体外培养诱导人肝脏星形细胞表型激活及其鉴定

In vitro culture-induced activation of human hepatic satellite cells and characterization of activated phenotype

目的 :观察常规培养条件下正常人肝脏星形细胞 (HSCs)表型激活并对其进行鉴定。方法 :由正常人肝脏中分离HSCs ,在未包被的玻璃及塑料培养支持物上 ,以常规的含小牛血清的DMEM培养 ,相差显微镜下观察培养过程中细胞形态改变 ,荧光显微镜下观察维生素A自发荧光 ,免疫细胞化学染色观察α SMA、Ⅰ型胶原、FN及PCNA改变。结果 :正常人HSCs未包被的玻璃及塑料培养支持物上培养时 ,其表型由刚分离时及原代培养初期的静息型转变为原代培养后期及传代后的激活型。激活的人HSCs呈现典型的成纤维细胞样形态 ,胞质中无明显脂滴 ,维生素A自发荧光明显减弱或消失 ,同时明显表达α SMA、Ⅰ型胶原、FN及PCNA。结论 :体外常规培养可诱导人HSCs激活 ,激活的人HSCs具有典型的形态及功能特点。

Objective: To induce activation of normal human hepatic satellite cells (HSCs) by in vitro culture and characterize the activated phenotype Methods: HSCs were isolated from normal human liver and cultured on uncoated plastic and glass materials in DMEM containing bovine serum HSCs were observed with phase contrast and fluroscence microscopy at various intervals in primary and secondary cultures α smooth muscle actin (α SMA), type I collagen, fibronectin (FN) and PCNA expression in the cells were detected with immunohistochemistry Results: Freshly isolated HSCs during early days of primary culture maintained a quiescent phenotype During late days of primary culture or after subculture, HSCs assumed an activated phenotype Activated HSCs were characterized by a typical fibroblast like morphology under the phase contrast microscope, a decrease or loss in fat droplets and vitamin A autofluroscence, and increase or obvious positive immunohistochemical staining for α SMA, type I collagen, FN and PCNA Conclusion: Routine in vitro culture on uncoated plastic or glass in DMEM containing bovine serum could induce phenotype activation of normal human HSCs and this activation was associated with characteristic morphological and functional changes