雌激素对小鼠心脏成纤维细胞增殖的影响研究
发布时间：2019-03-05 14:25
【摘要】：目的:探讨雌激素抑制心脏成纤维细胞(cardiac fibroblasts,CFS)增殖是否与中电导钙激活钾通道(intermediate-conductance Ca2+-activated K+channel,KCa3.1)有关。方法:1、新生小鼠心脏成纤维细胞的培养与鉴定:取新生雄性小鼠,经手术取其心脏心室,酶消化差速贴壁法培养,特异性抗体荧光免疫法鉴别CFS。2、心脏成纤维细胞增殖模型的建立:培养的CFS加入以下浓度的AngII:10-4、10-5、10-6、10-7、10-8mol/L分别培养12h、24h、48h、72h后,并以不加AngII作对照组,使用MTT法检测CFS增殖情况。3、观察雌二醇(estradiol,E2)和KCa3.1通道特异性阻断剂TRAM-34对小鼠CFS增殖活性的影响:(1)对照组:AngII:10-6mol/L,无E2,无TRAM-34;(2)AngII+E2组:先使用浓度为10-6mol/L的AngII预处理30min后,分别加入10-5、10-6、10-7、10-8mol/L浓度的E2;(3)AngII+TRAM-34组:先使用浓度为10-6mol/L的AngII预处理30min后,分别加入10-4、10-5、10-6、10-7mol/L浓度的TRAM-34;各组均处理24、48、72小时后,使用MTT法检测细胞增殖情况。4、普通PCR检测KCa3.1mRNA表达情况,ELISA法检测p38-MAPK磷酸化水平:CFS预先用AngII(10-6mol/L)处理30min后备用,实验分组为:(1)正常对照组(无AngII,无E2,无TRAM-34);(2)AngII组;(3)AngII+E2×10-5mol/L组;(4)AngII+TRAM-34×10-4mol/L组孵育48h,MTT检测细胞增殖活性,普通PCR检测细胞KCa3.1mRNA的表达,ELISA检测细胞p38-MAPK的磷酸化水平。结果:AngII可诱导小鼠CFS增殖,并具有时间及浓度依赖性,与空白组对照,10-6、10-5、10-4mol/L浓度AngII增殖效果显著,具有统计学意义(P0.01);10-5、10-4mol/L浓度增殖效果与10-6mol/L比较无显著差异(P0.05),故选用10-6mol/L作为增殖模型的浓度;与AngII组比较,E2和TRAM-34对小鼠CFS的增殖活性抑制作用明显,并具有浓度及时间依赖性;10-5mol/LE2与10-4mol/LTRAM-34干预48h对CFS增殖作用的影响无显著差异(P0.05)。与正常对照组比较,AngⅡ组可显著提高KCa3.1mRNA表达,具有统计学意义(P0.01);与正常对照组比较,AngⅡ+E2×10-5 mol/L组和AngⅡ+TRAM-34×10-4mol/L组,均可显著抑制KCa3.1mRNA表达,并具有统计学意义(P0.05);与AngⅡ组比较,AngⅡ+E2×10-5 mol/L组和AngⅡ+TRAM-34×10-4mol/L组,均可显著抑制KCa3.1mRNA表达,并具有统计学意义(P0.01);AngⅡ+E2×10-5 mol/L组和AngⅡ+TRAM-34×10-4mol/L组比较无显著差异(P0.05)。正常对照组比较,AngⅡ能够显著提高p38-MAPK磷酸化水平,具有统计学意义(P0.01);与AngⅡ组比较,AngII+E2×10-5mol/L组能够显著降低p38-MAPK磷酸化水平,具有统计学意义(P0.01);与AngⅡ组比较,AngII+TRAM-34×10-4mol/L组对p38-MAPK磷酸化水平影响不显著(P0.05);AngII+E2×10-5mol/L组与AngII+TRAM-34×10-4mol/L组间有显著差异,具有统计学意义(P0.01)。结论:AngII可诱导小鼠CFS增殖;E2与TRAM-34均能抑制CFS增殖,10-5mol/LE2与10-4mol/LTRAM-34对CFS增殖作用的影响无显著差异;雌激素抑制心脏成纤维细胞增殖的机制可能是降低p38-MAPK磷酸化水平,从而抑制KCa3.1mRNA表达。
[Abstract]:Aim: to investigate whether estrogen inhibits the proliferation of cardiac fibroblasts (cardiac fibroblasts,CFS) and whether estrogen inhibits the proliferation of cardiac fibroblasts (cardiac fibroblasts,CFS) with medium conductance calcium activated potassium channel (intermediate-conductance Ca2-activated K channel,KCa3.1). Methods: 1. Culture and identification of cardiac fibroblasts in neonatal mice: the cardiac ventricles of newborn male mice were obtained by operation and cultured by enzyme digestion differential adherent method, and the specific antibody fluorescence immunoassay was used to identify CFS.2,. Establishment of cardiac fibroblasts proliferation model: cultured CFS was cultured with the following concentrations of AngII:10-4,10-5,10-6,10-7,10-8mol/L for 12 h, 24 h, 48 h, 72 h, and no AngII was used as the control group, and the cells were cultured for 12 h, 24 h, 48 h, 72 h, respectively. The proliferation of CFS was detected by MTT method. 3. The effects of estradiol (estradiol,E2) and KCa3.1 channel specific blocker TRAM-34 on the proliferation of CFS in mice were observed. (1) the control group: AngII:10-6mol/L, had no E2 and no TRAM-34;. (2) AngII E2 group: the 30min was pretreated with AngII at the concentration of 10-6mol/L, and then E _ 2 was added at the concentrations of 10 ~ 5, 10 ~ 6, 10 ~ 7 and 10 ~ 8 mol / L, respectively. (3) AngII TRAM-34 group: 30min was pretreated with AngII at the concentration of 10-6mol/L, then TRAM-34; was added at concentrations of 10, 10, 6, 10, 7 mol / L, respectively. All groups were treated with 24,48,72 hours later, cell proliferation was detected by MTT method. 4, KCa3.1mRNA expression was detected by ordinary PCR, phosphorylation level of p38-MAPK was detected by ELISA method. CFS was pre-treated with AngII (10-6mol/L) for reserve, while 30min was treated with AngII (10-6mol/L) in advance. The experimental groups were as follows: (1) normal control group (no AngII, without E2, no TRAM-34); (2) AngII group, (3) AngII E2 脳 10-5mol/L group, (4) AngII TRAM- 34 脳 10-4mol/L group incubated for 48 hours, cell proliferation activity was detected by MTT, KCa3.1mRNA expression was detected by ordinary PCR, phosphorylation level of p38-MAPK was detected by ELISA. Results: AngII could induce the proliferation of CFS in a time-and concentration-dependent manner. Compared with the control group, the proliferation of AngII at the concentrations of 10? 