肝脱细胞基质溶液包被对HepG2细胞增殖及基质金属蛋白酶活性的影响

摘要

Objective:To establish a new method to prepare decellularized liver matrix solution using porcine liver tissues and investigate the effects of decellularized liver matrix solution coating on the proliferation and matrix metalloproteinase activity of in vitro cultured HepG2 cells.Methods:Decellularized liver matrix solution was obtained through decellularization of porcine liver slices,grind,lyophilization and pepsin digestion.HepG2 cells were cultured on the matrix coated polystyrene substrates.The proliferation,gene expression and activity of matxix metalloproteinases (MMPs) were evaluated by cell counting kit-8,real-time PCR and gelatin zymography,respectively.Results:The new method for the preparation of decellularized liver matrix solution was established.The proliferation of matrix coated HepG2 cells was significantly increased during culture.The cyclin D1 gene expression had a 1.95-fold increase compared to uncoated group (P＜0.01);the metastasis related MMP14 gene expression had a 2.01-fold increase over the uncoated group(P＜0.05),and a 6.66-fold increase of MMP9 activity were found in matrix coated group (P＜0.01).Conclusion:Decellularized liver matrix solution coating significantly promotes the proliferation and the MMP9 activity of in vitro cultured HepG2 cells,and has the potential to be applied in HCC cell cultures to simulate in vivo HCC microenvironment.%目的:建立由猪肝组织制备肝脱细胞基质溶液的新方法,并研究基质溶液包被对人肝癌细胞HepG2增殖及基质金属蛋白酶活性的影响.方法:对猪肝脏进行脱细胞处理,经研磨、冻干及胃蛋白酶消化制备肝脱细胞基质溶液,利用此基质溶液包被聚苯乙烯培养表面,进行HepG2细胞体外培养,并使用CCK-8、实时定量PCR、明胶酶谱等检测细胞增殖以及基质金属蛋白酶的基因表达和活性.结果:建立了由猪肝组织制备肝脱细胞基质溶液的新方法.与未包被组相比,脱细胞基质溶液包被培养显著促进HepG2细胞增殖,其细胞周期蛋白cyclin D1的基因表达增高,为未包被组的1.95倍(P＜0.01).此外,脱细胞基质溶液包被组HepG2细胞的MMP14基因表达为未包被组的2.01倍(P＜0.05),明胶酶谱检测基质溶液包被组细胞的MMP9活性为未包被组的6.66倍(P＜0.01).结论:肝脱细胞基质溶液包被培养可显著促进HepG2细胞增殖并提高其MMP9的活性,在构建模拟肝癌微环境培养体系中具有一定的应用潜力.