纤溶酶原激活物抑制因子-1对肝星状细胞活化及细胞外基质合成的影响

摘要

Objective To study the effect of plasminogen activator inhibitor type-1 (PAI-1) on the activation of hepatic stellate cells(LX-2) and extrocellar matrix. Methods The effect of PAI-1 on LX-2 proliferation was detected by Methyl thiazolyl tetrazolium(MTT). After incubation with PAI-1 for 12 h, the level of hyaluronic acid(HA) and transforming growth factor-β1 (TGFβ1) in LX-2 supernatant were detected with ELISA, and the level of smooth muscle α-actin (α-SMA),matrix metalloproteinase-2(MMP-2),tissue inhibitor of metalloproteinase-1 (TIMP-1), urokinase-type plasminogen activator(PLAU)and PAI-1 in LX-2 were detected by immunocytochemistry and the level of PAI-1 mRNA, TGFβ1 mRNA were investigated by reverse transcription polymerase chain reaction (RT-PCR). Results After incubation with PAI-1 for 12 h, the level of HA and TGFβ1 in the LX-2 supernatant increased significantly(t = 5. 474, t′= 5. 785, P＜0. 01 ). The protein expression of α-SMA, TIMP-1 and PAI-1 in LX-2 in the PAI-1 treatment group increased remarkally when compared to normal group as well (t = 5.438,9.511,4. 857, P＜0.01 ). The protein expression of MMP-2 and PLAU remain unchanged (t= 0. 473,0. 581 ,P＞0.05), while the level of PAI-1mRNA and TGFβ1mRNA in LX-2 increased significantly (t = 4. 683, t′ = 4. 135, P＜0. 01 ). There was a positive correlation between PAI-1 mRNA and TGFβ1mRNA(r = 0. 827,P＜0. 05). Conclusion PAI-1 may accelerate the formation and development of hepatic fibrosis and cirrhosis through activating LX-2, upregulating TGFβ1 mRNA and protein and promoting synthesis of extrocellar matrix.%目的 探讨外源性纤溶酶原激活物抑制因子-1(PAI-1)对人肝星状细胞(LX-2)活化及细胞外基质合成的影响.方法 采用四甲基偶氮唑盐法(MTT)检测培养液中加入PAI-1后LX-2细胞的增殖变化,确定PAI-1的最佳干预浓度.将LX-2培养液中加入PAI-1培养12 h,ELISA法检测细胞上清液中转化生长因子(TGFβ1)、透明质酸(HA)的变化;免疫细胞化学法检测LX-2细胞内α-平滑肌肌动蛋白(α-SMA)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶抑制剂-1(TIMP-1)、尿激酶型纤溶酶原刺激物(PLAU)及PAI-1蛋白变化;逆转录聚合酶链反应(RT-PCR)检测LX-2细胞内TGFβ1 mRNA和PAI-1 mRNA变化,分析其相关关系.结果 PAI-1对LX-2细胞增殖刺激作用明显,PAI-1浓度为10 ng/mL时刺激作用最为明显(F=11.697,P<0.01).加入PAI-1 12 h后(刺激组)较未加入PAI-1(对照组),细胞上清液中TGFβ1、HA表达显著增加(t=5.474,t'=5.785,P<0.01);LX-2细胞内ɑ-SMA、TIMP-1、PAI-1蛋白表达均显著增加(t=5.438、9.511、4.857,P<0.01),而MMP-2、PLAU蛋白表达差异无统计学意义(t=0.473、0.581,P>0.05);LX-2内TGFβ1 mRNA、PAI-1 mRNA增加显著(t=4.683,t'=4.135,P<0.01),TGFβ1与PAI-1 mRNA呈显著正相关(r=0.827,P<0.05).结论 PAI-1可通过活化LX-2,刺激LX-2 TGFβ1蛋白及mRNA的表达,促进细胞外基质生成,从而促进肝纤维化、肝硬化的形成和发展.