Isolation, cultivation and identification of endothelial progenitor cells from rabbit bone marrow by in vitro amplification method / 中国组织工程研究

ABSTRACT

AIM: To isolate and culture endothelial progenitor cells (EPCs) in blood vessel by in vitro amplification from bone marrow of rabbits to provide cytology basis for the implantation of autologous EPCs in the repair of blood vessel endothelium. METHODS: The experiment was performed at the Department of Cardiology, Changzheng Hospital, Second Military Medical University of Chinese PLA from March 2005 to February 2006. ①Dil labeled acetylated low density lipoprotein, FITC labeled BS-1 lectin, mouse anti-human CD34 antibody, rabbit anti-human FIK-1 antibody, mouse anti-human CD133 monoclonal antibody were purchased from molecular probes company, vector company, Biolegend company, Biolegeng company and R&D company. ②Totally 8 New Zealand rabbits were selected to extract the bone barrow. Mononuclear cell was isolated from bone marrow by density centrifugation. With the inoculated density of 1?106/cm2, it was put in the M199 medium containing vascular endothelial growth factor and Basic fibroblast growth factor (bFGF) for in vitro cultivation for 7 days. Cell growth and reproductive activity were observed. ③EPCs were identified by Dil labeled acetylated low density lipoprotein and FITC labeled BS-1 lectin. The cells showed red fluorescence were cells phagocytized acetylated low density lipoprotein, while those with green gluorescence were cells bind with BS-1, and the cells double stained showed orange fluorescence. ④Expressions of CD133, CD34 and Flk-1 were detected with immunofluorescent staining and flow cytometry. RESULTS: ①Observation of cell morphous New isolated mononuclear cells were round. After cultivation for 72 hours, adherent cells showed colony-like growth, and the cells were round or irregular, and the caryocinesis was relatively obvious. Till the 7th day after cultivation, cell colony was connected each other, showing fusiform endothelioid cells. ②Reproductive activity of EPCs in blood vessel At days 2-4, the reproduction of EPCs was rapid, and then became slow gradually. Growth curve showed typical "S" shape. At days 6 and 7, the EPCs grew rapidly. The absorbance (A) reached 0.58?0.15 and 0.62?0.23, respectively. ③Result of EPCs identification by Dil labeled acetylated low density lipoprotein and FITC labeled BS-1 lectin In kytoplasm of EPCs, red fluorescent concentration bind with acetylated low density lipoprotein appeared, with the positive rate of over 95%. Combined rate with BS-1 lectin reached 100% nearly. Double staining rate reached over 90%. ④Result of EPCs immunohistochemical method and flow cytometry The cell-surface marker CD133, FlK-1 and CD34 were positive. CONCLUSION: Cell colony with the feature of EPCs can be isolated and cultured from rabbit bone marrow by in vitro amplification method successfully.