中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2013年
1期
48-53
,共6页
王守力%黄明方%孙飞%殷召%陈力量%冯国斌
王守力%黃明方%孫飛%慇召%陳力量%馮國斌
왕수력%황명방%손비%은소%진역량%풍국빈
肌,平滑,血管%细胞增殖%西洛他唑
肌,平滑,血管%細胞增殖%西洛他唑
기,평활,혈관%세포증식%서락타서
Muscle,smooth,vascular%Cell proliferation%Cilostazol
目的 探讨不同浓度西洛他唑对体外培养的大鼠血管平滑肌细胞(VSMC)增殖的影响及其可能的调控机制.方法 实验分为6组,分别为DMEM阴性对照组(对照组),不同浓度西洛他唑组(共5组,分别给予1.0 ×10-7、2.5 ×10-7、5.0×10-7、7.5 ×10-7和1.0×10-6 mol/L西洛他唑干预).细胞计数和四唑盐(MTT)比色试验分析西洛他唑对VSMC增殖能力的影响,流式细胞计数仪分析细胞周期分布,TUNEL染色检测细胞凋亡,RT-PCR和Western blot方法分析给药前后视网膜母细胞瘤基因Rb、p53和p21基因的mRNA和蛋白表达水平的变化.结果 西洛他唑可抑制血管平滑肌细胞增生,使血管平滑肌细胞阻滞于G1期,且该效应呈现明显的剂量依赖性.高浓度的西洛他唑(7.5×10-和1.0×10-6mol/L)可诱导血管平滑肌细胞凋亡.西洛他唑各浓度组p53基因的mRNA 和蛋白表达水平均高于对照组,其中2.5 ×10-7、5.0×10-7和7.5 ×10-7 mol/L组mRNA表达水平与对照组比较差异有统计学意义(P均＜0.05),1.0×10-6 mol/L组差异更为显著(3.22±0.45比1.75±0.32,P＜0.01),西洛他唑7.5×10-7 mol/L组蛋白表达水平与对照组比较差异有统计学意义(P＜0.05),1.0×10-6 mol/L组差异更为显著(0.53 ±0.11比0.18 ±0.06,P＜0.01).西洛他唑1.0×10-7、2.5 ×10-7和5.0 × 10-7 mol/L组p21 mRNA表达水平均显著高于对照组(1.86±0.19、2.20±0.24和2.10 ±0.18比1.21±0.18,P均＜0.05),西洛他唑1.0×10-7 mol/L和2.5 ×10-7 mol/L组Rb mRNA表达水平均显著高于对照组(P均＜0.05),5.0×10-7 mol/L组则更为显著(0.89±0.07比0.38±0.04,P＜0.01).而西洛他唑7.5×10-和1.0×10-6mol/L组p21的mRNA表达水平则显著低于对照组(0.81 ±0.09比1.21±0.18,P＜0.05和0.36 ±0.10比1.21±0.18,P＜0.01),Rb mRNA的表达水平亦均显著低于对照组(0.12±0.02和0.11 ±0.02比0.38±0.04,P均＜0.01).随着西洛他唑浓度的增加,p21和Rb基因的蛋白表达水平亦呈现先升高后下降的态势.结论 西洛他唑可显著抑制体外培养的大鼠VSMC增殖,机制可能为通过Rb-p53-p21通路抑制VSMC增殖,并通过p53基因过表达诱导VSMC凋亡.
