肝细胞生长因子的抗鼠心肌细胞凋亡作用与抑制钙敏感受体有关
간세포생장인자적항서심기세포조망작용여억제개민감수체유관
Hepatocyte growth factor attenuates ischemia/reperfusion induced cardiomyocyte apoptosis via downregulating calcium sensing receptor expression
  • 万方数据
    中华心血管病杂志 중화심혈관병잡지
    Chinese Journal of Cardiology
    2010年 11期 1019-1024 ,共6页

    严凌%朱铁兵%王连生%陶正贤%潘世阳%杨志健%曹克将

    엄릉%주철병%왕련생%도정현%반세양%양지건%조극장

    心肌再灌注损伤%肝细胞生长因子%受体,钙敏感%细胞凋亡 心肌再灌註損傷%肝細胞生長因子%受體,鈣敏感%細胞凋亡
    심기재관주손상%간세포생장인자%수체,개민감%세포조망
    Myocardial reperfusion injury%Hepatocyte growth factor%Receptors,calciumsensing%Apoptosis
    目的 探讨肝细胞生长因子(HGF)在缺血再灌注损伤中的抗细胞凋亡作用与钙敏感性受体(CaSR)mRNA表达下调的关系.方法 分离的新生鼠心肌细胞,随机分为对照组、模拟心肌缺血再灌注组(I/R组)、特异性CaSR激活剂GdCl3组(GdCl3组)、GdCl3+Na+/Ca2+交换抑制剂NiCl2+L-型钙通道阻滞剂CdCl2组(GdCl3+NiCl2+CdCl2组)、GdCl3+选择性PI3K阻断剂LY294002组(GdCl3+LY294002组)、GdCl3+肝细胞生长因子HGF组(GdCl3+HGF组)、GdCl3+HGF+LY294002组.新生鼠心肌细胞经缺氧处理后在缺血缓冲液中培养2 h,然后在标准培养液中培养24 h,建立模拟心肌缺血再灌注(I/R)模型.TUNEL法检测各组心肌细胞凋亡情况.逆转录聚合酶链反应(RT-PCR)测定各组CaSR mRNA表达.Western blot测定促凋亡蛋白Caspase-3,抗凋亡蛋白Bcl-2和磷脂酰肌醇-3激酶(PI3K).结果 模拟的I/R增强了CaSR mRNA的表达(I/R组:2.62±0.41,对照组:1.00±0.31,P＜0.01)及心肌细胞的凋亡[I/R组:(15.32±2.54)%,对照组:(2.90±1.45)%,P＜0.01].GdCl3进一步增强了CaSR mRNA的表达(GdCl3组:4.46±0.62,I/R组:2.62±0.41,P＜0.01)及心肌细胞的凋亡[GdCl3组:(25.36±2.60)%,L/R组:(15.32±2.54)%,P＜0.01],同时上调了Caspase-3(GdCl3组:1.93±0.28,I/R组:1.50±0.21,P＜0.01),下调了Bcl2的表达(GdCl3组:0.82±0.18,I/R组:1.71±0.30,P＜0.01),抑制了PI3K的磷酸化(I/R组:0.87±0.08,GdCl3组:0.61±0.07,P＜0.01).GdCl3+LY294002组心肌细胞凋亡率显著高于GdCl3组[(32.60±3.42)%比(25.36±2.60)%,P＜0.01],但两组间CaSR mRNA的表达差异无统计学意义.HGF通过抑制Caspase-3(GdCl3+HGF组:1.12±0.23,GdCl3组:1.93±0.28,P＜0.05;GdCl3+HGF+LY294002组:1.87±0.31,GdCl3+LY294002组:3.86±0.47,P＜0.05)和促进Bcl-2的表达(GdCl3+HGF组:2.56±0.54,GdCl3组:0.82±0.18,P＜0.05;GdCl3+HGF+LY294002组:1.68±0.28,GdCl3+LY294002组:0.68±0.13,P＜0.05)减少了I/R和GdCl3诱导的心肌细胞凋亡[GdCl3+HGF组:(11.8±1.89)%,GdCl3组:(25.36±2.60)%,P＜0.05],同时促进了PI3K的磷酸化(GdCl3+HGF组:2.87±0.21,GdCl3组:0.61±0.07,P＜0.05;GdCl3+HGF+LY294002组:2.01±0.14,GdCl3+LY294002组:0.44±0.10,P＜0.05)、下调了CaSR mRNA的表达(GdCl3+HGF组:1.46±0.37,GdCl3组:4.46±0.62,P＜0.01).结论 HGF对心肌细胞的保护作用在I/R诱导的新生鼠心肌细胞凋亡中至少部分与抑制CaSR mRNA表达、促进PI3K的磷酸化途径活化从而下调Caspase-3和上调Bcl-2有关.
