[Effect of porcine small intestinal submucosa extracellular matrix in promoting vitality and functional gene expression of hepatocyte].

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 ( ) 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 ( >0.05). After cultured with PSISM hydrogels, the values of groups A2, B2, and C2 were significantly higher than those of group D2 at 3 and 5 days ( <0.05), the value of group A2 was significantly higher than that of groups B2 and C2 at 5 days ( <0.05), but there was no significant difference between groups at other time points ( >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 ( <0.05). The relative expression of CK18 mRNA in group C1 was significantly lower than that in groups A1 and B1 ( <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 ( <0.05); the relative expression of CK18 mRNA in group A2 was significantly higher than that in group B2 ( <0.05), and the relative expression of AFP mRNA in group A2 was significantly lower than that in group C2 ( <0.05), but no significant difference was found between other groups ( >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.