肾细胞共培养体系中miR-29b对OPN/TGF-β通路的调控作用及高糖环境下该通路的变化  

Regulatory effect of miR-29b on OPN/TGF-βpathway and the change of this pathway in a high glucose environment in a renal cell co-culture system

作　　者：刘娟 杨明正 李肖瑛 姬婷婷 熊晓燕 朱迎春 白寿军 LIU Juan;YANG Ming-zheng;LI Xiao-ying;JI Ting-ting;XIONG Xiao-yan;ZHU Ying-chun;BAI Shou-jun(Department of Nephrology,Qingpu Branch of Zhongshan Hospital,Fudan Univerisity,Shanghai 201700,China)

机构地区：[1]复旦大学附属中山医院青浦分院肾内科,上海201700

出　　处：《复旦学报（医学版）》2024年第6期921-930,共10页Fudan University Journal of Medical Sciences

基　　金：上海市自然科学基金(21ZR1413900);复旦大学附属中山医院青浦分院院级基金(QYM2020-05)。

摘　　要：目的建立模拟肾脏屏障系统的肾细胞共培养体系,探讨miR-29b-3p对骨桥蛋白(osteopontin,OPN)/转化生长因子β(transforming growth factor-β,TGF-β)通路的调控作用及高糖环境下该通路的变化。方法建立肾小管上皮细胞、肾小球系膜细胞和肾足细胞共培养体系,测试不同葡萄糖浓度(5、8、12、16 mmol/L)下的细胞活性和糖消耗值,并测定细胞上清中TGF-β和OPN的含量。转染表达OPN的重组质粒或OPN siRNA,采用Q-PCR和Western blot方法检测TGF-β和OPN的表达。结果在细胞共培养体系中,高糖(12 mmol/L)时OPN、TGF-β和miR-29b的mRNA表达均明显增加。Western blot结果显示,高糖时OPN蛋白增加,而TGF-β蛋白变化并不明显;加入miR-29b-3p激活剂后,细胞上清中OPN和TGF-β的mRNA水平明显增加;加入miR-29b-3p抑制剂后,细胞上清中OPN和TGF-β的mRNA水平明显下降。在基础糖浓度(5 mmol/L)下,miR-29b-3p激活剂使OPN和TGF-β的蛋白表达增加;miR-29b-3p抑制剂使OPN和TGF-β的蛋白表达减少。转染OPN重组质粒后,细胞上清中TGF-β蛋白表达增加,细胞中的OPN和TGF-β的mRNA表达明显增加;转染OPN siRNA至细胞后,细胞上清中TGF-β蛋白减少,细胞中的OPN mRNA表达明显减少,而TGF-βmRNA表达未见明显上升。结论肾细胞共培养体系可模拟体内肾脏环境,高糖诱导可抑制细胞增殖,使糖消耗增加,细胞上清中TGF-β含量增加,在该环境下miR-29b-3p对OPN/TGF-β信号通路具有调控作用。Objective To establish a renal cell co-culture system to simulate the renal barrier system,and to test its responsiveness to different glucose concentrations,and to investigate the regulatory effect of miR-29b-3p on osteopontin(OPN)/transforming growth factorβ(TGF-β)pathway and the changes of this pathway under high glucose condition.Methods The three-cell co-culture system consisting of human renal podocytes,human glomerular mesangial cells and human renal tubular epithelial cells was established to test the cell viability and glucose consumption value at glucose concentrations of 5,8,12 and 16 mmol/L.The content of TGF-βand OPN in cell supernatant was measured.The recombinant plasmid and siRNA of OPN were transfected,and the expressions of TGF-βand OPN were detected by Q-PCR and Western blot.Results The mRNA expressions of OPN,TGF-βand miR-29b were significantly increased at 12 mmol/L glucose conditions.Western blot results showed that the protein expression of OPN increased in high glucose conditions,while the protein expression of TGF-βdid not change significantly.After adding miR-29b-3p activator,the mRNA levels of OPN and TGF-βin the cell supernatant were significantly increased.After adding miR-29b-3p inhibitor,the mRNA levels of OPN and TGF-βin the cell supernatant were significantly decreased.Western blot results showed that compared with 5 mmol/L glucose,the protein expressions of OPN and TGF-βwere increased by miR-29b-3p activator,and the protein expressions of OPN and TGF-βwere decreased by miR-29b-3p inhibitor.After transfection with OPN recombinant plasmid,the content of TGF-βin the cell supernatant was significantly increased,and the mRNA expressions of OPN and TGF-βin the cells were significantly increased.After transfection with OPN siRNA,the content of TGF-βin the cell supernatant was decreased,and the expression of OPN mRNA in the cells was significantly decreased,but the expression of TGF-βmRNA was not significantly increased.Conclusion The renal cell co-culture system can mimic

关 键 词：糖尿病肾病(DN) 肾细胞 miR-29b-3p 骨桥蛋白(OPN) 转化生长因子β(TGF-β) 高糖 共培养 

分 类 号：Q291[生物学—细胞生物学] R692[医药卫生—泌尿科学]