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
2014年
7期
534-540
,共7页
王育蓉%李艳艳%卢仁隆%孙辉
王育蓉%李豔豔%盧仁隆%孫輝
왕육용%리염염%로인륭%손휘
肝肾综合征(HRS)%肿瘤坏死因子α(TNF-α)%人肾小球系膜细胞(HMCs)%Ⅰ型1,4,5-三磷酸肌醇受体(IP3R1)%转录因子Sp1
肝腎綜閤徵(HRS)%腫瘤壞死因子α(TNF-α)%人腎小毬繫膜細胞(HMCs)%Ⅰ型1,4,5-三燐痠肌醇受體(IP3R1)%轉錄因子Sp1
간신종합정(HRS)%종류배사인자α(TNF-α)%인신소구계막세포(HMCs)%Ⅰ형1,4,5-삼린산기순수체(IP3R1)%전록인자Sp1
Hepatorenal syndrome%TNF-α%Human mesangial cells%IP3R1%Sp1
目的探讨转录因子Sp1在肿瘤坏死因子（ TNF-α）上调人肾小球系膜细胞（ HMCs ） IP3R1表达中的作用，进一步阐明肝肾综合征（ HRS）肾小球滤过率（ GFR）下降的分子机制。方法用实时定量PCR和免疫印迹检测TNF-α刺激HMCs后，IP3R1 mRNA、IP3R1蛋白及Sp1蛋白表达的情况，重组质粒PGL3-IP3R1 promoter瞬时转染HMCs，应用荧光素酶报告基因检测TNF-α对IP3R1启动子活性的影响；应用Sp1和DNA结合抑制剂光辉霉素（ Mithramycin A ）及Sp1-siRNA质粒瞬时转染预处理HMCs后，检测IP3R1蛋白表达的变化。此外，免疫印迹法检测肿瘤坏死因子受体（ TNFR）对Sp1的活化及IP3R1蛋白表达的影响。结果 TNF-α作用HMCs后，IP3R1 mRNA、IP3R1蛋白和Sp1蛋白表达均明显增加，TNF-α显著增强IP3R1基因启动子的活性，Mithramycin A呈剂量依赖性阻断TNF-α诱导IP3R1蛋白表达上调，Sp1-siRNA预处理HMCs后，IP3R1蛋白表达明显下降。 TNFR1抗体＋TNF-α组和TNFR2抗体＋TNF-α组，IP3R1蛋白表达均有不同程度的下降。 TNFR1抗体＋TNF-α组Sp1蛋白表达明显下降，而TNFR2抗体＋TNF-α组，Sp1蛋白表达无明显变化。结论 TNF-α能上调人系膜细胞IP3R1的表达，TNF-α可能通过TNFR1/Sp1上调IP3R1的表达。
目的探討轉錄因子Sp1在腫瘤壞死因子（ TNF-α）上調人腎小毬繫膜細胞（ HMCs ） IP3R1錶達中的作用，進一步闡明肝腎綜閤徵（ HRS）腎小毬濾過率（ GFR）下降的分子機製。方法用實時定量PCR和免疫印跡檢測TNF-α刺激HMCs後，IP3R1 mRNA、IP3R1蛋白及Sp1蛋白錶達的情況，重組質粒PGL3-IP3R1 promoter瞬時轉染HMCs，應用熒光素酶報告基因檢測TNF-α對IP3R1啟動子活性的影響；應用Sp1和DNA結閤抑製劑光輝黴素（ Mithramycin A ）及Sp1-siRNA質粒瞬時轉染預處理HMCs後，檢測IP3R1蛋白錶達的變化。此外，免疫印跡法檢測腫瘤壞死因子受體（ TNFR）對Sp1的活化及IP3R1蛋白錶達的影響。結果 TNF-α作用HMCs後，IP3R1 mRNA、IP3R1蛋白和Sp1蛋白錶達均明顯增加，TNF-α顯著增彊IP3R1基因啟動子的活性，Mithramycin A呈劑量依賴性阻斷TNF-α誘導IP3R1蛋白錶達上調，Sp1-siRNA預處理HMCs後，IP3R1蛋白錶達明顯下降。 TNFR1抗體＋TNF-α組和TNFR2抗體＋TNF-α組，IP3R1蛋白錶達均有不同程度的下降。 TNFR1抗體＋TNF-α組Sp1蛋白錶達明顯下降，而TNFR2抗體＋TNF-α組，Sp1蛋白錶達無明顯變化。結論 TNF-α能上調人繫膜細胞IP3R1的錶達，TNF-α可能通過TNFR1/Sp1上調IP3R1的錶達。
목적탐토전록인자Sp1재종류배사인자（ TNF-α）상조인신소구계막세포（ HMCs ） IP3R1표체중적작용，진일보천명간신종합정（ HRS）신소구려과솔（ GFR）하강적분자궤제。방법용실시정량PCR화면역인적검측TNF-α자격HMCs후，IP3R1 mRNA、IP3R1단백급Sp1단백표체적정황，중조질립PGL3-IP3R1 promoter순시전염HMCs，응용형광소매보고기인검측TNF-α대IP3R1계동자활성적영향；응용Sp1화DNA결합억제제광휘매소（ Mithramycin A ）급Sp1-siRNA질립순시전염예처리HMCs후，검측IP3R1단백표체적변화。차외，면역인적법검측종류배사인자수체（ TNFR）대Sp1적활화급IP3R1단백표체적영향。