组织块法培养人胃成纤维细胞及其结缔组织生长因子的表达

Expression of connective tissue growth factor in primary-cultured human gastric fibroblasts

关键词:  细胞培养  人胃成纤维细胞  结缔组织生长因子

Key words:  connective tissue growth factor  human gastric fibroblasts  cell culture

摘要:

[目的] 探索人胃成纤维细胞体外培养的技术方法及其结缔组织生长因子表达情况。[方法] 采用组织块贴壁培养法进行人胃成纤维细胞体外培养并传代，倒置相差显微镜下观察成纤维细胞形态，并用免疫细胞化学法检测培养细胞的波形蛋白与角蛋白表达。用RT-PCR法检测人胃成纤维细胞结缔组织生长因子（CTGF）mRNA表达，免疫细胞荧光染色法检测人胃成纤维细胞CTGF蛋白表达情况。[结果] 人胃成纤维细胞培养成功传代，表现为长梭形或星形细胞，免疫细胞化学鉴定细胞波形蛋白阳性，角蛋白阴性。RT-PCR法与免疫细胞荧光染色法证实体外培养的人胃纤维细胞表达CTGF。[结论] 组织块法培养的人胃成纤维细胞可在体外稳定培养、传代，人胃成纤维细胞能合成CTGF。

Abstract:

[Objective] To explore a method of the isolation, culture and identification of human fibroblasts in vitro, and study the expression of connective tissue growth factor (CTGF) in human gastric fibroblas. [Methods] Tissue explants were used to culture the human gastric fibroblast in vitro, and then the primary cultured cells were purified, passaged and stored in liquid nitrogen. We observed their growth and morphology under inverted phase contrast microscope, and the expressions of vimentin and keratin in the cultured cells by immunocytochemical staining in order to identify the origin of the cell. We survey the expression of CTGF in the cultured human gastric fibroblast by RT-PCR and immunocyte fluorescent staining. [Results] Human gastric fibroblasts cultured by tissue explant grew well and passaged stably. The cultured cells present long spindle or star shape. The results of immunocytochemical staining show that staining of vimentin is positive and the staining of keratin is negative. The primary cultured human gastric fibroblasts express CTGF mRNA and protein by RT-PCR and immunocyte fluorescent staining methods. [Conclusions] The human gastric fibroblasts cultured from tissue explant can stably passage and secrete CTGF.