Sublytic C5b⁃9刺激上调KLF4促进肾小球系膜细胞生成IL⁃23的作用
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国家自然科学基金(31500701,81471626);江苏省自然科学青年基金(BK20140910)


Sublytic C5b ⁃ 9 induces IL ⁃ 23 production of rat glomerular mesangial cells via up ⁃regulated KLF4 expression
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    摘要:

    目的:研究转录因子KLF4调控sublytic C5b-9刺激肾小球系膜细胞(glomerular mesangial cell,GMC)诱导促炎因子白介素-23(IL-23)生成的作用。方法:构建KLF4的短发夹状小干扰RNA(shKLF4)及过表达质粒(pIRES2-KLF4)。将pIRES2-KLF4或shKLF4 转染GMC后再行sublytic C5b-9刺激,用qPCR、Western blot和ELISA法检查沉默或过表达KLF4基因后对GMC产生IL-23的影响。此外,构建IL-23基因近端启动子全长质粒,行荧光素酶报告基因实验测定沉默或过表达KLF4基因后对IL-23启动子活性的影响。结果:①Sublytic C5b-9刺激GMC后能明显促进IL-23的生成,而沉默KLF4基因后,由sublytic C5b-9诱导GMC产生的IL-23明显减少,但过表达KLF4后IL-23的水平则显著增加。②Sublytic C5b-9刺激GMC能显著上调IL-23的启动子活性,而沉默KLF4基因后由sublytic C5b-9诱导的IL-23启动子活性明显降低,但过表达KLF4后又能显著升高IL-23的启动子活性。结论:Sublytic C5b-9刺激诱导IL-23的生成可通过其刺激上调转录因子KLF4的表达而实现。

    Abstract:

    Objective:To explore the regulatory role of KLF4,as a transcription factor,in the production of pro-inflammation cytokine IL-23 gene in rat glomerular mesangial cells(GMC)induced by sublytic C5b-9. Methods:The plasmids of KLF4 short hairpin RNA(shKLF4)and KLF4 overexpression(pIRES2-KLF4)were first generated and transfected rat GMC respectively,then the cells were treated with or without sublytic C5b-9. The roles of silencing KLF4 gene in IL-23 synthesis induced by sublytic C5b-9 or overexpressing KLF4 gene in IL-23 production were detected by qPCR,Western blot and ELISA. Moreover,the plasmid of IL-23 promoter(full-length,FL)was also constructed. After co-transfection,the activity of IL-23 promoter with or without sublytic C5b-9 after KLF4 gene knockdown or overexpression was measured by luciferase reporter assay. Results:①Sublytic C5b-9 could obviously elevate IL-23 production in rat GMC,and after KLF4 gene knochdown with shKLF4,the increase of IL-23 expression upon sublytic C5b-9 was remarkably diminished,but KLF4 overexpression in the GMC could markedly elevate IL-23 synthesis. ②The IL-23 promoter activity incubated with sublytic C5b-9 was significantly up-regulated. Meanwhile,after KLF4 gene knockdown,the IL-23 promoter activity in the GMC stimulated by sublytic C5b-9 was obviously declined,but KLF4 overexpression greatly increased IL-23 promoter activivy. Conclusion:Sublytic C5b-9 could induce IL-23 production of GMCs through the increase of KLF4 expression.

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虞天一,赵晨卉,何风霞,刘玉,宫雅娟,赵聃,邱文,张婧,王迎伟. Sublytic C5b⁃9刺激上调KLF4促进肾小球系膜细胞生成IL⁃23的作用[J].南京医科大学学报(自然科学版),2018,(10):1331-1336,1387

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  • 收稿日期:2018-02-24
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  • 在线发布日期: 2018-11-08
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