目的 探討不同濃度西洛他唑對體外培養的大鼠血管平滑肌細胞(VSMC)增殖的影響及其可能的調控機製.方法 實驗分為6組,分彆為DMEM陰性對照組(對照組),不同濃度西洛他唑組(共5組,分彆給予1.0 ×10-7、2.5 ×10-7、5.0×10-7、7.5 ×10-7和1.0×10-6 mol/L西洛他唑榦預).細胞計數和四唑鹽(MTT)比色試驗分析西洛他唑對VSMC增殖能力的影響,流式細胞計數儀分析細胞週期分佈,TUNEL染色檢測細胞凋亡,RT-PCR和Western blot方法分析給藥前後視網膜母細胞瘤基因Rb、p53和p21基因的mRNA和蛋白錶達水平的變化.結果 西洛他唑可抑製血管平滑肌細胞增生,使血管平滑肌細胞阻滯于G1期,且該效應呈現明顯的劑量依賴性.高濃度的西洛他唑(7.5×10-和1.0×10-6mol/L)可誘導血管平滑肌細胞凋亡.西洛他唑各濃度組p53基因的mRNA 和蛋白錶達水平均高于對照組,其中2.5 ×10-7、5.0×10-7和7.5 ×10-7 mol/L組mRNA錶達水平與對照組比較差異有統計學意義(P均＜0.05),1.0×10-6 mol/L組差異更為顯著(3.22±0.45比1.75±0.32,P＜0.01),西洛他唑7.5×10-7 mol/L組蛋白錶達水平與對照組比較差異有統計學意義(P＜0.05),1.0×10-6 mol/L組差異更為顯著(0.53 ±0.11比0.18 ±0.06,P＜0.01).西洛他唑1.0×10-7、2.5 ×10-7和5.0 × 10-7 mol/L組p21 mRNA錶達水平均顯著高于對照組(1.86±0.19、2.20±0.24和2.10 ±0.18比1.21±0.18,P均＜0.05),西洛他唑1.0×10-7 mol/L和2.5 ×10-7 mol/L組Rb mRNA錶達水平均顯著高于對照組(P均＜0.05),5.0×10-7 mol/L組則更為顯著(0.89±0.07比0.38±0.04,P＜0.01).而西洛他唑7.5×10-和1.0×10-6mol/L組p21的mRNA錶達水平則顯著低于對照組(0.81 ±0.09比1.21±0.18,P＜0.05和0.36 ±0.10比1.21±0.18,P＜0.01),Rb mRNA的錶達水平亦均顯著低于對照組(0.12±0.02和0.11 ±0.02比0.38±0.04,P均＜0.01).隨著西洛他唑濃度的增加,p21和Rb基因的蛋白錶達水平亦呈現先升高後下降的態勢.結論 西洛他唑可顯著抑製體外培養的大鼠VSMC增殖,機製可能為通過Rb-p53-p21通路抑製VSMC增殖,併通過p53基因過錶達誘導VSMC凋亡.
목적 탐토불동농도서락타서대체외배양적대서혈관평활기세포(VSMC)증식적영향급기가능적조공궤제.방법 실험분위6조,분별위DMEM음성대조조(대조조),불동농도서락타서조(공5조,분별급여1.0 ×10-7、2.5 ×10-7、5.0×10-7、7.5 ×10-7화1.0×10-6 mol/L서락타서간예).세포계수화사서염(MTT)비색시험분석서락타서대VSMC증식능력적영향,류식세포계수의분석세포주기분포,TUNEL염색검측세포조망,RT-PCR화Western blot방법분석급약전후시망막모세포류기인Rb、p53화p21기인적mRNA화단백표체수평적변화.결과 서락타서가억제혈관평활기세포증생,사혈관평활기세포조체우G1기,차해효응정현명현적제량의뢰성.고농도적서락타서(7.5×10-화1.0×10-6mol/L)가유도혈관평활기세포조망.서락타서각농도조p53기인적mRNA 화단백표체수평균고우대조조,기중2.5 ×10-7、5.0×10-7화7.5 ×10-7 mol/L조mRNA표체수평여대조조비교차이유통계학의의(P균＜0.05),1.0×10-6 mol/L조차이경위현저(3.22±0.45비1.75±0.32,P＜0.01),서락타서7.5×10-7 mol/L조단백표체수평여대조조비교차이유통계학의의(P＜0.05),1.0×10-6 mol/L조차이경위현저(0.53 ±0.11비0.18 ±0.06,P＜0.01).서락타서1.0×10-7、2.5 ×10-7화5.0 × 10-7 mol/L조p21 mRNA표체수평균현저고우대조조(1.86±0.19、2.20±0.24화2.10 ±0.18비1.21±0.18,P균＜0.05),서락타서1.0×10-7 mol/L화2.5 ×10-7 mol/L조Rb mRNA표체수평균현저고우대조조(P균＜0.05),5.0×10-7 mol/L조칙경위현저(0.89±0.07비0.38±0.04,P＜0.01).이서락타서7.5×10-화1.0×10-6mol/L조p21적mRNA표체수평칙현저저우대조조(0.81 ±0.09비1.21±0.18,P＜0.05화0.36 ±0.10비1.21±0.18,P＜0.01),Rb mRNA적표체수평역균현저저우대조조(0.12±0.02화0.11 ±0.02비0.38±0.04,P균＜0.01).수착서락타서농도적증가,p21화Rb기인적단백표체수평역정현선승고후하강적태세.결론 서락타서가현저억제체외배양적대서VSMC증식,궤제가능위통과Rb-p53-p21통로억제VSMC증식,병통과p53기인과표체유도VSMC조망.
