稳定过表达/下调表达miR-125b-5p Jurkat T 细胞株的建立及验证  

Establishment and validation of Jurkat T cell lines with stable overexpression or low expression of miR-125b-5p

作　　者：曹文婷[1] 罗雯[1] 刘鑫[1] 阿霄 谭晓智 袁李梅[1] 邓丹琪[1] CAO Wen-ting;LUO Wen;LIU Xin(Department of Dermatology, the Second Affiliated Hospital of Kunming Medical University,Kunming, Yunnan 650120, China)

机构地区：[1]昆明医科大学第二附属医院皮肤科

出　　处：《中华全科医学》2019年第10期1631-1633,1672,共4页Chinese Journal of General Practice

基　　金：国家自然科学基金项目(81460472,81660517,81860552);云南省医疗卫生单位内设研究机构科研项目(2017NS312)

摘　　要：目的构建稳定过表达/下调表达miR-125b-5p的Jurkat T细胞株,为miR-125b-5p在免疫性疾病中的发病机制研究奠定基础。方法扩增质粒,经293T细胞包装产生慢病毒颗粒,感染Jurkat T细胞,RT-qPCR检测miR-125b-5p表达情况,Western blotting检测下游靶基因UVRAG表达情况。采用单因素方差分析,2组之间比较应用LSD检验,P<0.05为差异具有统计学意义。结果 hsa-miR-125b-5p慢病毒过表达载体及对照转染Jurkat T细胞后均有绿色荧光表达,hsa-miR-125b-5p慢病毒干扰载体及对照转染Jurkat T细胞后均有红色荧光表达;RT-qPCR显示和对照组比较慢病毒过表达载体组miR-125b-5p表达水平明显增高(P<0.01),慢病毒干扰载体组miR-125b-5p表达水平明显降低(P<0.01),差异均有统计学意义。Western blotting显示和对照组比较,慢病毒过表达载体组下游靶基因UVRAG表达明显降低(P<0.001),慢病毒干扰载体组下游靶基因UVRAG表达明显升高(P<0.001),差异均有统计学意义。结论成功建立了稳定过表达/下调表达hsa-miR-125b-5p的Jurkat T细胞株。Objective To establish the Jurkat T cell line which stably overexpresses or down expresses miR-125 b-5 p. Methods The plasmid was amplified and packaged into 293 T cells to produce lentiviral particles. Jurkat T cells were infected with recombinant lentivirus. The expression level of miR-125 b-5 p in Jurkat T cells was detected by RT-qPCR. Expression level of target gene UVRAG was detected by Western blotting. All data were analyzed by one-way ANOVA with LSD test as post-hoc test. P<0.05 was regarded as statistically significant. Results The emition of green fluorescence was detected in both Jurkat T cells that transfected with lentivirus overexpress hsa-miR-125 b-5 p vector or control vector. Besides, the emition of red fluorescence was detected in both Jurkat T cells that transfected with lentivirus hsa-miR-125 b-5 p interferential vector and control vector. The RT-qPCR results showed that compared with the control vector group, the expression level of miR-125 b-5 p was significantly increased in Jurkat T cells which were transfected with hsa-miR-125 b-5 p lentivirus over expression vector(P<0.01). The expression level of miR-125 b-5 p was significantly decreased in Jurkat T cells which were transfected with hsa-miR-125 b-5 p lentivirus interferential vector(P<0.01). Western blotting results showed that compared with the control vector group, the protein level of UVRAG was significantly decreased in Jurkat T cells which were transfected with hsa-miR-125 b-5 p lentivirus over expression vector(P<0.001). The protein level of UVRAG was significantly increased in Jurkat T cells which were transfected with hsa-miR-125 b-5 p lentivirus interferential vector(P<0.001). Conclusion Jurkat T cell lines with stable overexpression or low expression of hsa-miR-125 b-5 p were successfully established.

关 键 词：慢病毒 JURKAT T细胞 miR-125b-5p 

分 类 号：R751[医药卫生—皮肤病学与性病学] R373[医药卫生—临床医学]