中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
12期
1061-1065
,共5页
王育蓉%孙辉%章欢%刘沛
王育蓉%孫輝%章歡%劉沛
왕육용%손휘%장환%류패
肝肾综合征%肿瘤坏死因子α%人肾小球系膜细胞%Ⅰ型1,4,5-三磷酸肌醇受体(IP3R1)%蛋白激酶Cα
肝腎綜閤徵%腫瘤壞死因子α%人腎小毬繫膜細胞%Ⅰ型1,4,5-三燐痠肌醇受體(IP3R1)%蛋白激酶Cα
간신종합정%종류배사인자α%인신소구계막세포%Ⅰ형1,4,5-삼린산기순수체(IP3R1)%단백격매Cα
Hepatorenal syndrome%TNF-α%Human mesangial cells%IP3 R1%PKCα
目的 探讨肿瘤坏死因子(TNF-α)对人肾小球系膜细胞(HMCs) IP3R1蛋白和mRNA表达的影响及PKC在此信号通路中的作用,以期为肝肾综合征(HRS)肾小球滤过率下降的发生机制提供理论依据.方法 用实时定量PCR和免疫印迹检测IP3R1 mRNA和蛋白表达的情况,并分别用蛋白激酶C(PKC)激动剂PMA、PKCα、PKCδ特异性抑制剂及HA-DN-PKCα质粒转染干预上述诱导实验检测IP3 R1的表达变化.此外,免疫印迹检测TNF-α对p-PKCα蛋白的表达影响.结果 TNF-α明显上调IP3 R1蛋白和mRNA表达.PMA、PKCα抑制剂Safingol和HA-DN-PKCα质粒瞬时转染预处理HMCs后,均能明显阻断TNF-α诱导IP3 R1蛋白的表达,而PKCδ抑制剂Rottlerin预处理后,对IP3 R1蛋白表达无明显影响.此外,TNF-α刺激HMCs,各不同时间组总PKCα蛋白表达无明显差异,而TNF-α刺激8 h p-PKCα蛋白表达明显增加.结论 TNF-α能上调人系膜细胞IP3 R1蛋白及mRNA的表达,TNF-α活化PKCα是TNF-α上调IP3 R1表达的重要上游信号.
目的 探討腫瘤壞死因子(TNF-α)對人腎小毬繫膜細胞(HMCs) IP3R1蛋白和mRNA錶達的影響及PKC在此信號通路中的作用,以期為肝腎綜閤徵(HRS)腎小毬濾過率下降的髮生機製提供理論依據.方法 用實時定量PCR和免疫印跡檢測IP3R1 mRNA和蛋白錶達的情況,併分彆用蛋白激酶C(PKC)激動劑PMA、PKCα、PKCδ特異性抑製劑及HA-DN-PKCα質粒轉染榦預上述誘導實驗檢測IP3 R1的錶達變化.此外,免疫印跡檢測TNF-α對p-PKCα蛋白的錶達影響.結果 TNF-α明顯上調IP3 R1蛋白和mRNA錶達.PMA、PKCα抑製劑Safingol和HA-DN-PKCα質粒瞬時轉染預處理HMCs後,均能明顯阻斷TNF-α誘導IP3 R1蛋白的錶達,而PKCδ抑製劑Rottlerin預處理後,對IP3 R1蛋白錶達無明顯影響.此外,TNF-α刺激HMCs,各不同時間組總PKCα蛋白錶達無明顯差異,而TNF-α刺激8 h p-PKCα蛋白錶達明顯增加.結論 TNF-α能上調人繫膜細胞IP3 R1蛋白及mRNA的錶達,TNF-α活化PKCα是TNF-α上調IP3 R1錶達的重要上遊信號.
목적 탐토종류배사인자(TNF-α)대인신소구계막세포(HMCs) IP3R1단백화mRNA표체적영향급PKC재차신호통로중적작용,이기위간신종합정(HRS)신소구려과솔하강적발생궤제제공이론의거.방법 용실시정량PCR화면역인적검측IP3R1 mRNA화단백표체적정황,병분별용단백격매C(PKC)격동제PMA、PKCα、PKCδ특이성억제제급HA-DN-PKCα질립전염간예상술유도실험검측IP3 R1적표체변화.차외,면역인적검측TNF-α대p-PKCα단백적표체영향.결과 TNF-α명현상조IP3 R1단백화mRNA표체.PMA、PKCα억제제Safingol화HA-DN-PKCα질립순시전염예처리HMCs후,균능명현조단TNF-α유도IP3 R1단백적표체,이PKCδ억제제Rottlerin예처리후,대IP3 R1단백표체무명현영향.차외,TNF-α자격HMCs,각불동시간조총PKCα단백표체무명현차이,이TNF-α자격8 h p-PKCα단백표체명현증가.결론 TNF-α능상조인계막세포IP3 R1단백급mRNA적표체,TNF-α활화PKCα시TNF-α상조IP3 R1표체적중요상유신호.
Objective To explore the effects of TNF-α on the expression of IP3 R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the role of protein kinase C (PKC) in this signal pathway.Methods Quantitative real-time polymerase chain reaction and immunoblot assay were used to examine the effects of TNF-α on IP3R1 mRNA and protein expression.Depletion PKC,the selective inhibitor of PKCα Safingol and inhibitor of PKCδ Rottlerin,overexpression of dominant negative mutant of PKC to examine the mechanism of signal transduction of TNF-α-regulated IP3 R1 in HMCs.PKCα activation was assayed by Western blot.Results TNF-α increased IP3R1 mRNA and protein expression in HMCs,effects that were blocked by prolonged incubted chronic PMA,Safingol and also by domain negative PKCα construct.TNF-α promoted PKCα activation with maximal PKCα phosphorylation that occurred 8 h post-stimulation.Conclusion TNF-α increased the expression of IP3 R1 and this was mediated through the PKCα activation signaling pathways in HMCs.
