Cytokines in different combination plus total saponins of panax ginseng for inducing the differentiation of CD34+ cells cultured in vitro
- VernacularTitle:不同组合细胞因子与人参总皂苷对体外培养CD34+细胞分化的诱导效应
- Author:
Jianwei WANG
;
Hua ZHANG
;
Rong JIANG
;
Yaping WANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;11(42):8602-8606
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Hematopoietic growth factors (HGFs) can be involved in different hematopoietic cells and different phase of differentiation. Erythropoietin (EPO) can promote he proliferation and differentiation of erythroid progenitor cells in vivo and in vitro, Interleukin-3 (IL-3) appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. IL-3 acts to enhance the effect on the proliferation and differentiation of various hematopoietic cells.OBJECTIVE: To investigate the effect of HGFs and total saponins of panax ginseng (TSPG) on erythropoietic differentiation of purified CD34+ cells from human bone marrow.DESIGN: An observational comparative experiment.SETTING: Chongqing Medical University.MATERIALS: This experiment was performed in the Department of Histology and Embryology, Institute of Basic Medical Research and Key Laboratory of Biochemistry and Molecular Pharmacology in Chongqing Medical University from July 2002 to January 2004. Human bone marrow was collected from extracted ribs of patients without hematopoietic system diseases in the Department of Chest Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA. Informed contents were obtained from all the patients. TSPG (purified to more than 95%) was provided by Chongqing Institute of Chinese Traditional Medicine; Fetal bovine serum (FBS), fluorescein isothiocyanate (FITC)-conjugated anti-CD34 Ab from Becton Dickinson; FITC isotype-matched mouse IgG1 and rabbit anti-CD71 Ab from Santa Cruz Biotechnology Inc; Recombinant human erythropoietin (EPO) from Amgen. rHu Flt3 Ligand (FL), rHu thrombopoietin (TPO), rHu stem cell factor (SCF) and rHu interleukin-3 (IL-3) from Santa Cruz Biotechnology Inc.METHODS: CD34+ hematopoietic stem/progenitor cells (HSC/HPC) were isolated by StemsepTM according to the strategy of negative selection on immunomagnetic separation. The sorted CD34+ cells were incubated in various combinations with IL-3+EPO, SCF+IL-3+EPO and FL+TPO+SCF+IL-3+EPO. SCF+IL-3+EPO was taken as the control group, TSPG of 10, 20, 50, 70 and 100 mg/L, then the total cell number and ratio of CD71+ cells were detected. CD34+ culture systems were added by TSPG of different dosages, those un-added by TSPG as control group. Methylcellulose culture method was used to detect the capacity of TSPG in inducing the proliferation and differentiation of CD34+ HSC/HPC [burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E)].MAIN OUTCOME MEASURES: ① CD34+ cell proliferation in vitro in different cytokine groups; ② Effects of TSPG on inducing CD34+ cells to differentiation into CD34+ erythroid lineage hemocytes; ③ Effects of TSPG on the ability of CD34+ cells in forming erythroid lineage progenitor cells.RESULTS: ①For FL+TPO+SCF+IL-3+EPO, the mean increase in overall cell number corresponded to 546.38-fold expansion, and the ratio of CD71+ cells was increased to (61.20±5.31)%. ② TSPG 20 mg/L was identified as the most potent concentration of those tested, and the ratio of CD71+ cells was increased from (48.39±5.07)% to (56.20±1.40)%. ③ The addition of TSPG(10-50 mg/L) increased the colony production rates of BFU-E and CFU-E.CONCLUSION: A combination containing FL+TPO+SCF+IL-3+EPO may obviously induce CD34+ cells to proliferate and differentiate to erythroid lineage; TSPG may promote CD34+ cells to differentiate into erythroid lineage by cooperating with HGFs.
