高糖环境对人肾小球系膜细胞表达结缔组织生长因子及其受体蛋白的影响

Effects of high glucose on expressions of connective tissue growth factor and its receptor in cultured human mesangial cells

目的探讨高糖环境对体外培养的人肾小球系膜细胞(HMC)表达结缔组织生长因子(CTGF)及其受体——低密度脂蛋白受体相关蛋白(LRP)的影响以及相应的信号机制。方法以体外培养的HMC为研究对象,采用双抗体夹心酶联免疫吸附法(ABC-ELISA)测定正常糖组(NG组)、高糖组(HG组)和甘露醇组(MG组)培养液中CTGF、人磷酸化细胞外信号调节激酶(pERK)及LPR的浓度变化,分别观察高糖环境对系膜细胞CTGF蛋白表达、丝裂素激活蛋白激酶(MAPKS)信号通路的活化以及CTGF受体LRP表达的影响。结果 HMC本身存在基础水平的CTGF蛋白表达,HG组培养1 d后,CTGF蛋白表达升高,峰值出现在第3天,自第4天开始下降;而NG组、MG组的CTGF蛋白表达未随时间发生显著变化;高糖环境下HMC外信号调节激酶(ERK)出现动态活化:1 h即开始检测到pERK升高,峰值出现在第8至24小时,于48 h内降至基础水平,而NG组、MG组在各时点均未能检测到pERK表达;加入ERK活化特异抑制剂PD98059后发现高糖环境对HMC表达CTGF蛋白增加的影响被抵消;另外,高糖环境未影响LRP的表达。结论 高糖环境下HMC表达CTGF蛋白增加,但未增加CT-GF受体LRP的表达,初步明确CTGF的高表达是通过磷酸化激活ERK实现的。

Objective To explore effects of high glucose on expressions of connective tissue growth factor（CTGF） and its receptor-low density lipoprotein receptor related protein （ LRP）, and investigate the possible signaling pathway for modulation in cultured human mesangial ceils （HMC） exposed to high glucose. Methods Sandwich enzyme linked immunosorbent assay（ ELISA） was employed to determine concentration changes of CTGF, phospho-extracellular signal-regulated kinase（pERK） and LPR in HMC culture solution in the normal glucose group, the high glucose group and the mannitol glucose group. Effects of high glucose on expressions of CTGF protein and its receptor LRP, and activation of the mitogen activated protein kinase（MAPKS） signaling pathway were observed. Results Lower basal levels of CTGF protein were observed in cultured HMC. Higher levels of CTGF protein were detected in the high glucose group on the 1st day, reached a peak on the 3rd day（ P 〈0.05）, and then began to decrease on the 4th day（P 〉0.05）. The levels of CTGF in normal glucose and mannitol glucose groups＇did not notably change. A high glucose substratum induced the phosphorylation of ERK at the 1 st hour, reached a peak at 8 h, maintained active at 8-24 h, and then began to decrease to the basal level at 48 h. However, no expression of pERK was detected in the normal glucose and mannitol glucose groups. Effect of high glucose on expression of CTGF protein was counteracted by intercepting phosphorylation of ERK with PD98059 （ a specific ERK activation inhibitor ）. Also, high glucose did not affect expression of LRP prorein in each group. Conclusion High glucose influences expression of CTGF through the pERK-dependent signaling pathway in cultured HMC, while high glucose does not affect expression of LRP in HMC.