中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2013年
2期
153-157
,共5页
王育蓉%章欢%孙辉%刘沛
王育蓉%章歡%孫輝%劉沛
왕육용%장환%손휘%류패
肿瘤坏死因子-α%肝肾综合征%人肾小球系膜细胞%Ⅰ型1,4,5-三磷酸肌醇受体%蛋白激酶C-α%磷脂酰胆碱特异性磷脂酶C
腫瘤壞死因子-α%肝腎綜閤徵%人腎小毬繫膜細胞%Ⅰ型1,4,5-三燐痠肌醇受體%蛋白激酶C-α%燐脂酰膽堿特異性燐脂酶C
종류배사인자-α%간신종합정%인신소구계막세포%Ⅰ형1,4,5-삼린산기순수체%단백격매C-α%린지선담감특이성린지매C
TNF-α%Hepatorenal syndrome%HMCs%IP3R1%PKC-α%PC-PLC
目的 探讨肿瘤坏死因子(TNF-α)对人肾小球系膜细胞(HMCs)Ⅰ型1,4,5-三磷酸肌醇受体(IP3R1)蛋白和mRNA表达的影响及其分子机制,以期为肝肾综合征(HRS)肾小球滤过率下降的发生机制和防治思路提供理论依据.方法 用实时定量PCR和免疫印迹法检测TNF-α刺激HMCs 0、2、4、8、24 h的IP3R1mRNA和蛋白表达的情况.以TNF-α刺激HMCs 8 h为对照,应用D609,U73122,PP1,Safingol和Rottlerin预处理HMCs后,实时定量PCR法检测TNF-α对IP3R1 mRNA表达的影响；以TNF-α刺激HMCs 24 h组为对照,免疫印迹法检测上述抑制剂预处理后,TNF-α对IP3R1蛋白表达的影响.此外,应用非放射性测活法检测0、4、8、24 h TNF-α对PKC-α的活化影响,并予D609、Safingo干预后,检测TNF-α对PKC-α的活化的影响.结果 TNF-α刺激HMCs 2 h到8 h IP3R1mRNA表达明显增加,于8h达高峰(P＜0.01),而于24 h有所下降(P＜0.01)；而IP3 R1蛋白表达于TNF-α刺激4h开始升高,至24 h IP3R1蛋白升高达高峰(P＜0.01).与8h组比较,抑制剂+TNF-α各组中,Safingol+ TNF-α组和D609+ TNF-α组IP3R1mRNA的表达明显下降,差异具有统计学意义(3.30±0.81) vs.(1.95±0.13),P＜0.05； (2.10±0.49),P＜0.01.与24 h相比,Safingol+ TNF-α组和D609+ TNF-α组,IP3R1蛋白的表达亦明显下降(3.09±0.13) vs.(1.86 +0.39),P＜0.01；(1.98±0.02),P＜0.01.非放射性测活检测显示TNF-α处理8h使PKC-α自动磷酸化而活化,而D609或Safingol预处理后,可抑制TNF-α对PKC-α的活化.结论 TNF-α能上调IP3R1 mRNA及蛋白的表达,PC-PLC/PKC-α信号途径可能在TNF-α上调IP3R1的表达中起重要作用.
目的 探討腫瘤壞死因子(TNF-α)對人腎小毬繫膜細胞(HMCs)Ⅰ型1,4,5-三燐痠肌醇受體(IP3R1)蛋白和mRNA錶達的影響及其分子機製,以期為肝腎綜閤徵(HRS)腎小毬濾過率下降的髮生機製和防治思路提供理論依據.方法 用實時定量PCR和免疫印跡法檢測TNF-α刺激HMCs 0、2、4、8、24 h的IP3R1mRNA和蛋白錶達的情況.以TNF-α刺激HMCs 8 h為對照,應用D609,U73122,PP1,Safingol和Rottlerin預處理HMCs後,實時定量PCR法檢測TNF-α對IP3R1 mRNA錶達的影響；以TNF-α刺激HMCs 24 h組為對照,免疫印跡法檢測上述抑製劑預處理後,TNF-α對IP3R1蛋白錶達的影響.此外,應用非放射性測活法檢測0、4、8、24 h TNF-α對PKC-α的活化影響,併予D609、Safingo榦預後,檢測TNF-α對PKC-α的活化的影響.結果 TNF-α刺激HMCs 2 h到8 h IP3R1mRNA錶達明顯增加,于8h達高峰(P＜0.01),而于24 h有所下降(P＜0.01)；而IP3 R1蛋白錶達于TNF-α刺激4h開始升高,至24 h IP3R1蛋白升高達高峰(P＜0.01).與8h組比較,抑製劑+TNF-α各組中,Safingol+ TNF-α組和D609+ TNF-α組IP3R1mRNA的錶達明顯下降,差異具有統計學意義(3.30±0.81) vs.(1.95±0.13),P＜0.05； (2.10±0.49),P＜0.01.與24 h相比,Safingol+ TNF-α組和D609+ TNF-α組,IP3R1蛋白的錶達亦明顯下降(3.09±0.13) vs.(1.86 +0.39),P＜0.01；(1.98±0.02),P＜0.01.非放射性測活檢測顯示TNF-α處理8h使PKC-α自動燐痠化而活化,而D609或Safingol預處理後,可抑製TNF-α對PKC-α的活化.結論 TNF-α能上調IP3R1 mRNA及蛋白的錶達,PC-PLC/PKC-α信號途徑可能在TNF-α上調IP3R1的錶達中起重要作用.
