目的：建立一种方法简单、重复性好、培养周期短的小鼠肺细小动脉平滑肌细胞（ PASMCs ）原代培养及传代方法，并初步探索低氧下小鼠PASMCs的增殖与凋亡情况。方法：无菌条件显微镜下分离小鼠肺细小动脉，经胶原酶I消化结合血清铺底法培养PASMCs。传代过程中弃除对细胞损伤大的离心步骤。倒置相差显微镜观察细胞形态，免疫细胞化学法和免疫荧光染色法进行α-平滑肌肌动蛋白鉴定，CCK-8显法及TUNEL法测定低氧下PASMCs的增殖与凋亡情况。结果：形态学观察、免疫细胞化学法和免疫荧光染色法鉴定均表明培养的细胞为PASMCs。与大鼠传代后的PASMCs典型的“峰-谷”样生长相比，传代后的小鼠PASMCs形态各异，没有此典型“峰-谷”规律。原代培养后5～7 d即传代，依据细胞活性可传3～5代。 CCK-8法显示，与常氧组24 h比较，低氧组A值升高（P＜0．05）。 TUNEL法结果显示低氧24 h后凋亡率较常氧组下降（P＜0．05）。结论：胶原酶I消化结合血清铺底法培养小鼠PASMCs，方法简单，培养周期短，重复性好，是一种值得推广的小鼠PASMCs体外培养方法。低氧可以促进小鼠PASMCs的增殖，抑制小鼠PASMCs的凋亡。%AIM:To establish a fast , accurate and economical technique for culturing mouse pulmonary arte-riolar smooth muscle cells ( PASMCs ) , and to explore the effects of hypoxia on the proliferation and apoptosis of the PASMCs.METHODS:In sterile condition, the pulmonary artery was isolated from the male BALB/c mice by digesting with collagenase I, and the cells were cultured in fetal bovine serum-coated flask.Centrifugal procedure was not used dur-ing the cell passage .The cell morphology was observed under an inverted phase-contrast microscope .α-Smooth muscle ac-tin was identified by immunocytochemistry and immunofluorescence .The effects of hypoxia on the proliferation and apopto-sis of the PASMCs were detected by CCK-8 assay and TUNEL assay .RESULTS:PASMCs were identified by the methods of immunocytochemistry , immunofluorescence staining and observation of morphology .Unlike the rat PASMCs with typical subcultured peak-vally pattern, the mouse PASMCs showed a lot different without a peak-vally pattern.The cells could be subcultured after 5 d to 7 d and there was 3 to 5 generations depending on the activity of the cells .CCK-8 assay demonstra-ted that the A values of PASMCs exposed to hypoxia increased after 24 h ( P<0.05) as compared with normoxia .TUNEL result showed that the apoptotic index of the PASMCs in hypoxia decreased after 24 h (P<0.05).CONCLUSION:This technique for obtaining cultured mouse PASMCs is simple , fast, accurate and economical .The digestion time is easy to control.Hypoxia promotes the proliferation and inhibits the apoptosis of PASMCs .
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