【摘要】：目的:探討雌激素抑制心臟成纖維細胞(cardiac fibroblasts,CFS)增殖是否與中電導鈣激活鉀通道(intermediate-conductance Ca2+-activated K+channel,KCa3.1)有關(guān)。方法:1、新生小鼠心臟成纖維細胞的培養(yǎng)與鑒定:取新生雄性小鼠,經(jīng)手術(shù)取其心臟心室,酶消化差速貼壁法培養(yǎng),特異性抗體熒光免疫法鑒別CFS。2、心臟成纖維細胞增殖模型的建立:培養(yǎng)的CFS加入以下濃度的AngII:10-4、10-5、10-6、10-7、10-8mol/L分別培養(yǎng)12h、24h、48h、72h后,并以不加AngII作對照組,使用MTT法檢測CFS增殖情況。3、觀察雌二醇(estradiol,E2)和KCa3.1通道特異性阻斷劑TRAM-34對小鼠CFS增殖活性的影響:(1)對照組:AngII:10-6mol/L,無E2,無TRAM-34;(2)AngII+E2組:先使用濃度為10-6mol/L的AngII預處理30min后,分別加入10-5、10-6、10-7、10-8mol/L濃度的E2;(3)AngII+TRAM-34組:先使用濃度為10-6mol/L的AngII預處理30min后,分別加入10-4、10-5、10-6、10-7mol/L濃度的TRAM-34;各組均處理24、48、72小時后,使用MTT法檢測細胞增殖情況。4、普通PCR檢測KCa3.1mRNA表達情況,ELISA法檢測p38-MAPK磷酸化水平:CFS預先用AngII(10-6mol/L)處理30min后備用,實驗分組為:(1)正常對照組(無AngII,無E2,無TRAM-34);(2)AngII組;(3)AngII+E2×10-5mol/L組;(4)AngII+TRAM-34×10-4mol/L組孵育48h,MTT檢測細胞增殖活性,普通PCR檢測細胞KCa3.1mRNA的表達,ELISA檢測細胞p38-MAPK的磷酸化水平。結(jié)果:AngII可誘導小鼠CFS增殖,并具有時間及濃度依賴性,與空白組對照,10-6、10-5、10-4mol/L濃度AngII增殖效果顯著,具有統(tǒng)計學意義(P0.01);10-5、10-4mol/L濃度增殖效果與10-6mol/L比較無顯著差異(P0.05),故選用10-6mol/L作為增殖模型的濃度;與AngII組比較,E2和TRAM-34對小鼠CFS的增殖活性抑制作用明顯,并具有濃度及時間依賴性;10-5mol/LE2與10-4mol/LTRAM-34干預48h對CFS增殖作用的影響無顯著差異(P0.05)。與正常對照組比較,AngⅡ組可顯著提高KCa3.1mRNA表達,具有統(tǒng)計學意義(P0.01);與正常對照組比較,AngⅡ+E2×10-5 mol/L組和AngⅡ+TRAM-34×10-4mol/L組,均可顯著抑制KCa3.1mRNA表達,并具有統(tǒng)計學意義(P0.05);與AngⅡ組比較,AngⅡ+E2×10-5 mol/L組和AngⅡ+TRAM-34×10-4mol/L組,均可顯著抑制KCa3.1mRNA表達,并具有統(tǒng)計學意義(P0.01);AngⅡ+E2×10-5 mol/L組和AngⅡ+TRAM-34×10-4mol/L組比較無顯著差異(P0.05)。正常對照組比較,AngⅡ能夠顯著提高p38-MAPK磷酸化水平,具有統(tǒng)計學意義(P0.01);與AngⅡ組比較,AngII+E2×10-5mol/L組能夠顯著降低p38-MAPK磷酸化水平,具有統(tǒng)計學意義(P0.01);與AngⅡ組比較,AngII+TRAM-34×10-4mol/L組對p38-MAPK磷酸化水平影響不顯著(P0.05);AngII+E2×10-5mol/L組與AngII+TRAM-34×10-4mol/L組間有顯著差異,具有統(tǒng)計學意義(P0.01)。結(jié)論:AngII可誘導小鼠CFS增殖;E2與TRAM-34均能抑制CFS增殖,10-5mol/LE2與10-4mol/LTRAM-34對CFS增殖作用的影響無顯著差異;雌激素抑制心臟成纖維細胞增殖的機制可能是降低p38-MAPK磷酸化水平,從而抑制KCa3.1mRNA表達。
[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，

本文編號：2434999