一套系统培养鼠主动脉血管壁细胞简单可靠的方法  

The Simple and Reliable Methods of Systematically Culturing Rat Aortic Wall Cells

作　　者：宋方[1] 吴强[1] 陆德琴[2] 袁军[3] 杨永耀[1] 谭洪文 

机构地区：[1]贵州省心血管病研究所贵州省人民医院心内科,贵州省贵阳市550002 [2]贵阳医学院病理生理学教研室,贵州省贵阳市550004 [3]贵州省人民医院中心实验室,贵州省贵阳市550002

出　　处：《中国动脉硬化杂志》2011年第4期361-366,共6页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金资助项目(30871003);贵州省优秀科技教育人才省长基金项目(黔省专合字[2009]30号)

摘　　要：目的探讨一套系统培养鼠主动脉血管壁成分细胞的简单、可重复的方法。方法分别采用植块贴壁法进行主动脉血管平滑肌细胞和血管外膜成纤维细胞、结扎贴壁法进行血管内皮细胞的原代培养,胰酶消化传代;差速贴壁法及自然纯化法进行细胞纯化;转化生长因子β1诱导血管外膜成纤维细胞转化为肌成纤维细胞;相差显微镜及α-平滑肌肌动蛋白（α-SMA）、血小板/内皮细胞黏附分子（CD31）、波形蛋白（Vimentin）抗体两两联合的方式分别进行形态学和免疫细胞化学鉴定。结果组织及细胞活性良好,血管平滑肌细胞及血管外膜成纤维细胞原代培养周期为10-12天,血管内皮细胞为12-14天。传代培养周期为7-10天。经纯化传代后的细胞纯度达95%-100%。血管平滑肌细胞呈典型的＂峰-谷＂状生长,α-SMA（＋）/Vimentin（-）;血管内皮细胞呈＂铺路石＂样外观,CD31（＋）/α-SMA（-）;血管外膜成纤维细胞形态和血管平滑肌细胞不易区分,Vimentin（＋）/α-SMA（-）,诱导的肌成纤维细胞Vimentin（＋）/α-SMA（＋）。原代细胞传至10代以上仍未见生长活力减退。结论我们建立了一套系统培养纯度高、生长状态良好的大鼠主动脉血管内皮细胞、血管平滑肌细胞、血管外膜成纤维细胞及肌成纤维细胞的简单可靠、重复性好的方法。该法对培养小鼠主动脉壁成分细胞仍然适用。Aim To discuss a set of system to culture vascular smooth muscle cell（VSMC）,vascular adventitial fibroblast（VAF） and vascular endothelial cell（VEC） from murine aorta,which are simple,reliable and easy to replicate. Methods The primary culture of VSMC,VAF and VEC used the tissue adherent method,trypsin digestion for passage transfer. The cells were purified by differential adherent and natural growth purification. Myofibroblast（MF） was transformed from VAF by growth factor-β1（TGF-β1） induced. Phase contrast microscope and immunocytochemistry staining were used to identify the morphological and the immunological characteristics,respectively. Results The tissues and cells activity were excellent. The growth cycle of primary VSMC/VAF and VEC were 10-12 days and 12-14 days,respectively. And passage cycle was 7-10 days. After being purified and subculture,the cells purity achieved 95%-100%. The growth characteristics of VSMC assumed the model ＂peak and valley＂,and with anti α-SMA（＋） /Vimentin（-）;the VEC assumed ＂pebble-like＂ appearance,and anti CD31（＋）/α-SMA（-）;the staining of VAF were Vimentin（＋）/α-SMA（-）,while the induction MF were Vimentin（＋）/α-SMA（＋）. The primary cells were successfully passaged more than 10 generations,without growth vigor decreasing. Conclusion We have established a set of system methods to culture the primary VEC,VSMC,VAF and MF of rat aorta origin,the advantages of our approach are simple,reliable and easy to replicate,while the cells have high purity and excellent biological activity. This method is still suitable for culturing the ingredient cell of mouse aorta vessel wall.

关 键 词：主动脉 原代细胞培养 血管平滑肌细胞 外膜成纤维细胞 肌成纤维细胞 

分 类 号：Q2[生物学—细胞生物学]