摘要
目的基于生物信息学分析,筛选骨架蛋白Transgelin(SM22α)的上游调控基因-蛋白激酶B(Akt)和过氧化物酶体增殖物激活受体(PPAR),并通过构建过表达/敲除瞬时受体电位阳离子通道蛋白6(TRPC6)、Akt或PPAR的小鼠足细胞系(MPC5),结合小干扰RNA(siRNA)筛选,解析其对Transgelin的调控机制。方法本研究采用小鼠足细胞系(MPC5),通过siRNA技术特异性沉默TRPC6、Akt1和PPARα基因表达,建立嘌呤霉素氨基核苷(PAN)诱导的足细胞损伤模型。实验设置空白对照组、PAN处理组、基因沉默组(siRNA-TRPC6、siRNA-Akt1、siRNA-PPARα)及阴性对照组(siRNA-NC)。采用实时荧光定量聚合酶链式反应(qRT-PCR)检测Tagln及相关基因mRNA表达水平,蛋白质印迹法分析Transgelin、TRPC6、p-Akt和PPARα蛋白表达,并通过细胞骨架染色和流式细胞术评估细胞形态和凋亡情况。结果设计15种siRNA特异性沉默4个基因Akt1、Akt2、PPARα(phospho-S12)和TRPC6的表达。qRT-PCR验证显示,GAPDH、Akt1、Akt2、PPARα和TRPC6这5对引物的扩增效率符合实验要求(效率90%~110%,R^(2)>0.99)。结论引物满足基因表达分析要求,支持后续代谢-骨架机制研究。TRPC6异常提示需结合蛋白检测或替代基因编辑技术(如CRISPRi)以排除转录后调控干扰。
Objective Based on bioinformatics analysis,we screened the upstream regulatory genes of the cytoskeletal protein Transgelin(SM22α)-protein kinase B(Akt)and peroxisome proliferator-activated receptor(PPAR).By constructing mouse podocyte(MPC5)cell lines with overexpression or knockout of Transient receptor potential canonical 6(TRPC6),Akt or PPAR,combined with small interfering RNA(siRNA)screening,we elucidated their regulatory mechanisms on Transgelin.Methods In this study,we employed the mouse podocyte cell line(MPC5)and established a puromycin aminonucleoside(PAN)-induced podocyte injury model by specifically silencing TRPC6,Akt1,and PPARαgene expression using siRNA technology.The experimental groups included:blank control,PAN-treated group,gene-silenced groups(siRNA-TRPC6,siRNA-Akt1,siRNA-PPARα),and negative control(siRNA-NC).Gene expression levels of Tagln and related genes were measured by quantitative reverse transcription polymerase chain reaction(qRT-PCR),while protein expression of Transgelin,TRPC6,p-Akt,and PPARαwas analyzed via Western blotting.Additionally,cell morphology and apoptosis were assessed using cytoskeleton staining and flow cytometry.Results Fifteen siRNAs were designed to specifically silence the expression of four target genes:Akt1,Akt2,PPARα(phospho-S12),and TRPC6.qRT-PCR validation confirmed that all five primer pairs(GAPDH,Akt1,Akt2,PPARα,and TRPC6)met the experimental requirements,demonstrating amplification efficiencies of 90%-110%with R^(2)>0.99.Conclusion Primers meet the requirements of gene expression analysis and support subsequent metabolism-skeleton mechanism research.The abnormality of TRPC6 suggests that it is necessary to combine protein detection or alternative gene editing techniques(such as CRISPRi)to exclude post-transcriptional regulation interference.
作者
魏田力
李白虹
杨丽君
WEI Tian-li;LI Bai-hong;YANG Li-jun(Department of Pediatrics,Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China)
出处
《临床和实验医学杂志》
2025年第10期1009-1012,共4页
Journal of Clinical and Experimental Medicine
基金
国家自然科学基金资助项目(编号:81400718)
首都医科大学附属北京友谊医院启动基金(编号:Grant No.yyqdkt2018-21)。
