大鼠肺细小动脉平滑肌细胞培养新方法的建立及血小板衍生因子对其增殖迁移的影响

目的：建立一种操作简单、实验仪器要求低的大鼠肺细小动脉平滑肌细胞（PASMCs）分离和培养的方法，并且探索血小板衍生因子（PDGF）介导的增殖、迁移的情况。方法：向右心注射铁及琼脂糖，利用琼脂糖能同时粘附血管内皮细胞、平滑肌细胞及铁粉，再结合胶原酶I的消化，通过磁力架吸引铁，特异性地分选出带血管的肺组织，经过3~4周左右的培养及纯化，得到肺细小动脉平滑肌细胞。用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。MTT实验和划痕实验检测PDGF诱导的肺动脉细小平滑肌细胞的增殖和迁移。结果：分离后第14天、第21天及传代后进行鉴定，均表明分离培养的细胞为PASMCs。MTT结果表明，与不加PDGF组相比，PDGF增殖明显增加（P<0.05）。划痕实验结果显示PDGF刺激组比不刺激组迁移显著增多。结论：本方法分离培养大鼠的PASMCs，操作方便，实验仪器要求低。PDGF能够促进肺细小动脉平滑肌细胞的增殖、迁移。

A method of primary cell culture of rat pulmonary artery smooth muscle cells and effects of platelet-derived growth factor induced proliferation and migration

Objective:To establish an easy, not depending on advanced laboratory apparatus method to isolate and culture rat pulmonary artery smooth muscle cells (PASMCs), and to explore the effects of platelet-derived growth factor (PDGF) on cell proliferation and migration. Methods:The right ventricle was perfused with the mixture of iron, agarose, and the PASMCs and iron could adhere to agarose. The iron-con-taining tissue would move to side of the tube next to the magnet and could be digested by collagenase I. By the method, vessel-containing tissue could be attained. With 3-4 weeks' purification, the PASMCs could be obtained. The PASMCs morphology was observed by an inverted micro-scope, and identified by immunocytochemistry and immunofluorescence. The effects of PDGF on cell proliferation and migration was detected by MTT assay and scratch wound assay. Results:14 days、21 days and primary culture after isolation, the PASMCs was identified, and the re-sult showed that isolation and primary culture of the cells were PASMCs. Compared with the cells with no stimulation, the proliferation of PASMCs exposed to PDGF was increased significantly(P<0.05), and scratch wound assay demonstrated that PDGF induced the significant increase of migration of PASMCs. Conclusion:This method to isolate and culture rat PASMCs is simple, not depending on advanced laborato-ry. PDGF can promote the proliferation and migration of PASMCs.