결과 TNF-α작용HMCs후，IP3R1 mRNA、IP3R1단백화Sp1단백표체균명현증가，TNF-α현저증강IP3R1기인계동자적활성，Mithramycin A정제량의뢰성조단TNF-α유도IP3R1단백표체상조，Sp1-siRNA예처리HMCs후，IP3R1단백표체명현하강。 TNFR1항체＋TNF-α조화TNFR2항체＋TNF-α조，IP3R1단백표체균유불동정도적하강。 TNFR1항체＋TNF-α조Sp1단백표체명현하강，이TNFR2항체＋TNF-α조，Sp1단백표체무명현변화。결론 TNF-α능상조인계막세포IP3R1적표체，TNF-α가능통과TNFR1/Sp1상조IP3R1적표체。
Objective To investigate the effects of specific protein 1 ( Sp1 ) on the TNF-αin-duced expression of inositol 1, 4, 5 trisphosphate receptor type 1 ( IP3R1 ) in human mesangial cells ( HMCs) and to further elucidate the molecular mechanism regarding the decreased glomerular filtration rate ( GFR ) during hepatorenal syndrome .Methods Quantitative real-time polymerase chain reaction and Western blot assay were used to analyze the effects of TNF-αon the expression of IP3R1 at mRNA level and the expression of IP3R1 and Sp1 at protein level in HMCs , respectively.HMCs were transfected with a re-combinant plasmid PGL3-IP3R1 promoter to determine the effects of TNF-αon the activity of IP3R1 promot-er.HMCs were treated with Mithramycin A , an inhibitor of Sp1 binding, and transfected with Sp1-siRNA plasmid respectively to evaluate the expression of IP 3R1 regulated by TNF-α.The role of TNFR1 and TNFR2 in the TNF-αinduced expression of Sp 1 and IP3R1 proteins were detected by Western blot .Results TNF-αincreased the expression of IP3R1 at mRNA level and the expression of IP3R1 and Sp1 at protein lev-el in HMCs.Moreover, the activity of IP3R1 promoter in HMCs was remarkably increased by TNF-αas well.TNF-αinduced expression of IP3R1 was inhibited by Mithramycin A in a concentration dependent manner.HMCs transfected with Sp1-siRNA plasmid showed a significantly decreased expression of IP 3R1 protein.Both anti-TNFR1 and anti-TNFR2 antibodies blocked the TNF-αinduced IP3R1 expression, while only anti-TNFR1 antibodies inhibited the TNF-αinduced Sp1 expression.Conclusion TNF-αmight in-crease the expression of IP3R1 through the TNFR1/Sp1 signaling pathways in HMCs .
