改良大隐静脉平滑肌细胞的培养

Modified culture of saphenous vein smooth muscle cells

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 an effective method for culturing of human vascular smooth muscle cells （hVSMCs） in tissue engineered blood vessels （TEBVs） in vitro.Methods A 3-cm segment of human long saphenous vein was harvested under sterile conditions.The primary culture and subculture were done by modified tissue-piece inoculation and trypsin digestion respectively.The cells were purified by mechanical treatment and differential attachment.PDGF was combined with high concentration of glucose DMEM as the culture medium.The cultured cells were identified by morphology and immunohistochemistry with contrast microscope and SABC kit,and RT-PCR method detected the expression of α-SMA and calponin 1.Results The cultured cells possessed＂peak and valley＂characteristics for VSMCs under microscope.Immunohistochemical staining showed positive expression of intracytoplasmic α-actin,and RT-PCR detected positve expression of α-SMA and calponin 1.Conclusions Culture medium of PDGF combined with high concentration of glucose DMEM,and with differential attachment method can provide highly purified hVSMCs with good structure and function.