Toll样受体3抑制胰岛β细胞生长的分子机制研究
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国家自然科学基金(81170714)


Effect of Toll-like receptor 3 on the proliferation of pancreatic β-cell
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    摘要:

    目的:探索Toll样受体3 (Toll-like receptor 3,TLR3)在调节大鼠胰岛β细胞株INS-1细胞生长中的作用及其相关机制-方法:采用1%牛血清白蛋白(BSA)代替血清培养12 h使INS-1细胞同步化处于G0期-TLR3的特异性激动剂聚肌胞(polyinosinic-polycytidylic acid,PIC)激活TLR3,26S蛋白酶体抑制剂MG132抑制蛋白酶体活性-MTT法检测细胞活力,流式细胞术测定细胞周期,实时定量RT-PCR-免疫印迹(Western blot)法检测细胞周期相关蛋白Cyclin D1的表达-结果:和对照组相比,PIC呈剂量依赖性抑制INS-1细胞生长活力(P < 0.05);PIC能显著阻滞INS-1细胞周期于G1期(P < 0.05);PIC处理组Cyclin D1 的mRNA水平无明显变化(P > 0.05),但其蛋白水平显著下调(P < 0.05);给予蛋白酶体抑制剂MG132预处理能缓解此下调作用-结论:激活的TLR3可能通过蛋白酶体介导的Cyclin D1降解,导致胰岛β细胞G1期阻滞,从而抑制其增殖,参与机体胰岛β细胞整体功能障碍的调节-

    Abstract:

    Objective:To investigate the effect of elevated Toll-like receptor 3(TLR3) on the growth of INS-1 pancreatic β-cell and possible cell cycle changes during this process. Methods:INS-1 cells were synchronized at the G0 phase by serum deprivation for 12 h. Polyinosinic-polycytidylic acid (PIC) was used to activate TLR3,and the proteasome was inhibited with MG132. Cell viability was assessed by MTT colorimetric assay. Cell cycle distribution was measured by FACS analysis,and cell cycle protein Cyclin D1 was further detected using real-time RT-PCR and Western blot analysis. Results:PIC significantly inhibited the viability of INS-1 cells in a dose-dependent manner (P < 0.05). Furthermore,PIC induced an increase in the proportion of cells at the G1 phase and a corresponding decrease in the number of cells at the S phase(P < 0.05). Additionally,PIC treatment caused a significant reduction in Cyclin D1 levels (P < 0.05),but without a parallel decrease in Cyclin D1 mRNA levels(P > 0.05). Inhibition of the proteasome with MG132 rescued Cyclin D1 protein from degradation. Conclusion:The results suggest that TLR3 activation with PIC directly inhibits pancreatic β-cell proliferation through cell cycle arrest at the G1 phase,possibly by downregulation of Cyclin D1 in a proteasome-dependent manner,which ultimately results in an overall β-cell mass deficiency.

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王 怡,祁岳坤,姚旺林,刘银龙,郭 军. Toll样受体3抑制胰岛β细胞生长的分子机制研究[J].南京医科大学学报(自然科学版),2012,(5):621-625

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  • 收稿日期:2012-01-03
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