摘要
目的:改良大鼠心肌微血管内皮细胞(MMVECs)培养方法,提高细胞收获量和细胞纯度。方法:选择6～8周龄Wistar雄性大鼠,腹腔内注射200U肝素,摘取左心室剪成若干2mm3小块接种于培养皿中,待其贴壁后,加入含有肝素的DMEM高糖培养液,换液、去除组织块、消化、传代,取第二代MMVECs进行细胞形态学和免疫组化、免疫荧光染色鉴定。结果:贴块法培养48h可见组织块周围有细胞长出,70h长出较多,去组织块后再培养36h,细胞生长至80%～90%融合,呈典型铺路石样。免疫组化和免疫荧光染色证实培养细胞CD31、CD34和Ⅷ因子相关抗原染色均呈阳性,且纯度较高。结论:采用心肌组织贴块及培养液中加入肝素培养的方法可获得高纯度的MMVECs,方法简单,结果可靠。
Objective: To increase the quantity and purity of rat myocardial microvascular endothelial cells(MMVECs) with reformative culture method. Method: Wistar male rats (6~8 weeks of age) were used for primary MMVECs isolation. Peritoneal cavities of rats were injected with 200U heparin and the left ventricle tissues were cut into pieces of 2mm3. Myocardial tissues were then seeded on culture plates pre-coated with rat-tail tendon gelatin. After a 40min attachment period, the tissues were cultured in DMEM supplemented with 20% fetal bovine serum, 20Uml heparin.MMVECs at second passage were used for experiments and MMVECs were identified by morphology, immunofluorescence and immunohisto-chemistry methods. Results: MMVECs migrated from myocardial pieces approximately 48h and a great quantity of MMVECs appeared about 70h. The tissue pieces were discarded about 70h and MMVECs grown to 80%~90% confluence after 36h. MMVECs were identified by its cobblestone appearances, positive of Ⅷ, CD31 and CD34. Conclusion: This results proved that MMVECs cultured by reformative mounting method and cultured with DMEM of heparin were a simple and reliable method.
出处
《微循环学杂志》
2013年第1期39-40,F0004,I0002,共4页
Chinese Journal of Microcirculation
关键词
微血管内皮细胞
贴块法
肝素
大鼠
Microvascular endothelial cells
Mounting method
Heparin
Rat
