人红细胞生成素对高糖诱导肾近曲小管上皮细胞转分化过程中炎性因子的影响及其可能机制  

Effect and Possible Mechanism of rhEPO in the Transdifferentiation of Human Kidney Proximal Tubular Epithelial Cells Induced by High Glucose and the Changes of Inflammatory Cytokines

作　　者：陈艳霞[1] 杨丽萍[2] 吴险峰[1] 秦晓华[1] 黄翀[1] 房向东[1] 涂卫平[1] 

机构地区：[1]南昌大学第二附属医院肾内科,江西省南昌市330006 [2]江西省肿瘤医院

出　　处：《中国全科医学》2015年第20期2433-2438,共6页Chinese General Practice

基　　金：江西省自然科学基金资助项目(20122BAB205006)

摘　　要：目的探讨重组人红细胞生成素(rhEPO)对高糖诱导的正常人肾小管上皮细胞(HK-2细胞)转分化过程中炎性因子的变化及其可能机制。方法体外培养HK-2细胞,采用随机数字表法分为空白对照组(未加任何刺激物)、高糖诱导组(高糖终浓度为30 mmol/L)、甘露醇对照组(甘露醇为24.5 mmol/L)、rhEPO对照组(rhEPO终浓度为20 U/ml)、不同浓度rhEPO干预组(rhEPO终浓度分别为5、10、20 U/ml+高糖)及Rho激酶抑制剂(Y27632)组(Y27632终浓度为30μmol/L,加入Y27632 30 min后加高糖,高糖终浓度为30 mmol/L),以上各组均培养24 h。采用RT-PCR检测各组细胞RhoA mRNA、ROCK1 mRNA表达水平;采用细胞免疫荧光法检测E-钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)表达水平;采用酶联免疫吸附实验(ELISA)检测人纤维连接蛋白(FN)、白介素6(IL-6)、肿瘤坏死因子α(TNF-α)的蛋白表达水平。结果各组RhoA mRNA、ROCK1 mRNA表达比较,差异均有统计学意义(P<0.05);其中高糖诱导组与5 U/ml rhEPO组RhoA mRNA、ROCK1 mRNA表达均高于空白对照组,10 U/ml rhEPO组、20 U/ml rhEPO组及Rho激酶抑制剂组RhoA 、ROCK1 mRNA表达均低于空白对照组(P<0.05);不同浓度rhEPO组RhoA mRNA、ROCK1 mRNA表达均低于高糖诱导组(P<0.05),Rho激酶抑制剂组ROCK1 mRNA表达低于高糖诱导组(P<0.05);10 U/ml rhEPO组与20 U/ml rhEPO组RhoA mRNA、ROCK1 mRNA表达均低于5 U/ml rhEPO组(P<0.05);20 U/ml rhEPO组RhoA mRNA、ROCK1 mRNA表达均低于10 U/ml rhEPO组(P<0.05)。各组α-SMA、E-cadherin、FN、IL-6及TNF-α蛋白表达比较,差异均有统计学意义(P<0.05);其中高糖诱导组、不同浓度rhEPO组、Rho激酶抑制剂组α-SMA、FN、IL-6及TNF-α蛋白表达均高于空白对照组,E-cadherin均低于空白对照组(P<0.05);不同浓度rhEPO组、Rho激酶抑制剂组α-SMA、FN、IL-6及TNF-α蛋白表达均低于高糖诱导组,E-cadherin均高于高糖诱导组(P<0.05);10 U/ml rhEPO组、20 U/ml rhEPO组α-SMA、FN、IL-6及TNF-α�Objective To investigate the effect and possible mechanism of rhEPO in the transdifferentiation of human kidney proximal tubular epithelial cells（HK-2 cells） induced by high glucose and the changes of inflammatory cytokines.Methods By using random number table,HK-2 cells cultured in vitro were divided into several groups：blank control group（with no irritants）,high glucose induced group（with a high glucose final concentration of 30 mmol/L）,mannitol control group（with a mannitol concentration of 24.5 mmol/L）,rhEPO control group（with a rhEPO final concentration of 20 U/ml）,three rhEPO intervention groups（with a rhEPO final concentration of 5,10 and 20 U/ml respectively and high glucose added）,Rho kinase inhibitor（Y27632） group（in which Y27632 with a final concentration of 30 μmol/L was added and high glucose was added 30 minutes later with a final concentration of 30 mmol/L）.After 24 h in virto culture,reverse transcription polymerase chain reaction（RT-PCR） was used to evaluate the mRNA levels of RhoA and ROCK.The levels of E-cadherin and α-smooth muscle actin（α-SMA） proteins were assessed by immunofluorescent test.ELISA was used to measure the levels of Fibronectin（FN）,IL-6 and TNF-αproteins.Results The groups were all significantly different（P 0.05） in the mRNA levels of RhoA and ROCK; the high glucose induced group and the 5 U/ml rhEPO intervention group were higher（P 0.05）than the blank control group in the mRNA levels of RhoA and ROCK; the 10 U/ml rhEPO intervention group and the 20 U/ml rhEPO intervention group and the Y27632 group were lower（P 0.05） than the blank control group in the mRNA levels of RhoA and ROCK; the three rhEPO intervention groups were lower（P 0.05） than the high glucose induced group in the mRNA levels of RhoA and ROCK; the Y27632 group was lower（P 0.05） than the high glucose induced group in the mRNA levels of ROCK; the 10 U/ml rhEPO intervention group and the 20 U/ml rhEPO intervention group were lower（P 0.05） than

关 键 词：糖尿病肾病 红细胞生成素 细胞转分化 白细胞介素6 肿瘤坏死因子α RhoA/ROCK信号通路 

分 类 号：R587.24[医药卫生—内分泌]