Gremlin对小鼠原代肝星状细胞增殖和凋亡及细胞外基质分泌的影响

Effects of Gremlin on proliferation,apoptosis and extracellular matrix secretion of primary hepatic stellate cells in mice

目的探讨Gremlin对小鼠原代肝星状细胞增殖和凋亡及细胞外基质分泌的影响。方法采用经典肝脏二步酶灌注法分离小鼠原代肝星状细胞并培养。随机将肝星状细胞分为对照组、DMSO组及Gremlin低、高剂量组。对照组与DMSO组分别加入DMEM完全培养基、含0.05%DMSO的DMEM培养基培养,Gremlin低、高剂量组分别加入含100、200μg/L Gremlin的DMEM培养基培养。培养24 h,采用CCK-8法、流式细胞术检测各组细胞增殖率、细胞凋亡率,采用RT-q PCR法、Western blotting法检测各组CollagenⅠ、CollagenⅢ、a-SMA mRNA和蛋白表达。结果 Gremlin低、高剂量组细胞增殖率均高于对照组和DMSO组,细胞凋亡率均低于对照组和DMSO组(P均<0.05);Gremlin低剂量组与Gremlin高剂量组、对照组与DMSO组细胞增殖率、细胞凋亡率比较P均>0.05。Gremlin低、高剂量组CollagenⅠ、CollagenⅢ、a-SMA mRNA相对表达量均高于对照组、DMSO组(P均<0.05),且Gremlin高剂量组高于Gremlin低剂量组(P均<0.05),而对照组与DMSO组比较P均>0.05。Gremlin高剂量组CollagenⅠ、CollagenⅢ、a-SMA蛋白相对表达量均高于Gremlin低剂量组、对照组、DMSO组(P均<0.05),Gremlin低剂量组、对照组、DMSO组组间两两比较P均>0.05。结论 Gremlin能诱导小鼠原代肝星状细胞活化,使其分泌细胞外基质增加,从而参与肝纤维化的发生、发展。

Objective To investigate the effects of Gremlin on the proliferation,apoptosis,and extracellular matrix secretion of primary hepatic stellate cells（ HSCs） in mice. Methods Primary HSCs were isolated from mouse liver by two-step enzyme perfusion method. HSCs were randomly divided into the control group,DMSO group,and low-dose and high-dose Gremlin groups. The cells in the control group and the DMSO group were added with the DMEM complete medium and the DMEM medium containing 0. 05% DMSO,respectively. The cells in the low-dose and high-dose Gremlin groups were added with 100 and 200 ug/L Gremlin DMEM medium,respectively. The cell proliferation rate and apoptosis rate of each group were detected by CCK-8 and flow cytometry. The expression levels of Collagen I,Collagen III,a-SMA mRNA and protein were detected by RT-q PCR and Western blotting. Results The proliferation rate of low-dose and highdose group Gremlin groups was higher than that of control group and DMSO group,and the apoptotic rate was lower than that of control group and DMSO group（ both P〈0. 05）. There was no significant difference in the cell proliferation rate and apoptosis rate between low-dose group and high-dose Gremlin group and between the control group and DMSO group（ all P〈0. 05）. The relative mRNA expression of Collagen I,Collagen III,and a-SMA in the low-dose and high-dose Gremlin groups were higher than those in the control group and DMSO group（ all P〈0. 05）,and it was higher in the highdose Gremlin group than in the low-dose Gremlin group（ P〈0. 05）,but there was no significant difference between the control group and the DMSO group（ P〈0. 05）. The relative protein expression levels of Collagen I,Collagen III,and aSMA in the high-dose Gremlin group were higher than those in the low-dose Gremlin group,control group and DMSO group（ all P〈0. 05）. There was no significant difference between the low-dose Gremlin group,the control group,and the DMSO group（ P〉0. 05）. Conclusion Gremlin can induce the activation of primary HSCs and increase the secretion of extracellular matrix,thereby participating in the occurrence and development of liver fibrosis.