목적 탐토종류배사인자(TNF-α)대인신소구계막세포(HMCs)Ⅰ형1,4,5-삼린산기순수체(IP3R1)단백화mRNA표체적영향급기분자궤제,이기위간신종합정(HRS)신소구려과솔하강적발생궤제화방치사로제공이론의거.방법 용실시정량PCR화면역인적법검측TNF-α자격HMCs 0、2、4、8、24 h적IP3R1mRNA화단백표체적정황.이TNF-α자격HMCs 8 h위대조,응용D609,U73122,PP1,Safingol화Rottlerin예처리HMCs후,실시정량PCR법검측TNF-α대IP3R1 mRNA표체적영향；이TNF-α자격HMCs 24 h조위대조,면역인적법검측상술억제제예처리후,TNF-α대IP3R1단백표체적영향.차외,응용비방사성측활법검측0、4、8、24 h TNF-α대PKC-α적활화영향,병여D609、Safingo간예후,검측TNF-α대PKC-α적활화적영향.결과 TNF-α자격HMCs 2 h도8 h IP3R1mRNA표체명현증가,우8h체고봉(P＜0.01),이우24 h유소하강(P＜0.01)；이IP3 R1단백표체우TNF-α자격4h개시승고,지24 h IP3R1단백승고체고봉(P＜0.01).여8h조비교,억제제+TNF-α각조중,Safingol+ TNF-α조화D609+ TNF-α조IP3R1mRNA적표체명현하강,차이구유통계학의의(3.30±0.81) vs.(1.95±0.13),P＜0.05； (2.10±0.49),P＜0.01.여24 h상비,Safingol+ TNF-α조화D609+ TNF-α조,IP3R1단백적표체역명현하강(3.09±0.13) vs.(1.86 +0.39),P＜0.01；(1.98±0.02),P＜0.01.비방사성측활검측현시TNF-α처리8h사PKC-α자동린산화이활화,이D609혹Safingol예처리후,가억제TNF-α대PKC-α적활화.결론 TNF-α능상조IP3R1 mRNA급단백적표체,PC-PLC/PKC-α신호도경가능재TNF-α상조IP3R1적표체중기중요작용.
Objective To explore the effects of TNF-α on the expression of IP33R1 mRNA and protein in human mesangial cells (HMCs) and elucidate the mechnism of TNF-α indnces the IP3R1 expression in the occurrence of hepatorenal syndrome (HRS).Methods HMCs was stimulated by tumor (TNF-α) with 100 ng/mL for different hours (2,4,8,24 hours).The expression change of IP3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblot assay.Several inhibitors including D609,U73122,PP1,Safingol,Rottlerin and non-radioactive PKC assay to examine the mechanism of signal transduction of TNF-α-regulated IP3R1 in HMCs.Results The levels of IP3R1 mRNA at 2 h post-TNF-α exposure were significantly enhanced and reached peak at 8 h in HMCs (P ＜ 0.01),then descened at 24 h (P ＜ 0.01).The levels of IP3R1 protein at 4 h post-TNF-α exposure were obviously increased and reached peak at 24 h post-TNF-α exposure (P ＜ 0.01).Compared with the control group,safingol (PKC-α inhibitor) and D609 (PC-PLC inhibitor) each significantly suppressed TNF-α-induced expression of IP3R1 mRNA (3.30 ± 0.81) vs.(1.95 ± 0.130,P ＜ 0.05 ; (2.10 ± 0.49),P ＜ 0.01 andIP3R1 protein (3.09±0.13) vs.(1.86+0.39),P＜0.01; (1.98±0.02),P＜0.01.TNF-αpromoted autophosphorylation,and hence the activation,of PKC-α with maximal phosphorylation that occurred 8 h post-stimulation measured by non-radioactive PKC assay,and the effect was marked attenuated by pretreated with D609 or safingol.Conclusions TNF-α increased the expression of IP3R1 and this was mediated,at least in part,through the PC-PLC/PKC-α signaling pathways in HMCs.
