OPN促进大鼠肝星状细胞黏附、迁移及黏着斑激酶活性

Osteopontin promotes adhesion and migration of hepatic stellate cells and FAK phosphorylation

目的探讨细胞外间质(ECM)成分骨桥蛋白(OPN)和精-甘-天冬-丝氨酸(Arg-G ly-Asp-Ser,RGDS)四肽对大鼠肝星状细胞(HSCs)黏附、迁移与黏着斑激酶(FAK)活性的影响。方法应用体外细胞培养技术,MTT法检测HSCs增殖,甲苯胺蓝法检测HSCs黏附情况,并观察HSCs迁移;采用免疫沉淀和W estern b lot技术测定黏着斑激酶活性变化。结果①不同浓度OPN和RGDS四肽作用于HSCs 2 h后,8、16、32 mg/L OPN组细胞黏附促进率均高于对照组(P<0.05);25 mg/L^100 mg/L RGDS四肽不同浓度组细胞黏附率明显下降(P<0.05)。②不同浓度OPN干预细胞48 h后,促进HSCs增殖,而RGDS四肽组抑制HSCs增殖。③16 mg/L OPN作用12、24和36 h,细胞迁移距离显著高于对照组(P<0.05);而100 mg/L RGDS四肽组则显著性低于对照组(P<0.05)。④经OPN作用后各浓度组HSCs中磷酸化FAK均有显著表达;RGDS四肽干预后,磷酸化FAK表达量降低。结论①OPN促进HSCs增殖、黏附和迁移;RGDS四肽对HSCs增殖、黏附和迁移具有抑制作用。②OPN可以诱导FAK活化,RGDS四肽可抑制磷酸化FAK的表达。

Objective To evaluate the effect of osteopontin （OPN） on the adhesion and migration of hepatic stellate cells （HSC） and focal adhesion kinase （FAK）phosphorylation. Methods The proliferation of HSCs was determined by MTT. The adhesion rates were shown by toluidine blue colorimetric assay. The HSC migration rates were examined by HE staining. The expression of FAK phosphorylation was examined by Western blotting analysis. Results （1）Compared with control group, OPN at concentrations of 8 mg/L, 16 mg/L and 32 mg/L promoted the adhesion of HSCs（P 〈0. 05 ） ; RGDS tetrapeptide at concentrations of 25 mg/L, 50 mg/L and 100 mg/L inhibited the adhesion of HSCs. The inhabition rates were 13.34%, 16. 55% and 36. 31%, respectively （ P 〈 0. 05 ）. （2）After adding OPN to HSC culture media for 48 h, OPN at different concentrations promoted proliferation rates; RGDS tetrapeptide inhabited HSC proliferation. （3） The HSC migrated distances of OPN at concentration of 16 mg/L at 12 h, 24 h, and 36 h were （93.00 ± 4. 83） μm, （ 114.00 ± 9. 66） μm and （ 121.00 ± 17. 29） μm, respectively. The HSC migrated distances of RGDS tetrapeptide （ at concentration of 100 mg/L） at 12 h, 24 h and 36 h were （51.00 ±9.94）μm, （46. 00 ±6. 99） μm and （24.00 ±9. 66） μm. （4）The expressions of phosphorylation of FAK in OPN groups at concentrations of 8 mg/L, 16 mg/L and 32 mg/L were increased by 6. 98%, 14. 81% ,11.56%, respectively, which were reduced by 8.46%, 35.69%, 57.29% when RGDS tetrapeptide was added at concentration of 25 mg/L, 50 mg/L, 100 mg/L. Conclusion （1）OPN promotes proliferation, adhesion and migration of HSCs and RGDS tetrapeptide inhabits proliferation, adhesion and migration of HSCs promoted by OPN.（2）Proliferation, adhesion and migration of HSCs stimulated by OPN may be mediated by upregulating pro- tein expression of FAK phosphorylation, but RGDS tetrapeptide could reduce it.