重组慢病毒沉默rictor基因对mTORC2/SGK1信号通路及肺泡上皮钠离子通道的调控作用

Regulating effect of recombinant lentivirus silencing rictor gene on mTORC2/SGK1 signal pathway and pulmonary alveolar epithelial sodium ion channel

目的构建针对mTORC2特异组成蛋白中rictor基因的重组慢病毒沉默载体,研究其对mTORC2/SGK1信号通路的调控机制及其对肺泡上皮细胞钠离子通道的影响,并探讨其在急性呼吸窘迫综合征及急性肺损伤中的作用。方法构建目的基因的rictor干扰质粒及空载质粒并与慢病毒包装体系共转染293T细胞,收集病毒上清液,经离心、浓缩及纯化获取重组慢病毒。测定病毒滴度,感染A549细胞并筛选细胞稳定株,RT-PCR验证目的基因rictor沉默情况。采用PCR和western blot检测该通路中各信号指标表达情况。结果成功构建沉默rictor基因的重组慢病毒并感染A549细胞和获得细胞稳定株。与空白组及对照组相比,shRNA-rictor组中rictor和下游SGK1、α-ENaC、β-ENaC、γ-ENaC mRNA水平均有明显下降(P<0.05)。同时,shRNA-rictor组中rictor和下游SGK1、P-SGK、α-ENaC、β-ENaC、γ-ENaC的蛋白水平较前两组均有明显下降(P<0.05)。结论沉默rictor基因对mTORC2/SGK1信号通路有明显的调控作用,同时从基因和蛋白水平影响肺泡上皮细胞α-ENaC、β-ENaC、γ-ENaC的表达。mTORC2/SGK1可能是调控肺泡上皮细胞对肺泡液的清除能力,同时影响肺水肿形成的重要信号通路。

Objective To construct recombinant lentivirus silence vector aiming at rictor gene in mTORC2 specific protein,and to investigate its regulation on mTORC2/SGK1 signal pathway and the effect on pulmonary alveolar epithelial sodium ion channel,as well as the role in acute respiratory distress syndrome（ARDS）and acute lung injury.Methods The interfering vector plasmid and empty vector plasmid of target gene rictor were constructed,which and the lentivirus packaging system were co-transfected to 293 Tcells.The viral supernatant was collected,centrifuged,concentrated and purified for obtaining recombinant lentivirus.The virus titer was detected and the virus was infected to A549 cells.Stable cell lines were screened.RT-PCR was used to confirm the silencing situation of target gene rictor.The expression situation of various signal indexes in this pathway was detected by PCR and Western blot.Results The recombinant lentivirus of silence gene rictor was successfully constructed and transfected to A549 cell for obtaining stable cell lines.Compared with blank and control groups,the mRNA levels of rictor,downstream SGK1andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased（P〈0.05）.Meanwhile,the protein levels of rictor,downstream SGK1,P-SGK andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased compared with the other two groups（P〈0.05）.Conclusion Silence rictor gene has the obvious regulation effect on mTORC2/SGK1 signal pathway,meanwhile affects the expression of pulmonary alveolar epithelial cellularα-,β-andγ-ENaC at gene and protein level.It is speculated that mTORC2/SGK1 may be an important signal pathway for regulating the clearance capacity of pulmonary alveolar epithelial cells on pulmonary alveolar fluid and simultaneously affecting the pulmonary edema formation.