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目的研究血管组织工程中血管平滑肌细胞体外培养的有效方法。方法无菌条件下取3cm左右人大隐静脉,采用机械刮除、差异贴壁等手段进行组织贴块法原代培养,胰蛋白酶消化传代,高糖DMEM中加入血小板衍生生长因子BB(PDGF-BB)进行培养。用倒置显微镜和即用型SABC免疫组化染色试剂盒对细胞进行形态学和免疫组化鉴定,RT-PCR法检测α-SMA和calponin1的表达。结果镜下可见培养细胞呈典型的“峰”“谷”生长;免疫组化染色显示胞浆内α-actin阳性表达,RT-PCR法检测α-SMA和calponin1呈阳性表达。结论联合应用PDGF和高糖DMEM培养基差异贴壁法;可获纯度高、结构和功能良好的人血管平滑肌细胞。
Objective To study the effective method of in vitro culture of vascular smooth muscle cells in vascular tissue engineering. Methods A large saphenous vein of about 3 cm in diameter was obtained under aseptic conditions. Primary culture with tissue scraping method and differential adherent method were used for primary culture. Trypsin digestion and passage were performed. Platelet-derived growth factor BB (PDGF-BB ). The cells were identified by inverted microscope and ready-to-use SABC immunohistochemical staining. The expression of α-SMA and calponin1 was detected by RT-PCR. Results The cultured cells showed the typical “peak” “valley ” growth under microscope. The positive expression of α-actin in cytoplasm was detected by immunohistochemistry. The expression of α-SMA and calponin1 was detected by RT-PCR. Conclusion The combination of PDGF and high glucose DMEM medium with different adherence methods; can be high purity, structure and function of human vascular smooth muscle cells.