中国修复重建外科杂志

中国修复重建外科杂志

猪小肠黏膜下层细胞外基质促进肝细胞活力和功能基因表达的研究

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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 μg/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 D1 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, theA values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days (P<0.05), theA 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 of AFP 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 of AFP 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.

关键词: 肝脏组织工程; 肝细胞; 猪小肠黏膜下层; 细胞外基质; 凝胶

Key words: Liver tissue engineering; hepatocyte; porcine small intestinal submucosa; extracellular matrix; hydrogel

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