改良组织块法成人主动脉血管平滑肌细胞的培养

Modified explant technique for the culture of human vascular smooth muscle cells

目的建立成人主动脉血管平滑肌细胞体外培养的有效方法。方法无菌条件下分离成人主动脉的平滑肌层，剪成1mm^3的碎片，0．1％I型胶原酶预处理1h，组织块贴壁法，以含胎牛血清20％的DMEM低糖培养基培养。倒置显微镜观察培养细胞的形态学特点，免疫荧光法检测平滑肌细胞肌动蛋白（α-SMA）的表达。结果培养至4-5d组织块边缘即有少量细胞爬出，呈长梭形，胞质丰富；培养至1-2周组织块边缘细胞融合，呈典型的“峰谷”样表现。免疫荧光染色结果显示胞质内α-SMA的表达丰富，荧光强度在＋＋--＋＋＋。结论胶原酶预处理组织的改良组织块贴壁法培养人主动脉血管平滑肌细胞是一种可行有效的实验方法。

Objective To establish an effective method of human vascular smooth muscle cells in vitro culture. Methods Medial layer of human aorta was separated under sterile conditions, then cut into small pieces （ about 1 mm3 ）. After pretreatment with 0. 1% type I collagenase, the tissues were trans- ferred into flask and cultured in DMEM containing 20% fetal bovine serum, penicillin （ 100 U/ml） and streptomycin （ 100 g/L）. The cell morphology was observed under the inverted microscopy. Smooth muscle cell specific protein （a-SMA） was identified by using immumofluorescence methods. Results Some smooth muscle cells started growing from explants in 4 to 5 days. One to 2 weeks later, the cells grew to confluence by migrated cells. Immumofluorescence staining showed positive expression of α-SMA （ fluores- cence intensity ＋＋ -＋＋＋）. Conclusion Modified explant technique by pretreating tissues with collagenase was an effective method to get vascular smooth muscle cells in vitro.