| objective: Hematopoietic stem cells(HSCs)are the source of all blood cells,and have the ability of self-renewal and multiple differentiation potential.They regulate the physiological function of HSCs depended on the stromal cells,cytokines and extracellular matrix in the bone marrow microenvironment.Expansion of HSCs in vitro is a hot and difficult research topic.Many studies including our previous study have showed that MSCs could promote the proliferation of HSC cell.Our in vivo experiments found that MSCs promoted the proliferation of bone marrow cells and angiogenesis.This study aims to further explore whether this effect is related to the secretion of vascular endothelial growth factor(VEGF).Also,we have found that bone morphogenetic protein(BMP-2),one of TGF-? family members,can act synergistically with MSCs to promote the proliferation of HSCs.If so,there will be a new way for the expansion of HSCs and will benefit the treatment of hematologic diseases.Methods :(1)Collect the bone marrow from the healthy volunteers,separate the MSCs and expand to the third passage(p3).The cells were identified by using FCM with CD34-ECD?CD105-FITC?CD45-PCy7 antibodies,also the purity and surface markers were checked;(2)Human bone marrow MNCs are isolated by density gradient centrifugation,CD34+ cells(HSCs)were isolated by immunomagnetic bead cell sorting system(MACS).The purity of CD34+ cells was determined by FCM.(3)HSCs were co-cultured with MSCs(P3 generation),the numbers of HSCs on the 3rd day,7th day and 10 th day were counted then the time point with the highest proliferation was set as the best one for the following studies.(4)Add BMP-2 to the co-culture system,we have the following six groups: HSC?MSC?HSC+BMP2?MSC+BMP2?MSC+HSC?MSC+HSC+BMP2,culture and collect cells at the best time point we have determined in step(3).(5)HSCs proliferation was assessed in different groups,the numbers of HSCs and MSCs were counted separately(n=3).Total RNA was extracted using trizol and fluorescent quantitative PCR was used to detect the m RNA expressions of Ki67 in HSC and VEGF?Ang-1 in MSC,using 2-??Ct to analyze the mRNA relative quantitative expression.(6)All the datas were analyzed by SPSS23.0 software,measurement datas were presented as the mean ? SEM and were analyzed by one-way ANOVA.P values of <0.05 were regarded as statistically significant.Results:(1)The morphology and growth cycle of BMMSCs are consistent with the previous studies.High expression of CD105 and negative expressions of CD45 and CD34 were observed.The purity of MSCs was 52.4%.(2)CD34 positive cells were isolated by miniMACS,and the purity was 81.9%.(3)The best time point for the proliferation assay was determined as the 7th day by counting the numbers of HSCs in the co-culture system;(4)The number of HSCs in Each group was:HSC+MSC+BMP-2> HSC+MSC >HSC+BMP-2 >HSC;The number of MSCs in each group was:HSC+MSC+BMP-2 > HSC+MSC >MSC+BMP-2>MSC,P<0.05.(6)By QT-PCR,the relative expression of VEGF and Ang-1 in MSC in each group was: MSC+HSC+BMP2 > MSC+HSC >MSC+BMP-2>MSC;P<0.05.Conclusions:(1)The experiment confirmed that we can successfully separate MSCs by adherence;(2)Both MSCs and BMP-2 can promote the proliferation of HSCs,and the co-culture of the two enhanced the effect;(3)The promotion of the proliferation of HSCs was related to the increased secretion of VEGF. |
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