Objective To explore the effects and related mechanisms of cilostazol on rat vascular smooth muscle cells (VSMCs)proliferation.Methods VSMCs were treated with DMEM (control) and various doses of cilostazol (1.0 × 10-7,2.5 × 10-7,5.0 × 10-7,7.5 × 10-7 and 1.0 × 10-6 mol/L) for 13 d (cell counting) or 72 h.Proliferation of VSMCs was investigated by cell-counting,MTT and flow cytometry analysis.Cell apoptosis was determined by TUNEL staining.mRNA and protein expressions of cell cycle regulatory proteins,such as Rb,p53 and p21 were detected by RT-PCR and Western blot,respectively.Results Cilostazol inhibited VSMCs proliferation and induced VSMCs arrest at G1 phase in a dose-dependent manner.High dose of cilostazol (7.5 × 10-7 and 1.0 × 10-6 mol/L) induced VSMCs apoptosis.p53 mRNA expression in 2.5 × 10-7 mol/L to 7.5 × 10-7 mol/L groups as well as 1.0 × 10-6 mol/L group (3.22 ±0.45 vs.1.75 ±0.32) and p53 protein expression in 7.5 × 10-7 mol/L group and 1.0 × 10-6 mol/L group (0.53 ± 0.11 vs.0.18 ± 0.06) were significantly upregulated after 72 h culture (all P＜0.05 vs.control).Low dose of cilostazol (1.0 × 10-7,2.5 × 10-7 and 5.0 × 10-7 mol/L)significantly upregulated p21 mRNA expression compared to control group (1.86 ± 0.19,2.20 ± 0.24 and 2.10 ± 0.18 vs.1.210 ± 0.18,all P ＜ 0.05).Similarly,Rb mRNA expression was significantly upregulated in 1.0 × 10-7,2.5 × 10-7 and 5.0 × 10 7 mol/L groups (0.89 ±0.07 vs.0.38 ±0.04)compared with control group (all P ＜ 0.05).However,high dose cilostazol (7.5 × 10-7 and 1.0 × 10-6mol/L) significantly downregulated p21 mRNA expression (0.81 ±0.09 vs.1.21 ±0.18,0.36 ±0.10 vs.1.2t ±0.18,all P ＜0.05 vs.control) and Rb mRNA expression (0.12 ±0.02 and 0.11 ±0.02 vs.0.38± ± 0.04,all P ＜ 0.05 vs.control).p21 and Rb protein expressions also upregulated at low concentrations of cilostazol and downregulated at high concentrations of cilostazol.Conclusion Cilostazol could inhibit the proliferation of rat VSMCs through modulating Rb-p53-p21 pathway and induce VSMCs apoptosis through upregulating p53.