6, 10? 5 and 10? 4 mol / L was significantly higher than that of the control group (P0.01). There was no significant difference in proliferation effect between 10 mol / L and 10 ~ 4 mol / L 10-6mol/L (P0.05). Therefore, 10-6mol/L was chosen as the concentration of proliferation model. Compared with AngII group, E2 and TRAM-34 significantly inhibited the proliferation of CFS in a concentration-and time-dependent manner, while the effects of 10-5mol/LE2 and 10-4mol/LTRAM-34 on the proliferation of CFS at 48h were not significantly different (P0.05). Compared with the normal control group, Ang鈪，
本文编号：2434999
[Abstract]:Aim: to investigate whether estrogen inhibits the proliferation of cardiac fibroblasts (cardiac fibroblasts,CFS) and whether estrogen inhibits the proliferation of cardiac fibroblasts (cardiac fibroblasts,CFS) with medium conductance calcium activated potassium channel (intermediate-conductance Ca2-activated K channel,KCa3.1). Methods: 1. Culture and identification of cardiac fibroblasts in neonatal mice: the cardiac ventricles of newborn male mice were obtained by operation and cultured by enzyme digestion differential adherent method, and the specific antibody fluorescence immunoassay was used to identify CFS.2,. Establishment of cardiac fibroblasts proliferation model: cultured CFS was cultured with the following concentrations of AngII:10-4,10-5,10-6,10-7,10-8mol/L for 12 h, 24 h, 48 h, 72 h, and no AngII was used as the control group, and the cells were cultured for 12 h, 24 h, 48 h, 72 h, respectively. The proliferation of CFS was detected by MTT method. 3. The effects of estradiol (estradiol,E2) and KCa3.1 channel specific blocker TRAM-34 on the proliferation of CFS in mice were observed. (1) the control group: AngII:10-6mol/L, had no E2 and no TRAM-34;. (2) AngII E2 group: the 30min was pretreated with AngII at the concentration of 10-6mol/L, and then E _ 2 was added at the concentrations of 10 ~ 5, 10 ~ 6, 10 ~ 7 and 10 ~ 8 mol / L, respectively. (3) AngII TRAM-34 group: 30min was pretreated with AngII at the concentration of 10-6mol/L, then TRAM-34; was added at concentrations of 10, 10, 6, 10, 7 mol / L, respectively. All groups were treated with 24,48,72 hours later, cell proliferation was detected by MTT method. 4, KCa3.1mRNA expression was detected by ordinary PCR, phosphorylation level of p38-MAPK was detected by ELISA method. CFS was pre-treated with AngII (10-6mol/L) for reserve, while 30min was treated with AngII (10-6mol/L) in advance. The experimental groups were as follows: (1) normal control group (no AngII, without E2, no TRAM-34); (2) AngII group, (3) AngII E2 脳 10-5mol/L group, (4) AngII TRAM- 34 脳 10-4mol/L group incubated for 48 hours, cell proliferation activity was detected by MTT, KCa3.1mRNA expression was detected by ordinary PCR, phosphorylation level of p38-MAPK was detected by ELISA. Results: AngII could induce the proliferation of CFS in a time-and concentration-dependent manner. Compared with the control group, the proliferation of AngII at the concentrations of 10? 6, 10? 5 and 10? 4 mol / L was significantly higher than that of the control group (P0.01). There was no significant difference in proliferation effect between 10 mol / L and 10 ~ 4 mol / L 10-6mol/L (P0.05). Therefore, 10-6mol/L was chosen as the concentration of proliferation model. Compared with AngII group, E2 and TRAM-34 significantly inhibited the proliferation of CFS in a concentration-and time-dependent manner, while the effects of 10-5mol/LE2 and 10-4mol/LTRAM-34 on the proliferation of CFS at 48h were not significantly different (P0.05). Compared with the normal control group, Ang鈪，
本文编号：2434999
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