In vitro isolation and culture of rabbit bone marrow-derived vascular endothelial progenitor cells
- VernacularTitle:体外扩增法分离培养兔骨髓来源的血管内皮祖细胞
- Author:
Yan GAO
;
Cheng MA
;
Dongyan Lü
;
Tongku LIU
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2008;12(51):10193-10196
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: The in vitro amplification is a primary method for harvesting endothelial progenitor cells (EPCs) due to its simple operation and low cost.OBJECTIVE: To isolate EPCs from rabbit bone marrow to further observe the effects of autologous EPCs on promoting vascular endothelial repair.DESIGN, TIME AND SETTING: An open experiment was performed at the laboratory of Department of Internal Medicine, Changzheng Hospital, Second Military Medical University of Chinese PLA between March 2005 and February 2006. MATERIALS: Eight New Zealand rabbits of either gender, aged 6-8 months, weighing (2.5:L-0.5) kg, were included in this study. Rabbit bone marrow was taken for isolation of bone marrow mononuclear cells by density centrifugation. METHODS: Bone marrow-derived mononuclear cells were inoculated at 1×106/cm2 and cultured for 7 days in M199 medium containing vascular endothelial growth factors and basic fibroblast growth factors. EPCs were identified by Dil-labeled acetylated low-density lipoprotein (Dil- Ac-LDL) and FITC-labeled lectin BS-1 staining. Cells that phagocytized Ac-LDL displayed red fluorescence, cells that combined with lectin BS-1 showed green fluorescence, and cells that were labeled with both exhibited orange fluorescence. Expression levels of CD133, CD134, and Flk-lwere detected using immunofluorescent staining and through the use of flow cytometer.MAIN OUTCOME MEASURES: ① Cellular morphology observation. ② Proliferative capacity of EPCs.③EPCs identified by Dil- Ac-LDL and FITC-labeled lectin BS-1. ④ lmmanohistocbemical identification of EPCs. ⑤Flow cytometry identification of EPC surface marker.RESULTS: ① Cellular morphological observation: the newly isolated bone marrow-derived mononuclear cells exhibited a round appearance. Following 72-hour culture, adherent cells grew in colony cluster, presenting with round or irregular appearance, and nuclear division was obvious. By day 7, flaky cell colonies mutually connected together, presenting with shuttle-shaped endothelioid cells.② Proliferative capability of EPCs: in the 2-4 days of culture, EPCs proliferated fast, and the proliferation slowed down thereafter, exhibiting a typical "S" -shaped appearance. By days 6 and 7, EPC proliferation accelerated again, with the absorbance values of 0.58±0.15 and 0.62±0.23, respectively. ③ Over 95% of EPC cytoplasm exhibited red fluorescence after stained with Ac-LDL, appropriately 100% of cytoplasm exhibited green fluorescence after stained with FITC-labeled lectin BS-1, and over 90% of cytoplasm exhibited orange fluorescence after double staining. ④ Immonohistochemistry and flow cytometry results revealed positive expression of EPC surface markers CD133, FIK-1, and CD34.CONCLUSION: Cell population with EPC characteristics can be successfully isolated from rabbit bone marrow by in vitro amplification.
