糜酶对大鼠心脏成纤维细胞增殖和胶原合成的影响

Effects of chymase on proliferation and collagen synthesis of rat cardiac fibroblasts

目的探讨糜酶对大鼠心脏成纤维细胞(CFs)增殖和胶原合成的影响。方法用胰酶消化法分离、培养新生SD大鼠的CFs,采用MTT比色法(A490值)测定细胞数目,流式细胞术分析细胞周期,3H-脯氨酸掺入法测定总胶原合成,RT-PCR检测Ⅰ型和Ⅲ型胶原mRNA的表达。结果①糜酶以剂量依赖方式增加CFs的数目。其中15、30和60μg/L组的A490值分别为0.263±0.033、0.348±0.031和0.387±0.026,均高于对照组的A490值(0.201±0.019),并有非常显著性差异(P<0.01)。②随着糜酶作用浓度的增加,CFs在G0/G1期的百分率逐渐减少,S期的百分率和细胞增殖指数逐渐增加。其中15、30和60μg/L组与对照组相比,上述各项指标均有显著或非常显著性差异(P<0.05或P<0.01)。③糜酶以剂量依赖方式增加CFs的3H-脯氨酸掺入量。其中15、30和60μg/L组的3H-脯氨酸掺入量分别为(520±75)、(684±62)和(769±58)cpm/孔,均高于对照组[(435±60)cpm/孔],差异有显著或非常显著性意义(P<0.05或P<0.01)。④随着糜酶作用浓度的增加,CFs的Ⅰ型和Ⅲ型胶原mRNA表达水平均呈递增趋势。其中15、30和60μg/L组的Ⅰ、Ⅲ型胶原mRNA表达的水平均显著高于对照组(P<0.01)。结论糜酶具有促进CFs增殖和胶原合成的作用,提示其可能在心肌纤维化过程中发挥重要作用。

AIM To investigate the effects of chymase on the proliferation and collagen synthesis of rat cardiac fibroblasts （CFs）. METHODS Cultured CFs from neonatal SD rats were isolated by trypsinization and cellular number and cellular cycle were evaluated by MTT colorimetry （A490 value） and flow cytometry （FCM）. The total collagen synthesis by CFs was examined by 3 H-proline incorporation and the expressions of Types Ⅰ and Ⅲ collagen mRNA in CFs were determined by RT-PCR. RESULTS ① Chymase increased CFs number in a dose-dependent manner. The A490 values of 15, 30 and 60 p.g/L chymase groups were 0. 263±0. 033,0. 348 ±0. 031 and 0. 387 ±0. 026, respectively, significantly higher than those in control group（0. 201 ±0. 019, P 〈 0. 01 ）.②Cellular cycle analysis showed that treatment with 15, 30 and 60μg/L chymase markedly decreased cellular percentage in G0/G1 stage and increased cellular percentage in S stage and proliferation index （PI） in a dose-dependent manner compared with those in control groups （P 〈 0.05 or P 〈 0.01 ）. ③Chymase increased the 3 H-proline incorporation in CFs in a dose-dependent manner. The 3H-proline incorporations induced by 15, 30 and 60 μg/L ehymase were （520±75 ）, （684 ± 62） and （769 ± 58） cpm/well, respectively, significantly higher than those in control group [ （435 ± 60） cpm/well, P 〈 0. 05 or P 〈 0. 01 ].④15, 30 and 60μg/L chymase obviously increased the expressions of Types Ⅰ and Ⅲ collagen mRNA in a dose-dependent manner compared with those in control groups （ P 〈0. 01 ）. CONCLUSION Chymase promotes the proliferation and collagen synthesis of rat CFs, which is involved in the process of myocardial fibrosis.