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肾脏病与透析肾移植杂志 ›› 2018, Vol. 27 ›› Issue (4): 336-341.DOI: 10.3969/j.issn.1006-298X.2018.04.008

• 论文 • 上一篇    下一篇

Krüppel样因子4抑制高糖刺激MES-13细胞炎症的机制

  

  • 出版日期:2018-08-28 发布日期:2018-08-31

Research of Krüppellike factor 4 inhibit inflammation in MES13 cells stimulated by high glucose  

  • Online:2018-08-28 Published:2018-08-31

摘要:

目的:观察Krüppel样因子4(KLF4)在小鼠肾小球系膜细胞(MES13细胞)中的表达。观察甲基化抑制剂5氮杂胞苷(5azacytidine,5Az)对高糖刺激的MES13细胞中KLF4、IL6、MCP1表达的影响。
方法:常规培养MES13细胞,经高糖(30 mmol/L)处理不同时间后,检测KLF4蛋白表达水平。将细胞分为低糖组、高渗组、高糖组、高糖+不同浓度5Az组,检测KLF4、IL6、MCP1 mRNA和蛋白表达水平。慢病毒质粒转染细胞后,高糖处理0h、48h,检测KLF4、IL6、MCP1 mRNA表达。
结果:高糖处理MES13细胞后,KLF4蛋白表达于24 h开始下调,于48h时下调明显。高糖处理细胞48h后,KLF4 mRNA和蛋白表达明显下调,MCP1、IL6 mRNA和蛋白表达水平明显上调(P<001),加用5Az(5 μmol/L)干预后,KLF4 mRNA和蛋白表达水平明显上调,而IL6、MCP1 mRNA和蛋白表达均较干预前下调。慢病毒质粒转染细胞后,KLF4 mRNA表达明显增加,IL6、MCP1 mRNA表达无明显变化。高糖处理转染细胞48h后,LvNC(hi)组与LvNC组相比,KLF4 mRNA表达水平明显下调,IL6、MCP1 mRNA表达水平明显上调。LvKLF4(hi)组与LvNC(hi)组相比,KLF4 mRNA表达水平明显上调,IL6、MCP1 mRNA表达水平明显下调。
结论:在MES13细胞中存在KLF4表达,且高糖处理可使KLF4表达下调,IL6、MCP1表达上调。高糖引起的MES13细胞炎症与KLF4低表达有关,恢复KLF4表达,可减轻细胞炎症,推测其机制部分与DNA甲基化相关。

关键词: 糖尿病肾病, Krü, ppel样因子4, 炎症反应, 5-氮杂胞苷

Abstract:

Objective:To observe the expression  of Krüppellike factor 4 (KLF4) in MES13 cells and to detect the expression  of KLF4,IL6,MCP1in MES13 cells stimulated by high glucose.
Methodology:MES13 cells were stimulated by high glucose(30 mmol/L) for different time.The expression of KLF4 protein was detected by Western blot respectively.MES13 cells were divided into four groups:normal glucose,hypertonic group,high glucose and high glucose with 5AzThe expressions of KLF4,IL6,MCP1 mRNA and protein were detected by RTPCR and Western blot separately.MES13 cells were divided into two groups:LvNC glucose and LvKLF4 group.The expressions of KLF4,IL6,MCP1 mRNA were detected by RTPCR respectively after stimulating by high glucose for 0 hour and 48 hours.
Results:In MES13 cells,the expression of KLF4 protein was decreased after stimulating by high glucose for 24 hours,and it was significantly decreased at 48 hours.The expressions of KLF4 protein and mRNA were significantly decreased in MES13 cells stimulated by high glucose for 48 hours,while the expressions of IL6,MCP1 protein and mRNA were increased obviously.After treatment with 5Az(5 μmol/L),the expressions of KLF4 protein and mRNA were upregulated in MES13 cells,while the expressions of IL6,MCP1 protein and mRNA were downregulated distinctly.After lentivirus plasmid transfected into MES13 cells,the expression level of KLF4 mRNA was significantly increased,while IL6 and MCP1 mRNA expressions didnt change obviously.After stimulating MES13 cells by high glucose for 48 hours,the expression of KLF4 mRNA was significantly decreased in lvnc(hi) group compared with lvnc group,and the expression  of IL6 and MCP1 mRNA were distinctly increased.Compared with lvnc (hi) group,the expression level of KLF4 mRNA was significantly upregulated in LVKLF4(hi) group,while the expression  of IL6 and MCP1 mRNA were downregulated obviously.
Conclusion:KLF4 is expressed in MES13 cells.High glucose can downregulate the expression of KLF4 and upregulate the expressions of IL6、MCP1Low expression of KLF4 is related with inflammation in MES13 cells stimulated by high glucose.Upregulate KLF4 expression can alleviate cell inflammation.Its mechanism probably is associated with DNA methylation partly.

Key words: diabetic kidney disease, Krüppel-like factor 4, inflammation, 5-azacytidine