中华心血管病杂志
中華心血管病雜誌
중화심혈관병잡지
Chinese Journal of Cardiology
2013年
4期
315-319
,共5页
裘琳%杜光%刘东%郑恒%曾和松%周宁%杨晓云
裘琳%杜光%劉東%鄭恆%曾和鬆%週寧%楊曉雲
구림%두광%류동%정항%증화송%주저%양효운
心肌病,肥厚性%缓激肽%信号传导%钙调神经磷酸酶
心肌病,肥厚性%緩激肽%信號傳導%鈣調神經燐痠酶
심기병,비후성%완격태%신호전도%개조신경린산매
Cardiomyopathy,hypertrophic%Bradykinin%Signal transduction%Calcineurin
目的 探讨缓激肽对机械刺激引起的心肌细胞肥大的抑制作用及其可能机制.方法 体外分离培养新生大鼠心肌细胞,并接种存特制的硅胶皿上,分为对照组、机械刺激组(牵张硅胶皿牵张120％ 30min)和机械刺激+缓激肽组(1×10-8 mol/L的缓激肽作用24 h后再牵张120％作用30 min),每组3个复孔,实验至少重复3次.以3H-亮氨酸掺入法检测各组心肌细胞蛋白合成率,心肌细胞α-MHC免疫荧光染色、以激光共聚焦成像测算心肌细胞表面积.提取各组心肌细胞RNA和总蛋白后分别以实时PCR和Western blot检测心肌细胞中心房利钠肽(ANP)、肌浆网Ca2+-ATP酶(SERCA2) mRNA表达和血管紧张素转换酶(ACE)、血管紧张素Ⅱ1型受体(AT1受体)、钙调神经磷酸酶(CaN)的蛋白表达水平.结果 机械刺激组和机械刺激+缓激肽组大鼠心肌细胞表面积均显著大于对照组[(973±103)和(603±74) μm2分别比(312 ±29) μm2,P分别＜0.01和0.05].机械刺激组和机械刺激+缓激肽组大鼠心肌细胞蛋白合成率均显著高于对照组,分别为对照组的(4.5±3.7)和(3.0±1.9)倍,ANP蛋白表达水平亦均显著高于对照组,分别为对照组的(4.1±0.4)和(2.3±0.2)倍,而SERCA2蛋白表达水平则均显著低于对照组,分别为对照组的(0.52±0.05)和(0.79±0.08)倍(P＜ 0.01或0.05).机械刺激组和机械刺激+缓激肽组大鼠心肌细胞ACE蛋白表达水平均显著高于对照组,分别为对照组的(3.2±0.4)和(2.7±0.4)倍(P均＜0.05),机械刺激组与机械刺激+缓激肽组组间比较差异无统计学意义.机械刺激组和机械刺激+缓激肽组大鼠心肌细胞AT1受体蛋白表达水平均显著高于对照组,分别为对照组的(3.7±0.3)和(2.1±0.2)倍(P分别＜0.01和0.05),且机械刺激+缓激肽组显著低于机械刺激组(P＜0.05).机械刺激组和机械刺激+缓激肽组大鼠心肌细胞磷酸化CaN表达水平均高于对照组,分别为对照组的(6.4±0.6)和(3.6±0.3)倍(P分别＜0.01和0.05),且机械刺激+缓激肽组显著低于机械刺激组(P＜0.05).结论 缓激肽可通过抑制Ca2+/CaN信号通路抑制机械刺激诱发的大鼠心肌细胞肥大.
目的 探討緩激肽對機械刺激引起的心肌細胞肥大的抑製作用及其可能機製.方法 體外分離培養新生大鼠心肌細胞,併接種存特製的硅膠皿上,分為對照組、機械刺激組(牽張硅膠皿牽張120％ 30min)和機械刺激+緩激肽組(1×10-8 mol/L的緩激肽作用24 h後再牽張120％作用30 min),每組3箇複孔,實驗至少重複3次.以3H-亮氨痠摻入法檢測各組心肌細胞蛋白閤成率,心肌細胞α-MHC免疫熒光染色、以激光共聚焦成像測算心肌細胞錶麵積.提取各組心肌細胞RNA和總蛋白後分彆以實時PCR和Western blot檢測心肌細胞中心房利鈉肽(ANP)、肌漿網Ca2+-ATP酶(SERCA2) mRNA錶達和血管緊張素轉換酶(ACE)、血管緊張素Ⅱ1型受體(AT1受體)、鈣調神經燐痠酶(CaN)的蛋白錶達水平.結果 機械刺激組和機械刺激+緩激肽組大鼠心肌細胞錶麵積均顯著大于對照組[(973±103)和(603±74) μm2分彆比(312 ±29) μm2,P分彆＜0.01和0.05].機械刺激組和機械刺激+緩激肽組大鼠心肌細胞蛋白閤成率均顯著高于對照組,分彆為對照組的(4.5±3.7)和(3.0±1.9)倍,ANP蛋白錶達水平亦均顯著高于對照組,分彆為對照組的(4.1±0.4)和(2.3±0.2)倍,而SERCA2蛋白錶達水平則均顯著低于對照組,分彆為對照組的(0.52±0.05)和(0.79±0.08)倍(P＜ 0.01或0.05).機械刺激組和機械刺激+緩激肽組大鼠心肌細胞ACE蛋白錶達水平均顯著高于對照組,分彆為對照組的(3.2±0.4)和(2.7±0.4)倍(P均＜0.05),機械刺激組與機械刺激+緩激肽組組間比較差異無統計學意義.機械刺激組和機械刺激+緩激肽組大鼠心肌細胞AT1受體蛋白錶達水平均顯著高于對照組,分彆為對照組的(3.7±0.3)和(2.1±0.2)倍(P分彆＜0.01和0.05),且機械刺激+緩激肽組顯著低于機械刺激組(P＜0.05).機械刺激組和機械刺激+緩激肽組大鼠心肌細胞燐痠化CaN錶達水平均高于對照組,分彆為對照組的(6.4±0.6)和(3.6±0.3)倍(P分彆＜0.01和0.05),且機械刺激+緩激肽組顯著低于機械刺激組(P＜0.05).結論 緩激肽可通過抑製Ca2+/CaN信號通路抑製機械刺激誘髮的大鼠心肌細胞肥大.
