安徽医科大学学报2024，Vol.59Issue(2)：193-199,7.DOI:10.19405/j.cnki.issn1000-1492.2024.02.002
采用慢病毒载体干扰TRAF2表达的MH7A细胞稳转株的构建及意义
Significance and successful construction of stable transfection of MH7A cells with lower TRAF2 expression using lentiviral vector
摘要
Abstract
Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 µ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.关键词
类风湿关节炎/MH7A/肿瘤坏死因子受体相关因子2/慢病毒载体Key words
rheumatoid arthritis/MH7A/tumor necrosis factor receptor related factor 2/lentivirus vector分类
医药卫生引用本文复制引用
陈露颖,蒋励萍,王伟康,左书俊,蒯佳婕,马旸,韩陈陈,魏伟..采用慢病毒载体干扰TRAF2表达的MH7A细胞稳转株的构建及意义[J].安徽医科大学学报,2024,59(2):193-199,7.基金项目
国家自然科学基金(编号:82104187、82173824、81973332) （编号:82104187、82173824、81973332）
安徽省自然科学基金(编号:2108085QH382) （编号:2108085QH382）
中国博士后科学基金(编号:2021T140002、2021M700184) （编号:2021T140002、2021M700184）
安徽省博士后基金(编号:2020B430) （编号:2020B430）
