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目的 探讨酪氨酸蛋白激酶抑制剂4、5、7-三羟基异黄酮(genistein)对肝窦内皮细胞(sinusoidalendothelial cell, SEC)窗孔、增殖及合成一氧化氮(nitric oxide, NO)的影响。方法 用胶原酶原位灌注,Percoll不连续密度梯度离心法分离正常及CCl_4实验性肝纤维化大鼠的SEC并进行体外培养。采用扫描电镜技术,MTT法及硝酸还原酶法,分别观察genistein对SEC细胞窗孔、增殖及合成NO的影响。结果 Genistein对肝纤维化各级 SEC的窗孔数目及大小均无明显的影响。经不同浓度的genistein作用24h后,肝纤维化各级大鼠SEC的生长均受抑制,以100 μmol/L浓度的genistein对肝纤维化Ⅰ级大鼠SEC的作用最为明显[细胞增殖率为-(15.38±6.26)% vs(4.91±2.16)%,t=13.7. P＜0.05]。100 μmol/L浓度的genistein作用24h,可明显促进肝纤维化Ⅰ级大鼠SEC NO的合成[NO合成为(25.4±3.8)μmol/L vs(16.6±3.3)μmol/L,t=6.79,P＜0.05];但对肝纤维化Ⅱ级、Ⅲ级大鼠SEC的NO合成的影响却不明显。结论 体外实验中genistein可以抑制肝纤维化Ⅰ级SEC增殖,促进SEC细胞NO的合成;对肝纤维化SEC细胞的功能有一定的调节作用。
Objective To investigate the effects of genistein, a tyrosine kinase inhibitor, on the proliferation, synthesis of nitric oxide (NO) in the sinusoidal endothelial cell (SEC) . Methods The SEC of normal and CCl 4 experimental hepatic fibrosis rats was isolated by collagenase in situ perfusion and Percoll discontinuous density gradient centrifugation. Using scanning electron microscopy, MTT and nitrate reductase method, the effect of genistein on the proliferation of cell and the synthesis of NO in SEC cells were observed. Results Genistein had no significant effect on the number and size of the SEC in each stage of liver fibrosis. After 24h of different concentrations of genistein, the growth of SEC in rats with hepatic fibrosis was inhibited. Genistein with the concentration of 100 μmol / L showed the most obvious effect on SEC in rats with grade Ⅰ hepatic fibrosis [cell proliferation rate was - (15.38 ± 6.26)% vs (4.91 ± 2.16)%, t = 13.7, P <0.05]. The genistein concentration of 100 μmol / L for 24 h significantly enhanced the SEC NO production in liver fibrosis rats [(25.4 ± 3.8) μmol / L vs (16.6 ± 3.3) μmol / L, t = 6.79, P <0.05]. However, the effect of NO on SEC synthesis in grade Ⅱ and grade Ⅲ hepatic fibrosis was not obvious. Conclusion Genistein can inhibit the proliferation of grade 1 SEC in hepatic fibrosis in vitro and promote the synthesis of NO in SEC cells. It can regulate the function of SEC cells in vitro.