大鼠远端肺动脉平滑肌细胞分离与原代培养

Isolation and primary culture of rat distal pulmonary artery smooth muscle cells

目的 探索简便、高效原代培养大鼠远端肺动脉平滑肌细胞的方法，为研究肺动脉高压发病机制提供实验材料。方法采用显微操作和分次酶消化法进行原代培养并与传统酶消化法比较。对培养的细胞进行形态学观察、平滑肌α-actin免疫荧光细胞化学法鉴定、激光扫描共聚焦显微镜计算纯度。结果 两种酶消化法均可获得高纯度的远端肺动脉平滑肌细胞。镜下细胞呈典型的“峰-谷”状生长，胞质特异的α-actin阳性表达，细胞纯度达98％。分次酶消化法获得的细胞数及细胞存活率均大于传统酶消化法，并且提前了3d得到足够进行实验的细胞数量。结论本法简便、可靠、低成本，短期内可获得大量高纯度、功能良好的远端肺动脉平滑肌细胞。

Objective To establish a simple and high performance method to isolate high purity pulmonary artery smooth muscle cells （PASMC） from rat distal pulmonary artery for researches of the pulmonary artery hypertension. Methods Microsurgery operations were performed to isolate rat distal pulmonary ＂artery. Step-by-step enzyme digestion method of isolated rat distal pulmonary artery to separate PASMCs was compared with traditional one step enzyme digestion method. Morphology of the isolated cells was observed by phase-contrast microscopy and identified by immunofluorescence assay using employed smooth muscle-α-actin antibody. The purity of the cell was checked by laser-scanning confocal fluorescence microscope. Results Both digesting methods produced high purity PASMCs, reached 98% purity. The PASMC developed typically ＂ peak and valley＂ growth pattern and expressed the smooth muscle specific differentiation marker α-actin. Compared with one-step method, step-by-step digestion of rat distal pulmonary artery produced more cells, and the cell vitality was also increased significantly. As a result of this, the days required to cultivate enough cell to conduct experiment decreased to 5 days, 3 days earlier than the traditional method. Conclusion The method presented here is a simple and efficient method to isolate and purify smooth muscle cells from distal pulmonary artery of rat.