组织块贴壁法行SD大鼠肺内动脉段平滑肌细胞的原代培养及其鉴定

Primary culture of SD-rats pulmonary artery smooth muscle cells and it's identification

目的研究采用组织块贴壁法进行SD大鼠肺内动脉段平滑肌细胞(PASMCs)原代培养的方法特点及其生物学特性。方法将SD大鼠消毒后剖开胸腹腔,无菌下取出心肺组织,然后分离出肺血管段,用组织块贴壁法培养,倒置相差显微镜观察细胞形态、普通免疫组织化学法(SP法)和免疫荧光法进行细胞鉴定。结果组织块贴壁法培养的SD大鼠PASMCs为典型的"峰-谷"样生长状态,细胞的胞核较大,免疫组织化学法和免疫荧光法检测α-actin均为强阳性表达,免疫荧光法检测传至三代以后的细胞纯度可达98%以上,传代细胞生长未见明显异常改变。结论在不使用显微技术情况下,用组织贴块法成功培养大鼠PASMCs,得到稳定生长、纯度高的PASMCs,且培养与纯化可以同时进行,可获得稳定传代的细胞。

Objective To study method and characteristics of SD-rats pulmonary artery smooth muscle cells（PASMCs） with primary culture by tissue-pieces inoculation,then identify their biological properties.Methods SD-rats was disinfected,dissected in turn.Then lung was catched from chest under aseptic condition.Pulmonary artery was separated and pulmonary artery tissue was planted with the adherent method of tissue explants.The cellular morphology and their typical growth condition were observed with inverted phase contrast microscope.Morphology of the isolated cells were observed by phase-contrast microscopy and identified by immunocytochemistry and immunofluorescence assay using smooth muscle-α-actin antibody.Results The PASMCs was successfully cultured by the adherent method of tissue explants and growed in the ＂peak and valley＂ mode under microscope,the round cell nucleus was big.Immunohistochemical method showed strong expression of α-actin,reached 98% purity after digestion three times.Conclusion Under no micro engineering factor,the primary culture PASMCs with the adherent method of tissue explants can stably grow.Culture and purification can perform in the same time.