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目的探讨环孢霉素A(CsA)对血管升压素(AVP)诱导心脏成纤维细胞(CFs)增殖和胶原合成作用的影响。方法采用胰酶消化法培养新生Sprague-Daw ley大鼠CFs,无血清培养基培养24 h以上的细胞在AVP刺激的基础上给予不同浓度的CsA。采用MTT法测定细胞数目,应用流式细胞仪分析细胞周期,羟基脯氨酸比色法测定细胞培养上清液羟基脯氨酸含量,钙调神经磷酸酶(CaN)活性通过分光光度计测定。结果环孢霉素A可剂量依赖性地抑制AVP诱导的心脏成纤维细胞数目的增加,0.05,0.5及5μmol.L-1CsA对CFs的MTT吸收度值的抑制率分别为12%,24%和29%(均P<0.05);细胞周期分析显示,0.5μmol.L-1CsA可降低AVP诱导的S期百分数和增殖指数(P<0.05);0.5及5μmol.L-1CsA可降低细胞培养上清液羟基脯氨酸含量,抑制率分别为29%和33%,有统计学差异(均P<0.05);CsA可完全阻断AVP诱导CFs细胞内CaN活性,而CsA对基础状态下的CaN活性和细胞存活率无明显影响。结论CsA通过阻断CaN信号通路抑制AVP诱导的CFs增殖和胶原合成,可抑制心肌纤维化进程,为心肌纤维化的防治提供了新的治疗靶点。
Objective To investigate the effects of CsA on the proliferation and collagen synthesis of cardiac fibroblasts (CFs) induced by vasopressin (AVP). Methods CFs of neonatal Sprague-Dawley rats were cultured by trypsin digestion. Cells cultured for 24 hours in serum-free medium were given different concentrations of CsA on the basis of AVP stimulation. Cell number was determined by MTT method. Cell cycle was analyzed by flow cytometry. Hydroxyproline content and calcineurin (CaN) activity of cell culture supernatant were measured by hydroxyproline colorimetric method. Results Cyclosporine A inhibited the AVP-induced increase of cardiac fibroblasts in a dose-dependent manner. The inhibitory rates of MTT absorption of CFs by 0.05, 0.5 and 5 μmol.L-1CsA were 12% and 24%, respectively (P <0.05). The cell cycle analysis showed that 0.5μmol.L-1CsA could reduce the S phase percentage and proliferation index induced by AVP (P <0.05); 0.5 and 5μmol.L-1CsA could reduce the cell culture supernatant (P <0.05). CsA completely blocked AVP-induced intracellular CaN activity in CFs, but CsA had no effect on CaN activity in basal state And cell survival rate had no significant effect. Conclusions CsA can inhibit the progression of myocardial fibrosis by inhibiting the CaN signal pathway and inhibiting AVP-induced CFs proliferation and collagen synthesis, providing a new therapeutic target for the prevention and treatment of myocardial fibrosis.