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目的 探讨猪小肠黏膜下层细胞外基质(porcine small intestinal submucosa extracellular matrix,PSISM)对肝细胞活力及相关基因表达的调控,为PSISM应用于肝脏组织工程提供实验依据.方法 实验分为两部分.①取PSISM添加至含10％FBS的H-DMEM培养基中,制备终浓度为50、100、200 μtg/mL的PSISM培养基;将大鼠肝细胞系BRL细胞分别用以上浓度PSISM培养基培养(A1、B1、C1组),以单纯H-DMEM培养基培养作为对照(D1组).②取PSISM溶解于含0.1 mol/L NaOH的PBS无菌溶液中,调整溶液浓度为1％、2％、3％,制备PSISM凝胶;将BRL细胞分别接种于以上浓度PSISM凝胶进行培养(A2、B2、C2组),鼠尾Ⅰ型胶原凝胶作为对照(D2组).培养后第1、3、5天,采用Live/Dead荧光染色观察细胞形态及存活情况,细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)检测细胞活力,实时荧光定量PCR检测肝细胞特异基因白蛋白(albumin,ALB)、细胞角蛋白18 (cytokeratin 18,CK18)和甲胎蛋白(alpha-fetoprotein,AFP)的表达.结果 Live/Dead荧光染色显示各组细胞均存活良好.细胞活力检测显示,PSISM培养基培养后,第5天C1组吸光度(A)值显著高于D1组(P＜0.05),其余各时间点各组间比较差异均无统计学意义(P＞0.05);PSISM凝胶培养后,A2、B2、C2组第3天和第5天A值显著高于D2组(P＜0.05),A2组第5天A值显著高于B2、C2组(P＜0.05),其余各时间点各组间比较差异均无统计学意义(P＞0.05).实时荧光定量PCR检测显示,A1、B1、C1组ALB、CK18基因相对表达量较D1组显著增高(P＜0.05)、AFP基因相对表达量降低(P＜0.05),C1组CK18基因相对表达量显著低于A1、B1组(P＜0.05);A2、B2、C2组ALB、CK18基因相对表达量较D2组显著增高(P＜0.05)、AFP基因相对表达量降低(P＜0.05),A2组CK18基因相对表达量显著高于B2组(P＜0.05)、AFP基因相对表达量显著低于C2组(P＜0.05).其余各组间各基因相对量表达比较,差异均无统计学意义(P＞0.05).结论 PSISM无毒性,对肝细胞有良好相容性,能促进肝细胞活力和功能基因表达,有望作为细胞培养添加物或细胞移植载体用于肝脏组织工程.“,”Objective To investigate the effect of porcine small intestinal submucosa extracellular matrix (PSISM) on the vitality and gene regulation of hepatocyte so as to lay the experimental foundation for the application of PSISM in liver tissue engineering.Methods The experiment was divided into two parts:① BRL cells were cultured with 50,100,and 200 μg/mL PSISM-medium which were prepared by adding PSISM into the H-DMEM-medium containing 10％FBS in groups A1,B1,and C1,and simple H-DMEM-medium served as a control (group D1);② BRL cells were seeded on 1％,2％,and 3％ PSISM hydrogel which were prepared by dissolving PSISM in sterile PBS solution containing 0.1 mol/L NaOH in groups A2,B2,and C2,and collagen type Ⅰ gel served as a control (group D2).At 1,3,and 5 days after culture,the morphology and survival of liver cells were detected by the Live/Dead fluorescent staining.The cell vitality was tested by cell counting kit-8 (CCK-8) assay.And the relative expressions of albumin (ALB),cytokeratin 18 (CK18),and alpha-fetoprotein (AFP) in hepatocytes were determined by real-time fluorescent quantitative PCR (RT-qPCR).Results The Live/Dead fluorescent staining showed the cells survived well in all groups.CCK-8 results displayed that the absorbance (A) value of group C1 was significantly higher than that of group D 1 at 5 days after culture with PSISM-medium,and there was no significant difference between groups at other time points (P＞0.05).After cultured with PSISM hydrogels,the A values of groups A2,B2,and C2 were significantly higher than those of group D2 at 3 and 5 days (P＜0.05),the A value of group A2 was significantly higher than that of groups B2 and C2 at 5 days (P＜0.05),but there was no significant difference between groups at other time points (P＞0.05).RT-qPCR showed that the relative expressions of ALB and CK18 mRNA significantly increased and the relative expression ofAFP mRNA significantly decreased in groups A1,B1,and C1 when compared with group D1 (P＜0.05).The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 (P＜0.05).The relative expressions of ALB and CK18 mRNA were significantly higher and the relative expression of AFP mRNA was significantly lower in groups A2,B2,and C2 than group D2 (P＜0.05);the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 (P＜0.05),and the relative expression ofAFP mRNA in group A2 was significantly lower than that in group C2 (P＜0.05),but no significant difference was found between other groups (P＞0.05).Conclusion PSISM has good compatibility with hepatocyte and can promote the vitality and functional gene expression of hepatocyte.PSISM is expected to be used as culture medium supplement or cell carrier for liver tissue engineering.