肝脏脱细胞基质微颗粒促进HepG2细胞球状体形成与肝相关功能分化的研究

Liver decellularized matrix microparticles promote the formation of HepG2 cell spheroids and the differentiation of liver-related function

目的探索肝脏脱细胞基质(dECM)微颗粒对3D培养HepG2细胞的肝相关功能状态的影响。方法通过肝脏门静脉灌流脱细胞液等成分,制得肝脏dECM。用组织学特殊染色、扫描电镜及免疫组织化学等方法鉴定脱细胞效果以及dECM成分。通过液氮冷冻研磨将dECM制备成微颗粒,通过差速离心分离不同粒径范围的微颗粒。利用超低吸附培养板进行HepG2细胞球状体3D培养,向培养基中加入dECM微颗粒,观察其对HepG2细胞球状体的形成及其肝相关功能状态的影响。利用ELISA、PCR检测相关蛋白的分泌和基因表达。结果制得的dECM无细胞结构,保留了细胞外基质的相关成分。在液氮研磨与差速离心分离后,可获得粒径在微米级的dECM微颗粒。dECM微颗粒可以促进HepG2细胞形成更致密的球状体;加入dECM微颗粒的HepG2球状体,细胞分泌白蛋白和尿素氮的水平增加(P<0.05);PCR结果表明,加入dECM微颗粒后,细胞的肝功能相关基因的表达水平显著提升(P<0.05)。结论肝脏脱细胞基质微颗粒可以促进HepG2细胞球状体的形成和HepG2细胞球状体的肝相关功能分化。

Objective To explore the effects of liver decellularized extracellular matrix(dECM)microparticles on liver-related functional status of HepG2 cells cultured in 3D spheroids.Methods Liver dECM was prepared by perfusing detergent fluids through portal vein.The decellularization effect and the components of dECM were determined by histological special staining,scanning electron microscopy and immunohistochemistry.The dECM was prepared into microparticles by liquid nitrogen freeze grinding,and the microparticles with different particle size range were separated by differential centrifugation.3D HepG2 cell spheroids were cultured by ultra-low attachment culture plate and dECM microparticles were added into the culture medium to observe their effects on the formation of HepG2 cell spheroids and the functional status of HepG2 cell spheroids.The secretion and gene expression of related proteins were detected by ELISA and PCR.Results Obtained dECM retained the components of the extracellular matrix and no cellular components were observed.dECM particles with a particle size of several microns could be obtained after liquid nitrogen grinding and differential centrifugation.dECM particles could promote the formation of HepG2 cell spheroids.And the levels of albumin and urea nitrogen in culture supernatant were increased after the addition of dECM particles(P<0.05);PCR result also showed that the expression levels of genes related to liver function were increased after the addition of dECM particles(P<0.05).Conclusion Liver decellularized matrix microparticles can promote the formation of HepG2 cell spheroids and the differentiation of liver-related function of HepG2 spheroids.