慢病毒载体介导过表达和敲低miR-155的AB 8/13细胞株的建立

Establishment of overexpression and knockdown of miR-155 genes of AB 8/13 cell lines by lentivirus vector

目的 利用慢病毒载体构建稳定过表达miR-155的AB 8/13细胞株(AB 8/13-LV-has-miR-155)和稳定敲低miR-155的AB 8/13细胞株(AB 8/13-LV-has-miR-155p inhibiton),为研究miR-155在足细胞焦亡和肾脏疾病中的作用奠定基础。方法 体外培养AB 8/13细胞,使用不同浓度嘌呤霉素刺激AB 8/13细胞48 h,在光学显微镜下观察AB 8/13细胞的死亡情况;使用LV-has-miR-155阴性对照病毒、LV-has-miR-155-5p inhibiton阴性对照病毒分别感染AB 8/13细胞,在荧光显微镜下观察AB 8/13细胞的状态和感染效率;在嘌呤霉素筛选出稳定细胞株后,通过实时荧光定量PCR法检测稳定细胞株的miR-155表达量。结果 在3.5μg/mL及以上浓度的嘌呤霉素作用于AB 8/13细胞48 h后,所有细胞全部死亡。LV-has-miR-155慢病毒感染AB 8/13细胞的感染效率达到80%的最佳条件为在含HiTransG P的完全培养基中进行感染,MOI为10;LV-has-miR-155-5p inhibiton慢病毒感染AB 8/13细胞的感染效率达到80%的最佳条件为在含HiTransG A的完全培养基中进行感染,MOI为100。qRT-PCR结果显示AB 8/13-LV-has-miR-155细胞株的miR-155表达量明显升高,AB 8/13-LV-has-miR-155p inhibiton细胞株的miR-155表达量降低。结论 成功构建AB 8/13-LV-has-miR-155稳定细胞株和AB 8/13-LV-has-miR-155p inhibiton稳定细胞株,为进一步探究miR-155在人足细胞焦亡中的作用机制奠定基础。

Objective To construct the stable overexpression of miR-155 of AB 8/13 cell line(AB 8/13-LV-has-miR-155) and stable knockdown of miR-155 of AB 8/13 cell line(AB 8/13-LV-has-miR-155 p inhibiton) by lentivirus vector, so as to lay a foundation for the study of the role of miR-155 in podocyte pyroptosis and kidney diseases. Methods AB 8/13 cells were cultured in vitro and stimulated with puromycin at different concentrations for 48 hours, and then the death of AB 8/13 cells was observed under optical microscope. After AB 8/13 cells were respectively infected with LV-has-miR-155 negative control virus and LV-has-miR-155 p inhibiton negative control virus, the state and infection efficiency of AB 8/13 cells were observed under fluorescence microscope. After the stable cell lines were screened by puromycin, the expression of miR-155 in the stable cell line was detected by real-time fluorescence quantitative PCR. Results After being treated with puromycin at the concentration of 3.5 μg/mL or above for 48 hours, all AB 8/13 cells died. The optimal conditions for AB 8/13 cells infected with LV-has-miR-155 lentivirus to achieve 80% infectious efficiency were as follows: infection was performed in complete medium containing HiTransG P, and MOI was 10. The optimal conditions for AB 8/13 cells infected with LV-has-miR-155 p inhibiton lentivirus to achieve 80% infectious efficiency were as follows: infection was performed in complete medium containing HiTransG A, and MOI was 100. QRT-PCR results showed that the expression of miR-155 in AB 8/13-LV-has-miR-155 cell line increased significantly, and the expression of miR-155 in AB 8/13-LV-has-miR-155 inhibiton cell line decreased. Conclusion To successfully construct AB 8/13-LV-has-miR-155 stable cell lines and AB 8/13-LV-has-miR-155 p inhibiton stable cell lines can lay a foundation for further exploring the mechanism of miR-155 in human podocyte pyroptosis.