    목적 탐토간세포생장인자(HGF)재결혈재관주손상중적항세포조망작용여개민감성수체(CaSR)mRNA표체하조적관계.방법 분리적신생서심기세포,수궤분위대조조、모의심기결혈재관주조(I/R조)、특이성CaSR격활제GdCl3조(GdCl3조)、GdCl3+Na+/Ca2+교환억제제NiCl2+L-형개통도조체제CdCl2조(GdCl3+NiCl2+CdCl2조)、GdCl3+선택성PI3K조단제LY294002조(GdCl3+LY294002조)、GdCl3+간세포생장인자HGF조(GdCl3+HGF조)、GdCl3+HGF+LY294002조.신생서심기세포경결양처리후재결혈완충액중배양2 h,연후재표준배양액중배양24 h,건립모의심기결혈재관주(I/R)모형.TUNEL법검측각조심기세포조망정황.역전록취합매련반응(RT-PCR)측정각조CaSR mRNA표체.Western blot측정촉조망단백Caspase-3,항조망단백Bcl-2화린지선기순-3격매(PI3K).결과 모의적I/R증강료CaSR mRNA적표체(I/R조:2.62±0.41,대조조:1.00±0.31,P＜0.01)급심기세포적조망[I/R조:(15.32±2.54)%,대조조:(2.90±1.45)%,P＜0.01].GdCl3진일보증강료CaSR mRNA적표체(GdCl3조:4.46±0.62,I/R조:2.62±0.41,P＜0.01)급심기세포적조망[GdCl3조:(25.36±2.60)%,L/R조:(15.32±2.54)%,P＜0.01],동시상조료Caspase-3(GdCl3조:1.93±0.28,I/R조:1.50±0.21,P＜0.01),하조료Bcl2적표체(GdCl3조:0.82±0.18,I/R조:1.71±0.30,P＜0.01),억제료PI3K적린산화(I/R조:0.87±0.08,GdCl3조:0.61±0.07,P＜0.01).GdCl3+LY294002조심기세포조망솔현저고우GdCl3조[(32.60±3.42)%비(25.36±2.60)%,P＜0.01],단량조간CaSR mRNA적표체차이무통계학의의.HGF통과억제Caspase-3(GdCl3+HGF조:1.12±0.23,GdCl3조:1.93±0.28,P＜0.05;GdCl3+HGF+LY294002조:1.87±0.31,GdCl3+LY294002조:3.86±0.47,P＜0.05)화촉진Bcl-2적표체(GdCl3+HGF조:2.56±0.54,GdCl3조:0.82±0.18,P＜0.05;GdCl3+HGF+LY294002조:1.68±0.28,GdCl3+LY294002조:0.68±0.13,P＜0.05)감소료I/R화GdCl3유도적심기세포조망[GdCl3+HGF조:(11.8±1.89)%,GdCl3조:(25.36±2.60)%,P＜0.05],동시촉진료PI3K적린산화(GdCl3+HGF조:2.87±0.21,GdCl3조:0.61±0.07,P＜0.05;GdCl3+HGF+LY294002조:2.01±0.14,GdCl3+LY294002조:0.44±0.10,P＜0.05)、하조료CaSR mRNA적표체(GdCl3+HGF조:1.46±0.37,GdCl3조:4.46±0.62,P＜0.01).결론 HGF대심기세포적보호작용재I/R유도적신생서심기세포조망중지소부분여억제CaSR mRNA표체、촉진PI3K적린산화도경활화종이하조Caspase-3화상조Bcl-2유관.
