雌激素对增殖期ATDC5细胞生长及C型利钠肽信号通路蛋白表达的影响
자격소대증식기ATDC5세포생장급C형리납태신호통로단백표체적영향
Estrogen stimulates cell proliferation and regulates the expression of proteins in C-type natriuretic peptide signaling pathway during chondrogenesis in ATDC5 cells
  • 万方数据
    中华儿科杂志 중화인과잡지
    Chinese Journal of Pediatrics
    2014年 8期 596-601 ,共6页

    郑丕媚%马华梅%苏喆%张璧涛%杜敏联

    정비미%마화매%소철%장벽도%두민련

    ATDC5细胞%雌二醇%利钠肽,C型%软骨发生 ATDC5細胞%雌二醇%利鈉肽,C型%軟骨髮生
    ATDC5세포%자이순%리납태,C형%연골발생
    ATDC5 cells%Estradiol%C-type natriuretic peptide%Chondrogenesis
    目的 观察雌二醇(E2)对增殖期ATDC5细胞的生长及C型利钠肽(CNP)信号通路蛋白[包括CNP、利钠肽B受体(NPR-B)及利钠肽C受体(NPR-C)]表达的影响.方法 以胰岛素(10 μg/ml)诱导分化第6天的ATDC5细胞为研究对象,观察E2对细胞生长的影响(MTT法)及CNP、NPR-B及NPR-C蛋白表达的影响(Western-blot法):(1)量效研究:分别加入7个不同浓度(10-11 ～10-5 mol/L)的E2(浓度组)及DMSO溶剂(对照组)共8组,比较作用24 h后各组细胞数吸光度(A)值及作用48 h后各组细胞的蛋白水平;(2)时效研究:加E2(10-8 mol/L)作用不同时间(0、24、48、72、96、120 h)(6组),除0h外,各时间点均设相应溶剂对照组.比较不同时间点细胞数与其对照组的差值;比较0 ～96 h细胞蛋白水平;(3)雌激素受体阻断试验:分别加入E2(10-8 mol/L)(E2组)、雌激素受体阻断剂ICI 182782(10-7 mol/L)(ICI组)、E2(10-8 mol/L)和ICI 182780(10-7mol/L)(E2+ ICI组)以及溶剂DMSO(对照组),比较作用24 h后4组细胞数.结果 (1)量效研究:①作用24 h后,与对照组(0.38±0.02)相比,随E2浓度增大,ATDC5细胞数呈增加趋势,至浓度为10-9和10-8 mol/L时最为显著(A值分别为0.56±0.06和0.52±0.02,P值分别＜0.05和＜0.01);②作用48 h后,与对照组相比,各浓度组细胞CNP、NPR-B及NPR-C蛋白水平均显著增加(P均＜0.01),CNP和NPR-B蛋白增加以10-10 mol/L组最为显著,NPR-C蛋白增加以10-9 mol/L组最为显著.(2)时效研究:①E2(10-8 mol/L)分别作用24 ～96 h后,各时间点细胞数均较对照组增多,48 h时最为显著(0.030±0.003),随后逐渐减少(P＜0.05或P＜0.01),至120 h时(0.007 ±0.001)与对照组差异无统计学意义.②CNP水平在24 h时显著增加(P＜0.05),48 h和72 h时有增加趋势,96 h时则有降低趋势.NPR-B和NPR-C蛋白水平在24 h时均有增加趋势(P分别为0.060和0.055),48 h则均有降低的趋势,至72 h和96 h时均低于对照组(P均＜0.05).(3)雌激素受体阻断试验:作用24 h时,E2组、ICI组、(E2 +ICI)组和对照组4组细胞数的差异有统计学意义(P＜0.05).E2组(0.470±0.032)和(E2+ICI)组(0.410±0.018)均多于对照组(0.370±0.011),E2组多于(E2+ ICI)组(P均＜0.05);ICI组(0.360-±0.035)与对照组差异无统计学意义.结论 E2能通过雌激素受体介导、以时效和量效作用方式促进增殖期ATDC5细胞生长.E2在一定浓度、不同作用时间时可以不同方式调节(上调或下调)CNP、NPR-B和NPR-C蛋白的表达,提示雌激素是CNP信号通路的调节因子之一.
    목적 관찰자이순(E2)대증식기ATDC5세포적생장급C형리납태(CNP)신호통로단백[포괄CNP、리납태B수체(NPR-B)급리납태C수체(NPR-C)]표체적영향.방법 이이도소(10 μg/ml)유도분화제6천적ATDC5세포위연구대상,관찰E2대세포생장적영향(MTT법)급CNP、NPR-B급NPR-C단백표체적영향(Western-blot법):(1)량효연구:분별가입7개불동농도(10-11 ～10-5 mol/L)적E2(농도조)급DMSO용제(대조조)공8조,비교작용24 h후각조세포수흡광도(A)치급작용48 h후각조세포적단백수평;(2)시효연구:가E2(10-8 mol/L)작용불동시간(0、24、48、72、96、120 h)(6조),제0h외,각시간점균설상응용제대조조.비교불동시간점세포수여기대조조적차치;비교0 ～96 h세포단백수평;(3)자격소수체조단시험:분별가입E2(10-8 mol/L)(E2조)、자격소수체조단제ICI 182782(10-7 mol/L)(ICI조)、E2(10-8 mol/L)화ICI 182780(10-7mol/L)(E2+ ICI조)이급용제DMSO(대조조),비교작용24 h후4조세포수.결과 (1)량효연구:①작용24 h후,여대조조(0.38±0.02)상비,수E2농도증대,ATDC5세포수정증가추세,지농도위10-9화10-8 mol/L시최위현저(A치분별위0.56±0.06화0.52±0.02,P치분별＜0.05화＜0.01);②작용48 h후,여대조조상비,각농도조세포CNP、NPR-B급NPR-C단백수평균현저증가(P균＜0.01),CNP화NPR-B단백증가이10-10 mol/L조최위현저,NPR-C단백증가이10-9 mol/L조최위현저.(2)시효연구:①E2(10-8 mol/L)분별작용24 ～96 h후,각시간점세포수균교대조조증다,48 h시최위현저(0.030±0.003),수후축점감소(P＜0.05혹P＜0.01),지120 h시(0.007 ±0.001)여대조조차이무통계학의의.②CNP수평재24 h시현저증가(P＜0.05),48 h화72 h시유증가추세,96 h시칙유강저추세.NPR-B화NPR-C단백수평재24 h시균유증가추세(P분별위0.060화0.055),48 h칙균유강저적추세,지72 h화96 h시균저우대조조(P균＜0.05).(3)자격소수체조단시험:작용24 h시,E2조、ICI조、(E2 +ICI)조화대조조4조세포수적차이유통계학의의(P＜0.05).E2조(0.470±0.032)화(E2+ICI)조(0.410±0.018)균다우대조조(0.370±0.011),E2조다우(E2+ ICI)조(P균＜0.05);ICI조(0.360-±0.035)여대조조차이무통계학의의.결론 E2능통과자격소수체개도、이시효화량효작용방식촉진증식기ATDC5세포생장.E2재일정농도、불동작용시간시가이불동방식조절(상조혹하조)CNP、NPR-B화NPR-C단백적표체,제시자격소시CNP신호통로적조절인자지일.
