不同贴壁法培养原代大鼠肺动脉平滑肌细胞比较
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摘要
目的比较改良贴壁法和传统贴壁法对大鼠肺动脉平滑肌细胞(PASMCs)原代培养的差异,为研究肺血管疾病提供良好的体外模型。方法分别用传统贴壁法和改良贴壁法分离大鼠PASMCs,倒置显微镜比较两种方法的细胞形态;α-肌动蛋白细胞(α-SMA)免疫组织化学和细胞免疫荧光法对细胞进行鉴定;细胞计数法获得细胞生长曲线观察细胞生长情况;MTS法检测低氧或血小板源性生长因子处理48 h对原代培养的PASMCs增殖的影响。结果显微镜下两种方法获得的PASMCs细胞为长梭形,呈典型的"峰-谷"状结构;改良贴壁法细胞爬出数量高于传统贴壁法。α-SMA细胞免疫化学和细胞免疫荧光检测显示α-SMA主要表达在胞浆,第3代细胞阳性率达100%。MTS检测显示低氧或血小板源性生长因子刺激48 h可显著刺激PASMCs增殖(P<0.01)。结论虽然两种组织块贴壁培养方法均简单易行,获得的细胞性状稳定;但改良贴壁法较传统贴壁法培养周期更短,获得细胞数量更多。
Objective To provide better in vitro models to study pulmonary vascular diseases by comparing the difference in rat pulmonary arterial smooth muscle cells(PASMCs) cultured by improved and traditional tissue block anchorage. Methods Rat PASMCs were extracted by improved and traditional method of tissue block anchorage, and the cell morphology was observed under inverted microscope. The cells were identified by alpha-smooth muscle actin(α-SMA) immunohistochemistry and immunofluorescence. The cell growth was analyzed by plotting cell growth curve. MTS assay determined the influence of hypoxia or platelet-derived growth factor(PDGF) on the proliferation of primary PASMCs. Results The cells obtained by both methods tended to be long spindle and grew in the "peak-valley" mode under inverted microscope. The number of cells around the tissue by the improved method was higher than that by the traditional method. Immunology showed that endochylema was stained in brownish yellow, and positive rate was 100%. MTS assay demonstrated that exposure to hypoxia and PDGF for 48 h significantly promoted the PASMCs proliferation(P < 0.01). Conclusion Both methods are simple and convenient, with highly stable rat PASMCs, but the improved method obtains more cells with shorter culture cycle.
Objective To provide better in vitro models to study pulmonary vascular diseases by comparing the difference in rat pulmonary arterial smooth muscle cells(PASMCs) cultured by improved and traditional tissue block anchorage. Methods Rat PASMCs were extracted by improved and traditional method of tissue block anchorage, and the cell morphology was observed under inverted microscope. The cells were identified by alpha-smooth muscle actin(α-SMA) immunohistochemistry and immunofluorescence. The cell growth was analyzed by plotting cell growth curve. MTS assay determined the influence of hypoxia or platelet-derived growth factor(PDGF) on the proliferation of primary PASMCs. Results The cells obtained by both methods tended to be long spindle and grew in the "peak-valley" mode under inverted microscope. The number of cells around the tissue by the improved method was higher than that by the traditional method. Immunology showed that endochylema was stained in brownish yellow, and positive rate was 100%. MTS assay demonstrated that exposure to hypoxia and PDGF for 48 h significantly promoted the PASMCs proliferation(P < 0.01). Conclusion Both methods are simple and convenient, with highly stable rat PASMCs, but the improved method obtains more cells with shorter culture cycle.
引文
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[2]GalièN,Humbert M,Vachiery JL,et al.2015 ESC/ERS Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension[J].Rev Esp Cardiol(Engl Ed),2016,69(2):177-177.
[3]朱丹,江振洲,张陆勇.野百合碱致肺动脉高压相关机制研究进展[J].中南药学,2011,9(6):456-459.
[4]Crosswhite P,Sun Z.Molecular mechanisms of pulmonary arterial remodeling[J].Mol Med,2014,20(1):191-201.
[5]王静,戴爱国.原代大鼠肺动脉平滑肌细胞的提取和鉴定以及缺氧对其增殖的影响[J].中国呼吸与危重监护杂志,2012,11(2):147-152.
[6]肖欣荣,陈文彬,程德云.大鼠肺动脉平滑肌细胞的分离及鉴定[J].华西医科大学学报,1998,29(3):241-243.
[7]钱国清,王良兴,陈婵,等.大鼠细小肺动脉平滑肌细胞原代培养和鉴定方法的研究[J].中国应用生理学杂志,2010,26(1):125-128.
[8]易斌,钱桂生,白莉,等.不同方法培养大鼠肺动脉平滑肌细胞的差异[J].第三军医大学学报,2005,27(24):2425-2427.
[9]Morrell NW,Adnot S,Archer SL,et al.Cellular and molecular basis of pulmonary arterial hypertension[J].J Am Coll Cardiol,2009,54(1 Suppl):S20-S31.
[10]张卫芳,熊爱珍,吴卫华,等.肺动脉高压时肺血管内皮间质转化相关mi RNAs网络调控的生物信息学分析[J].中国药理学通报,2016,32(9):1294-1230.
[11]Ranchoux B,Antigny F,Rucker-Martin C,et al.Endothelial-to-mesenchymal transition in pulmonary hypertension[J].Circulation,2015,131(11):1006-1018.
[12]Lin F,Wang N,Zhang TC.The role of endothelial-mesenchymal transition in development and pathological process[J].IUBMB Life,2012,64(9):717-723.