    Objective To examine whether the anti-apoptotic effect of hepatocyte growth factor (HGF) in cardiomyocytes underwent ischemia/reperfusion (I/R) is associated with downregulation of calcium sensing receptor (CaSR) mRNA expression. Methods Neonatal rat cardiomyocytes were isolated and randomly divided into 7 groups: control, I/R, GdCl3, GdCl3 + NiCl2 + CdCl2, GdCl3 + LY294002,GdCl3 + HGF, GdCl3 + HGF + LY294002. L/R was established by incubating primary neonatal rat ventricular cardiomyocytes in ischemia-mimetic solution for 2 h, then reincubated in normal culture medium for 24 h.Cardiomyocyte apoptosis was detected by TUNEL. The expression of CaSR mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of Caspase-3, Bcl-2 and Phosphoinositide-3 Kinase (PI3K) was analyzed by Western blot. Results I/R enhanced the expression of CaSR mRNA ( I/R: 2. 62 ± 0. 41, control: 1.00 ± 0. 31, P ＜ 0. 01 ) and cardiomyocyte apoptosis [ I/R:( 15.32 ± 2. 54) %, control: (2. 90 ± 1.45 ) %, P ＜ 0. 01 ]. GdCl3 further increased the expression of CaSR mRNA (GdCl3:4.46 ±0.62, I/R:2.62±0.41, P＜0.01) and cardiomyocyte apoptosis [GdCl3:(25.36 ±2. 60) %, I/R: ( 15.32 ± 2. 54) %, P ＜ 0. 01 ], along with upregulation of Caspase-3 ( GdCl3: 1.93 ± 0. 28,I/R:1. 50 ± 0. 21, P ＜ 0. 01 ), downregulation of Bcl-2 (GdCl3:0. 82 ± 0. 18, I/R: 1.71 ± 0. 30, P ＜ 0. 01 )and PI3 K phosphorylation inhibition ( I/R:0. 87 ± 0. 08, GdCl3:0. 61 ± 0. 07, P ＜ 0. 01 ). Combination of GdCl3 with LY294002 furthex enhanced cardiomyocytes apoptosis [ GdCl3 + LY294002: ( 32. 6 ± 3.42 ) %,GdCl3: (25.36 ± 2. 60) %, P ＜ 0. 01 ] but did not affect CaSR mRNA expression ( GdCl3 + LY294002:4. 27 ± 0. 56, GdCl3:4. 46 ± 0. 62, P ＞ 0. 05). HGF decreased I/R- and GdCl3-induced apoptosis [ GdCl3 +HGF: ( 11.8 ± 1.89 ) %, GdCl3: ( 25.36 ± 2. 60 ) %, P ＜ 0. 05 ] by suppressing Caspase-3 ( GdCl3 +HGF:1.12±0.23, (GdCl3:1.93 ±0.28, P＜0. 05;GdCl3 + HGF+LY294002:1.87±0.31,GdCl3 +LY294002:3.86 ± 0. 47, P ＜ 0. 05 ) and promoting Bcl-2 ( GdCl3 + HGF: 2. 56 ± 0. 54, GdCl3: 0. 82 ±0. 18, P＜0.05;GdCl3 + HGF + LY294002:1.68 ±0.28,GdCl3 + LY294002:0.68 ±0. 13, P＜0.05)and PI3K phosphoration expression (GdCl3 + HGF:2. 87 ±0. 21 ,GdCl3 :0. 61 ±0. 07, P ＜0. 05;GdCl3 +HGF+ LY294002:2.01 ± 0. 14, GdCl3 + LY294002:0.44 ± 0. 10, P ＜ 0.05) in accordance with downregulation of CaSR mRNA expression ( GdCl3 + HGF: 1.46 ± 0. 37, GdCl3:4. 46 ± 0. 62, P ＜ 0. 01 ).Conclusion HGF exerts protective role in I/R-induced apoptosis at least in part by inhibiting CaSR mRNA expression along with promoting Bcl-2, suppressing Caspase-3 expression and stimulating PI3K phosphorylation signaling pathway.
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