    Objective To investigate the effect of estrogen on cell proliferation and expression of proteins of C-type natriuretic peptide (CNP),natriuretic peptides B receptor (NPR-B) and natriuretic peptides C receptor (NPR-C) in ATDC5 cells during chondrogenesis.Method ATDC5 cells were induced for differentiation with insulin 10 μg/ml (day 0),and were started to be investigated on day 6.They were incubated with:(1) Estradiol (E2) at different concentrations (10-n-10-5 mol/L) for 24 hours (for studying cell proliferation),or for 48 hours (for studying CNP,NPR-B and NPR-C protein expression) ;(2) E2(10-s mol/L) for 24,48,72,96 and 120 h (for studying cell proliferation),or for 24,48,72 and 96 hours (for studying CNP,NPR-B and NPR-C protein expression) ; (3) E2 (10-8 mol/L),and/or ICI 182782 (estrogen receptor antagonist) (10-7 mol/L) for 24 hours (for studying cell proliferation).ATDC5 cells proliferation were determined by MTT (OD value).Western-blotting was performed to identify the protein levels of CNP,NPR-B and NPR-C.Result (1) After incubation with E2 (10-11-10-5 mol/L) for 24 h,ATD5 cell number increased with the increasing E2 concentration,peak in E2 concentrations of 10-9 and 10-8 mol/L (0.56 ±0.06 and 0.52 ±0.02,P ＜0.05 and ＜0.01,respectively),while significantly decreased in E2 (10-5 mol/L) (0.30 ±-0.02) compared with DMSO-control (0.38 ± 0.02) (P ＜ 0.05).After incubation with E2 (10-11-10-5 mol/L) for 48 h,the protein level of CNP,NPR-B and NPR-C increased significantly,with the greatest effect seen at a concentration of 10-10 mol/L E2 for CNP and NPRB,10-9 moL/L E2 for NPR-C (P ＜ 0.05).(2) After incubation with E2 (10-8 mol/L) for 24 to 96 hours:① The cell number in each of the four time points was significantly increased compared with DMSO-control,with the greatest effect in 48 h (0.030-± 0.003) (P ＜ 0.05 or ＜ 0.01,respectively).While the cell number at 120 h was similar to that in DMSO-control.② The protein level of CNP increased significantly at 24 h (P ＜ 0.05),seemed to be increased at 48 h and 72 h and decreased at 96 h.Both NPR-B and NPR-C level seemed to be increased at 24 h (P =0.060 and 0.055,respectively) and seemed to decrease at 48 h,with decreasing significantly at both 72 h and 96 h (P ＜ 0.05).(3) After incubation for 24 h,there was significant difference among the cell number of the four groups (P ＜ 0.05).Cell number of group E2 (0.470-±0.032) was increased compared with group (E2 + ICI) (0.410 ±0.018),both being increased compared with group DMSO-control (0.370-±0.011,P ＜0.05,respectively).There was no difference in cell number between group ICI 182782 (0.360 ± 0.035) and group DMSO-control.Conclusion E2 promotes the proliferation of ATDC5 cells i.e.chondrogenesis via estrogen receptor mediated mechanism,in both concentration-dependent and time-dependent manner.E2 (10-11-10-8 mol/L) up-regulates protein expression of CNP,NPR-B and NPR-C of ATDC5 cells during chondrogenesis,and regulate the expression of the three proteins mentioned above positively or negatively at different time point,which implied that estrogen is one of the regulators of CNP signaling pathway.
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