, 2014, 34(11): 60-66.

MA Yi, LUO Tian-jie, HONG An. Preparation of the Novel Recombinant VPAC2 Receptor Agonist RD and Its Molecular Mechanism of Promoteing Insulin Fuction. China Biotechnology, 2014, 34(11): 60-66.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20141109 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I11/60

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