小鼠主动脉平滑肌细胞的分离培养及鉴定

目的:建立分离培养小鼠原代主动脉血管平滑肌细胞(VSMC)的方法并检测其生长特性。方法:剥离小鼠主动脉中膜层,分别采用组织块培养法及胶原酶消化法分离培养小鼠主动脉来源的原代VSMC,免疫荧光法检测细胞的纯度和分化状态;3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四氮唑蓝(MTT)法测定小鼠主动脉VSMC传代细胞的生长、增殖特性。结果:组织块培养法培养组织块8d后,细胞从组织块边缘爬出,18 d后细胞汇合度达到80%以上后传代;胶原酶消化法分离培养的细胞生长7 d后,汇合度可达80%,此时进行传代;2种方法获得的细胞进行免疫荧光染色,结果显示细胞传至第3代时纯度在95%以上,传至第8代时分化状态并没有改变;MTT法显示细胞生长3~5 d时处于指数生长期。结论:本研究建立了2种可靠稳定的分离和培养小鼠主动脉VSMC的方法,VSMC纯度高,多次传代后细胞特征稳定。

Isolated Culture and Identification of Mouse Aortic Vascular Smooth Muscle Cell

Objective:To establish the technical method of isolated culture mouse aorta vascular smooth muscle cell (VSMC) in vitro and observe their growth characteristics.Methods:The mouse aorta middle layer was isolated, VSMC of original generation separation from aorta in mice by tissue piece inoculation and collagenase digestion method were compared and characterized by immunofluorescence; The growth and proliferation characteristics of passage VSMC were measured by growth curve method, 3-(4,5-dimethyl-2-thiahiazo)- 2,5-diphenyltetrazolium bromide (MTT).Results:After cells were cultured for 8 days with tissue piece inoculation method, VSMC were slowly grown from the tissue edge and confluence reached to 80 %at 18 days and could be passaged. After cells were cultured for 7 days with collagenase digestion method, the cells confluence reached to more than 80 % and were passaged. Immunofluorescence staining showed the purity was more than 95 % after 3 passages, keeping their differentiation state until 8 passages; MTT assay showed that optical density change of cell growth was obvious at 3~5 days.Conclusion:Two kinds of efficient isolation methods were established to culture pure and stable mouse aortic VSMC.