| 稳定过表达/下调表达miR-125b-5p Jurkat T细胞株的建立及验证 |
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| 引用本文： | 曹文婷,罗雯,刘鑫,阿霄,谭晓智,袁李梅,邓丹琪.稳定过表达/下调表达miR-125b-5p Jurkat T细胞株的建立及验证[J].中华全科医学,2019,17(10):1631. |
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| 作者姓名： | 曹文婷 罗雯 刘鑫 阿霄 谭晓智 袁李梅 邓丹琪 |
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| 作者单位： | 昆明医科大学第二附属医院皮肤科, 云南 昆明 650101 |
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| 基金项目： | 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) |
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| 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。
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| 关 键 词： | 慢病毒 Jurkat T细胞 miR-125b-5p |
| 收稿时间： | 2019-03-16 |
Establishment and validation of Jurkat T cell lines with stable overexpression or low expression of miR-125b-5p |
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| Affiliation: | Department of Dermatology, the Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650120, China |
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| Abstract: | Objective To establish the Jurkat T cell line which stably overexpresses or down expresses miR-125b-5p. Methods The plasmid was amplified and packaged into 293T cells to produce lentiviral particles. Jurkat T cells were infected with recombinant lentivirus. The expression level of miR-125b-5p 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-125b-5p vector or control vector. Besides, the emition of red fluorescence was detected in both Jurkat T cells that transfected with lentivirus hsa-miR-125b-5p interferential vector and control vector. The RT-qPCR results showed that compared with the control vector group, the expression level of miR-125b-5p was significantly increased in Jurkat T cells which were transfected with hsa-miR-125b-5p lentivirus over expression vector (P<0.01). The expression level of miR-125b-5p was significantly decreased in Jurkat T cells which were transfected with hsa-miR-125b-5p 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-125b-5p 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-125b-5p lentivirus interferential vector (P<0.001). Conclusion Jurkat T cell lines with stable overexpression or low expression of hsa-miR-125b-5p were successfully established. |
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