卡维地洛对自发性高血压大鼠左室心肌肥厚作用的细胞和分子水平研究

Cellular and molecular level analysis of the effect of carvedilol on left ventricular hypertrophy in spontaneously hypertensive rats

目的:观察基础和卡维地洛干预条件下,高血压大鼠和Wistar大鼠心脏成纤维细胞增殖细胞核抗原蛋白表达及脱氧核糖核酸合成变化对心肌纤维化的影响。方法:实验于2001-10/2002-12在解放军空军总医院临床试验中心及解放军第四军医大学唐都医院高血压实验室完成。实验选用12周龄血压176～190mmHg(1mmHg=0.133kPa)的高血压大鼠10只,培养心脏成纤维细胞,按随机数字法将其分为空白对照组和0.01,0.10,1.00,10.00μmol/L卡维地洛干预72h组;选择血压120～132mmHg的Wis-tar健康大鼠10只,培养心脏成纤维细胞为正常对照组。消化法培养大鼠心脏成纤维细胞,采用免疫组化法观察增殖细胞核抗原蛋白在心脏成纤维细胞细胞核表达的变化,3H-胸腺嘧啶核苷掺入法测定脱氧核糖核酸合成。结果:在基础条件下培养72h,高血压大鼠空白对照组的心脏成纤维细胞增殖细胞核抗原含量犤(131.2±11.0)A·mm2犦及3H-胸腺嘧啶核苷掺入量犤(606.0±54.7)min-1犦明显高于正常大鼠空白对照组犤(107.2±16.3)A·mm2,(495.2±49.8)min-1,t=2.725,3.3489;P<0.05犦。0.10,10.00μmol/L卡维地洛分别干预72h,高血压大鼠心脏成纤维细胞增殖细胞核抗原含量显著低于高血压大鼠空白对照组犤(96.6±9.78),(87.0±12.5),(69.2±11.3)A·mm2t=5.258,5.926,8.770,P<0.01犦;1.00,10.00μmol/L卡维地洛分别干预72h时,Wistar大鼠心脏成纤维细胞增殖细胞核抗原含量分别为(81.4±10.6),(72.2±10.8)A·mm2显著低于正常大鼠空白对照组犤t=2.956,P<0.05;t=3.994,P<0.01犦。0.10,1.00,10.00μmol/L卡维地洛分别干预72h时,高血压大鼠心脏成纤维细胞3H-胸腺嘧啶核苷掺入量分别为犤(452.0±35.9),(317.2±36.2)和(279.6±24.7)min-1犦均显著低于高血压大鼠空白对照组(t=5.261,9.838,12.153,P<0.01);Wistar大鼠组3H-胸腺嘧啶核苷均明显低于正常大鼠空白对照组犤(388.2±35.0),(322.6±32.7),(277.8±25.3)min-1,t=3.931,6.478,8.705,P<0.01犦。在不同浓度卡维地洛作用下,高血压大鼠心脏成纤维细胞的3H-胸腺嘧啶核苷掺入量随增殖细胞核抗原蛋白表达的增强而增高,两者呈显著正相关(r=0.856,P<0.01)。结论:卡维地洛不仅能抑制与脱氧核糖核酸合成密切相关的胸腺嘧啶核苷酸的活性,而且也能抑制增殖细胞核抗原的表达,直接影响了脱氧核糖核酸的复制,进而抑制心脏成纤维细胞增殖后的左室重构。

AIM:To observe the effect of the changes of proliferating cell nuclear antigen expression and DNA synthesis on myocardial fibrosis in cardiac fibroblasts(CFs) derived from spontaneously hypertensive rats and Wistar rats under normal condition and intervention of carvedilol.METHODS:The experiment was carried out in the clinical trial center of Chinese PLA General Hospital of Air Forces and the hypertension laboratory of Tangdu Hospital of the Fourth Military Medical University of Chinese PLA between October 2001 and December 2002.Ten 12 week old hypertensive rats with the blood pressure of 176 to 190 mm Hg(1 mm Hg=0.133 kPa) were used in this study,and the cultured cardiac fibroblasts were divided into blank control group,0.01,0.10,1.00,10.00 μ mol/L carvedilol intervention groups(72 hours) with the method of random number. The cardiac fibroblasts of healthy Wistar rats with the blood pressure of 120 to 132 mm Hg were cultured as the normal control group.The cardiac fibroblasts were cultured with the method of digestion method.The changes of protein expression of the proliferating cell nuclear antigen in the nuclear of cardiac fibroblasts were observed with immunohistochemistry,and the DNA synthesis was determined with the method of 3H thymidine incorporation.RESULTS:After 72 hour culture under basic condition,the content of proliferating cell nuclear antigen and amount of 3H thymidine incorporation in cardiac fibroblasts were obviously higher in the hypertensive blank control group[(131.2± 11.0) A· mm2,(606.0± 54.7) min- 1] than in the Wistar rats normal blank control group[(107.2± 16.3) A· mm2, (495.2± 49.8) min- 1] (t=2.725,3.348 9;P< 0.05).After intervention of 0.10 to 10.00 μ mol/L carvedilol for 72 hours,the contents of proliferating cell nuclear antigen of the spontaneously hypertensive rats[(96.6± 9.78),(87.0± 12.5),(69.2± 11.3) A· mm2] were significantly lower than that in the hypertensive blank control group(t=5.258,5.926,8.770,P< 0.01). After intervention of 1.00 to 10.00 μ mol/L carvedilol for 72 hours, the contents of proliferating cell nuclear antigen of the spontaneously hypertensive rats[(81.4± 10.6),(72.2± 10.8) A· mm2] were significantly than that in the normal blank control group(t=2.956,P< 0.05;t=3.994,P< 0.01).After intervention of 0.10 to 10.00 μ mol/L carvedilol for 72 hours,the amount of 3H thymidine incorporation was significantly lower in the spontaneously hypertensive rats[(452.0± 35.9),(317.2± 36.2),(279.6± 24.7) min- 1] than in the hypertensive blank control group(t=5.261,9.838,12.153,P< 0.01),also obviously lower in the Wistar rats[(388.2± 35.0),(322.6± 32.7),(277.8± 25.3) min- 1] than in the normal blank control group(t=3.931,6.478,8.705,P< 0.01). Under carvedilol intervention of different concentration,the amount of 3H thymidine incorporation was increased with the enhancement of proliferating cell nuclear antigen protein expression in the cardiac fibroblasts of spontaneously hypertensive rats, and there was a significant positive correlation between them(r=0.856,P< 0.01).CONCLUSION:Carvedilol cannot only inhibit the thymidine activity,which is closely related with DNA synthesis,but also inhibit the expression of proliferating cell nuclear antigen.It directly affects the DNA duplication,and then inhibits the left ventricular remodeling after the proliferation of cardiac fibroblasts.