胶原水凝胶在HepG2.2.15细胞三维培养中的应用研究

Use of a collagen hydrogel to create a three-dimensional culture model of HepG2.2.15cells

目的了解用胶原水凝胶为支架构建HBV转染细胞HepG2.2.15的三维(three-dimensiona,3D)培养模型的应用情况。方法采用扫描电镜(SEM)和原子力显微镜(AFM)观察水凝胶内胶原纤维的结构特征,以胶原水凝胶为支架建立HepG2.2.15细胞3D培养模型。采用倒置显微镜观察细胞形态特征;采用MTS法和钙黄绿素-AM(Ca-AM)染色法检测细胞的增殖活性;采用ELISA法检测细胞HBsAg、HBeAg的分泌;采用荧光探针PCR法检测HBV DNA的表达。结果HepG2.2.15细胞在96孔板胶原水凝胶中能培养45d,在24孔板内培养则长达160d,且生长良好,并能维持转染细胞特性,持续分泌HBsAg、HBeAg和HBV DNA。结论建立的胶原水凝胶HepG2.2.15细胞3D培养模型能长时间维持细胞增殖。

Objective To understand the use of a three-dimensional(3D)culture model with which to construct HBVtransfected HepG2.2.15cells using collagen hydrogel as a scaffold in vitro.Methods The structural characteristics of collagen hydrogel were characterized using a scanning electron microscope(SEM)and atomic force microscope(AFM).HepG2.2.15cells were cultured in collagen hydrogel as a three-dimensional scaffold.The morphological characteristics of HepG2.2.15cells in 3Dculture were observed using an inverted microscope.The proliferative activity of HepG2.2.15cells in 3Dculture was detected using the MTS method and the calcein-AM(Ca-AM)method.The secretion of HBsAg and HBeAg by HepG2.2.15cells in 3Dculture was detected using ELISA.The expression of HBV DNA by HepG2.2.15cells in collagen hydrogel was detected using fluorescence probe PCR.Results HepG2.2.15cells were cultured in collagen hydrogel on 96-well plates for 45days and on 24-well plates for up to 160days.This allowed the transfected cells to retain their characteristics and to continue secreting HBsAg,HBeAg,and HBV DNA.Conclusion A model for 3Dculture of HepG2.2.15cells in collagen hydrogel sustained cell proliferation for a prolonged period.