小鼠肺细小动脉平滑肌细胞原代培养和鉴定以及低氧对其增殖与凋亡的影响

Primary culture and identification of mouse PASMCs and effects of hypoxia on proliferation and apoptosis of PASMCs

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 arteriolar 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 during the cell passage. The cell morphology was observed under an inverted phase-contrast microscope. α-Smooth muscle actin was identified by immunocytochemistry and immunofluorescence. The effects of hypoxia on the proliferation and apoptosis 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 demonstrated 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.