一种大鼠肺静脉平滑肌细胞快速培养方法的建立及评价

The establishment and evaluation of a rapid methed of rat＇s pulmonary vein smooth muscle cells culture

目的通过改进原代大鼠肺静脉平滑肌细胞（pulmonary vein smooth muscle cells,PVSMCs）的培养方案，以期建立快速、方便、高细胞活力的肺静脉平滑肌细胞培养方法，为肺静脉在肺血管相关疾病研究中提供适合的细胞模型。方法体视镜下显微分离大鼠肺静脉，胶原酶消化后获取原代PVSMCs，分别用低糖DMEM培养基、DMEM—F12培养基、高糖DMEM培养基作为培养液，观察PVSMCs的生长情况和细胞形态，对平滑肌a—actin进行免疫荧光鉴定，并用实时荧光法检测PVSMCs对高钾溶液引起的细胞内钙浓度升高的反应，评价细胞活力。结果PVSMCs在3种培养基中的生长速率比较：DMEM-F12〉高糖DMEM〉低糖DMEM。DMEM-F12中培养的PVSMCs可见细胞质中有典型的、并行排列的、绿色荧光显色的平滑肌α—actin蛋白，细胞核呈蓝色荧光，细胞纯度达95％以上，而阴性对照只见染蓝光的细胞核。细胞对高钾溶液具有良好反应。结论DMEM-F12培养基中PVSMCs生长速率最大，细胞符合平滑肌细胞免疫结构特征，并具有良好的细胞功能。DMEM-F12培养基更适合PVSMCs的培养。

Objective To optimize the procedure of primary rat＇s pulmonary vein smooth muscle cells （PVSMCs） culture in order to set up a rapid, convenient and high-vitalizing method and provide with a suitable cell model to the research in pulmonary vascular related diseases. Methods The primary PVSMCs were obtained from isolation of the rat＇s pulmonary veins by micromanipulation and digestion with collagenase. During the cultivation in DMEM with low glucose, DMEM-F12 and DMEM with high glucose, the growth rate and the morphological changes of PVSMCs were observed, PVSMCs were identified by immunofluorescence staining with a-smooth muscle actin, the vitality of PVSMCs was defined by high potassium solution on intracellular calcium concentration with fluorescence microscopy. Results PVSMCs growth rate in three kinds of cultural solutions showed DMEM-F12〉DMEM with high glucose〉DMEM with low glucose. Immunofluorescence demonstrated typically juxtaposed a-smooth muscle actin protein fiber in DMEM-F12, while blue dyed cell nucleus which had good response to high potassium solution were only seen in negative control group. And the purity can be up to 95%. The PVSMCs reacted well to the high potassium solution. Conclusions The PVSMCs cultured in DMEM-F12 showed high proliferated rate with healthy cellular function, conforming with the smooth muscle cellular immunity structure, which demonstrated it was the optimal method for PVSMCs culture.