高糖诱导下肾小管上皮细胞中细胞因子信号转导抑制因子-1/3的表达及意义

The expression of suppressor of cytokine signaling-1/3 in renal tubular epithelial cells induced by high glucose

目的 观察高糖诱导下肾小管上皮细胞形态学改变及细胞因子信号转导抑制因子（SOCS-1/3）在肾上管上皮细胞中的表达及意义. 方法 体外培养人肾近曲小管上皮细胞株（HKCs）,并分为空白对照组、高糖组、Janus激酶2抑制剂AG490组.空白对照组以无血清培养基（5.5 mmol/L葡萄糖）培养细胞8 h;高糖组在含1%胎牛血清培养基中加高糖（300.0 mmol/L葡萄糖）培养;AG490组在高糖组基础上加10 μmol/L AG490培养.后两组再按培养时间分为2、4、6、12、24、48 h 6个亚组,每组6孔.倒置显微镜和电镜下观察细胞形态学及超微结构改变,采用免疫细胞化学和蛋白质免疫印迹法（Western blotting）检测SOCS-1/3蛋白表达,用逆转录-聚合酶链反应（RT-PCR）检测SOCS-1/3 mRNA表达. 结果 与空白对照组比较,高糖组12 h后细胞呈梭形改变,有不规则突起,随时间延长细胞界限不清,胞质内颗粒增多;电镜下细胞表面微绒毛及细胞内线粒体明显减少,粗面内质网明显增加.AG490组细胞变化不明显.免疫细胞化学显示,SOCS-1/3蛋白表达以胞质为主,散在胞核表达.SOCS-1/3在正常HKC中有基础表达（0.218±0.023,0.337±0.009）;高糖组4～24 h SOCS-1/3蛋白表达均高于空白对照组,其中SOCS-1以4 h表达最高（1.022±0.072）,SOCS-3以6 h表达最高（1.256±0.105,均P〈0.01）;AG490组SOCS-1/3蛋白表达较高糖组明显下降（4 h SOCS-1：0.589±0.167,6 h SOCS-3：0.656±0.075,均P〈0.05）.高糖组2～12 h SOCS-1/3 mRNA表达高于空白对照组,其中SOCS-1以4 h表达最高（1.716±0.098比0.475±0.045,P〈0.05）,SOCS-3以6 h表达最高（2.848±0.116比0.749±0.086,P〈0.01）;AG490组则低于高糖组（4 h SOCS-1：0.865±0.075,6 h SOCS-3：0.923±0.116,均P〈0.05）. 结论 高糖可诱导肾小管上皮细胞发生形态学及超微结构改变,在早期即有SOCS-1/3表达上调,可能与Janus激酶（JAK）/信号转导和转录激活因子（STAT）/SOCS信号转导通路的负反馈调节途径有关.

Objective To observe the morphologic changes and the expression of suppressor of cytokine signaling-1/3（SOCS-1/3） in renal tubular epithelial cells induced by high glucose（HG） and to investigate their significance. Methods The renal tubular epithelial cell line（HKCs） cultured in vitro were divided into blank control group,HG group,and Janus kinase 2 inhibitor AG490 group.HKC of blank control group was cultured for 8 hours in 5.5 mmol/L glucose,and the other two groups were cultured in 300.0 mmol/L glucose or 300.0 mmol/L glucose＋10 μmol/L AG490 for 2,4,6,12,24,48 hours（n=6）.The morphology and ultrastructure were observed with inversion microscope and electron microscope at different time points.Protein expression of SOCS-1/3 was assayed by immunocytochemistry and Western blotting;SOCS-1/3 mRNA was assessed by reverse transcription-polymarese chain reaction（RT-PCR）. Results Under inversion microscope it was showed that 12 hours after being cultured with HG,the cells assumed a spindle-shape,with irregular protrusions,and cellular membrane became indistinguishable with prolongation of time,with increase of intracellular granules.Under the electron microscope,it was seen that there was distinct decrease in microvilli on the cell membrane and mitochondria,with an increase in rough endoplamic reticulum.The cellular changes were not obvious in AG490 group.Furthermore,immunocytochemistry and Western blotting showed that the immunoreactivity was localized in the cytoplasm as well as in the nuclei,and there was basic expression of SOCS-1/3 protein in normal HKC（0.218±0.023,0.337±0.009）.HG was shown to induce up-regulation of the expression of SOCS-1/3 protein at 4,6,12,24 hours compared with blank control group.The expression of SOCS-1 was highest at 4 hours（1.022±0.072）,and that of SOCS-3 was highest at 6 hours（1.256±0.105,both P〈0.01）,while the expression of SOCS-1/3 protein in AG490 group was lower than that in HG group（4 hours SOCS-1：0.589±0.167,6 hours SOCS-3：0.656±0.075,both P〈0.05）.However,HG induced a higher expression of SOCS-1/3 mRNA at 2,4,6,12 hours compared with blank control group.The expression of SOCS-1 was highest at 4 hours（1.716±0.098 vs.0.475±0.045,P〈0.05）,and that of SOCS-3 was highest at 6 hours（2.848±0.116 vs.0.749±0.086,P〈0.01）,while the expression of SOCS-1/3 mRNA in AG490 group was lower（4 hours SOCS-1：0.865±0.075,6 hours SOCS-3：0.923±0.116,both P〈0.05）. Conclusion HG could produce morphology and ultrastructure changes in renal tubular epithelial cell,and it induces up-regulation of SOCS-1/3 expression.These changes might be related with negative regulation of Janus kinase（JAK）/signal transducers and activators of transcription（STAT）/SOCS patyway.