목적 탐토완격태대궤계자격인기적심기세포비대적억제작용급기가능궤제.방법 체외분리배양신생대서심기세포,병접충존특제적규효명상,분위대조조、궤계자격조(견장규효명견장120％ 30min)화궤계자격+완격태조(1×10-8 mol/L적완격태작용24 h후재견장120％작용30 min),매조3개복공,실험지소중복3차.이3H-량안산참입법검측각조심기세포단백합성솔,심기세포α-MHC면역형광염색、이격광공취초성상측산심기세포표면적.제취각조심기세포RNA화총단백후분별이실시PCR화Western blot검측심기세포중심방리납태(ANP)、기장망Ca2+-ATP매(SERCA2) mRNA표체화혈관긴장소전환매(ACE)、혈관긴장소Ⅱ1형수체(AT1수체)、개조신경린산매(CaN)적단백표체수평.결과 궤계자격조화궤계자격+완격태조대서심기세포표면적균현저대우대조조[(973±103)화(603±74) μm2분별비(312 ±29) μm2,P분별＜0.01화0.05].궤계자격조화궤계자격+완격태조대서심기세포단백합성솔균현저고우대조조,분별위대조조적(4.5±3.7)화(3.0±1.9)배,ANP단백표체수평역균현저고우대조조,분별위대조조적(4.1±0.4)화(2.3±0.2)배,이SERCA2단백표체수평칙균현저저우대조조,분별위대조조적(0.52±0.05)화(0.79±0.08)배(P＜ 0.01혹0.05).궤계자격조화궤계자격+완격태조대서심기세포ACE단백표체수평균현저고우대조조,분별위대조조적(3.2±0.4)화(2.7±0.4)배(P균＜0.05),궤계자격조여궤계자격+완격태조조간비교차이무통계학의의.궤계자격조화궤계자격+완격태조대서심기세포AT1수체단백표체수평균현저고우대조조,분별위대조조적(3.7±0.3)화(2.1±0.2)배(P분별＜0.01화0.05),차궤계자격+완격태조현저저우궤계자격조(P＜0.05).궤계자격조화궤계자격+완격태조대서심기세포린산화CaN표체수평균고우대조조,분별위대조조적(6.4±0.6)화(3.6±0.3)배(P분별＜0.01화0.05),차궤계자격+완격태조현저저우궤계자격조(P＜0.05).결론 완격태가통과억제Ca2+/CaN신호통로억제궤계자격유발적대서심기세포비대.
Objective To evaluate the inhibitory effect and related mechanism of bradykinin on mechanical stress induced myocardial hypertrophy.Methods Neonatal rat cardiomyocytes were isolated and cultured in silicon plates.All cardiomyocytes were randomly divided into three groups:control group,mechanical stretch group (mechanical stretch of silicon plates to 120％ for 30 min) and mechanical stretch plus bradykinin group (1 × 10-8 mol/L for 24 h before stretch).The protein synthesis and surface area of cardiomyocytes were detected by [3H] leucine incorporation and immunofluorescence of α-MHC,respectively.mRNA expression of atrial natriuretic peptide (ANP) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2) was detected by real time-PCR,the phosphorylation of calcineurin (CaN),the expression of Angiotensin Ⅱ receptor 1 (AT1R) and angiotensin converting enzyme (ACE)by Western blot.Results The surface area of cardiomyocytes of mechanical stretch group [(973 ± 103) μm2] was significantly enlarged than in control group [(312-± 29) μm2] and this effect could be partly attenuated by bradykinin [(603-± 74) μm2,all P ＜ 0.05].Mechanical stretch also significantly increased the protein synthesis,upregulated the expression of ANP and decreased the expression of SERCA2,and these effects could be partly reversed by pretreatment with bradykinin.Moreover,bradykinin partly abolished the mechanical stretchinduced increases in CaN phosphorylation,up-regulation of AT1R but preserved the expression of ACE.Conclusions Bradykinin significantly attenuates mechanical stretch-induced myocardial hypertrophy through inhibition of Ca2+/CaN pathway.
