重庆医学
重慶醫學
중경의학
Chongqing Medicine
2015年
26期
3601-3604
,共4页
呼吸窘迫综合征,成人%rictor 基因%慢病毒属
呼吸窘迫綜閤徵,成人%rictor 基因%慢病毒屬
호흡군박종합정,성인%rictor 기인%만병독속
respiratory distress syndrome,adult%rictor gene%lentivirus
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。
目的：構建針對 mTORC2特異組成蛋白中 rictor 基因的重組慢病毒沉默載體,研究其對 mTORC2/SGK1信號通路的調控機製及其對肺泡上皮細胞鈉離子通道的影響,併探討其在急性呼吸窘迫綜閤徵及急性肺損傷中的作用。方法構建目的基因的 rictor 榦擾質粒及空載質粒併與慢病毒包裝體繫共轉染293T 細胞,收集病毒上清液,經離心、濃縮及純化穫取重組慢病毒。測定病毒滴度,感染 A549細胞併篩選細胞穩定株,RT-PCR 驗證目的基因 rictor 沉默情況。採用 PCR 和 western blot 檢測該通路中各信號指標錶達情況。結果成功構建沉默 rictor 基因的重組慢病毒併感染 A549細胞和穫得細胞穩定株。與空白組及對照組相比,shRNA-rictor 組中 rictor 和下遊 SGK1、α-ENaC、β-ENaC、γ-ENaC mRNA 水平均有明顯下降(P <0.05)。同時, shRNA-rictor 組中 rictor 和下遊 SGK1、P-SGK、α-ENaC、β-ENaC、γ-ENaC 的蛋白水平較前兩組均有明顯下降(P <0.05)。結論沉默 rictor 基因對 mTORC2/SGK1信號通路有明顯的調控作用,同時從基因和蛋白水平影響肺泡上皮細胞α-ENaC、β-ENaC、γ-ENaC 的錶達。mTORC2/SGK1可能是調控肺泡上皮細胞對肺泡液的清除能力,同時影響肺水腫形成的重要信號通路。
목적：구건침대 mTORC2특이조성단백중 rictor 기인적중조만병독침묵재체,연구기대 mTORC2/SGK1신호통로적조공궤제급기대폐포상피세포납리자통도적영향,병탐토기재급성호흡군박종합정급급성폐손상중적작용。방법구건목적기인적 rictor 간우질립급공재질립병여만병독포장체계공전염293T 세포,수집병독상청액,경리심、농축급순화획취중조만병독。측정병독적도,감염 A549세포병사선세포은정주,RT-PCR 험증목적기인 rictor 침묵정황。채용 PCR 화 western blot 검측해통로중각신호지표표체정황。결과성공구건침묵 rictor 기인적중조만병독병감염 A549세포화획득세포은정주。여공백조급대조조상비,shRNA-rictor 조중 rictor 화하유 SGK1、α-ENaC、β-ENaC、γ-ENaC mRNA 수평균유명현하강(P <0.05)。동시, shRNA-rictor 조중 rictor 화하유 SGK1、P-SGK、α-ENaC、β-ENaC、γ-ENaC 적단백수평교전량조균유명현하강(P <0.05)。결론침묵 rictor 기인대 mTORC2/SGK1신호통로유명현적조공작용,동시종기인화단백수평영향폐포상피세포α-ENaC、β-ENaC、γ-ENaC 적표체。mTORC2/SGK1가능시조공폐포상피세포대폐포액적청제능력,동시영향폐수종형성적중요신호통로。
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 chan-nel,as well as the role in acute respiratory distress syndrome(ARDS)and acute lung injury.Methods The interfering vector plas-mid and empty vector plasmid of target gene rictor were constructed,which and the lentivirus packaging system were co-transfected to 293T cells.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 SGK1 andα-,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 cellular α-,β-and γ-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.
