Rab32促进树突状细胞内BCG增殖的实验研究

Effects of Rab32 on enhancing BCG proliferation in dendritic cells via PI3K/AKT pathway

目的探讨Rab32(Ras-related proteins in brain 32)在树突状细胞(dendritic cells,DCs)内卡介苗(Bacillus Calmette-Guérin,BCG)感染中的作用。方法通过小鼠DC2.4细胞建立BCG感染模型,通过RNA-Seq技术检测BCG感染后DCs内Rab基因的表达变化;通过生信分析筛选BCG感染后差异表达的Rab蛋白;western blot法检测BCG感染后DC2.4细胞内Rab32蛋白的表达情况;采用慢病毒转染技术制备EYFP-Rab32过表达DC2.4细胞(EYFP-Rab32 DC2.4细胞),采用western blot法和流式细胞术进行转染效率验证;通过细胞载菌量实验检测EYFP-Rab32 DC2.4细胞内BCG的增殖;采用western blot法检测磷脂酰肌醇-3-激酶(phosphoinositide 3-kinase,PI3K)/蛋白激酶(protein kinase B,AKT)通路蛋白的变化。结果DC2.4细胞中存在55种Rab蛋白,BCG感染后10种Rab蛋白的表达水平发生明显变化;Rab32蛋白随着感染时间的增加而增加,感染0 h和48 h时Rab32表达显著增加(P<0.05)。BCG感染EYFP-Rab32 DC2.4细胞48 h时BCG载菌量为(60.16±4.69)×10^(4)CFU/孔,较DC2.4细胞明显增多[(21.09±1.56)×10^(4)CFU/孔](P<0.01);这可能与Rab32过表达导致BCG感染DC2.4细胞后p-PI3K/PI3K,p-AKT/AKT增多(P<0.05),即Rab32激活PI3K/AKT通路相关。结论Rab32可能通过调控PI3K/AKT通路促进DCs内BCG增殖。

Objective To explore the effect of Ras-related proteins in brain 32(Rab32)on Bacillus Calmette-Gu rin(BCG)infection in dendritic cells(DCs).Methods In murine DC2.4 cell model of BCG infection,The Changes of Rab genes expression in DCs after BCG infection were detected by RNA-Seq.Differentially expressed Rabs after BCG infection were characterized using bioinformatics tools.Then Rab32 expression in DC2.4 cells with BCG infection was measured by western blot assay.EYFP-Rab32 overexpressing DC2.4 cells(EYFP-Rab32 DC2.4 cells)were prepared by lentivirus transfection and the transfection efficiency was detected by western blot assay and flow cytometry.The proliferation of BCG in EYFP-Rab32 DC2.4 cells was counted by bacterial load assay.The expression of PI3K/AKT pathway was measured by western blot assay.Results Fifty-five Rab proteins were present in DC2.4 cells,and the expression levels of 10 Rab proteins changed significantly after BCG infection.Rab32 increased with increasing duration of infection,with significant differences at 0 h and 48 h(P<0.05).BCG loading was(60.16±4.69)10^(4)CFU/well at 48 h in BCG-infected EYFP-Rab32 DC2.4 cells,which was significantly higher than DC2.4 cells[(21.09±1.56)10^(4)CFU/well](P<0.01).This may be associated with Rab32 overexpression leading to increased p-PI3K/PI3K,p-AKT/AKT in BCG-infected DC2.4 cells(P<0.05),meaning that Rab32 activated the PI3K/AKT pathway.Conclusion Rab32 may promote BCG proliferation in DCs by regulating the PI3K/AKT pathway.