BMP-2对间充质干细胞促进造血干细胞增殖的影响及其机制

Synergistic effect of bone marrow mesenchymal stem cell and BMP-2 on proliferation of hematopoietic stem cells

目的探讨人骨形态发生蛋白2(BMP-2)对间充质干细胞(MSC)促进造血干细胞(HSC)增殖的影响及其机制。方法采用全骨髓贴壁法体外扩增成人骨髓MSC至第3代,采用流式细胞术鉴定MSC纯度为52.4%;通过miniM ACS磁珠分选仪从骨髓中分选人骨髓CD34^+细胞即HSC,并用流式细胞术鉴定其纯度为81.9%;利用Transwell非接触共培养骨髓CD34^+细胞与第3代MSC,计数造血干细胞,确定HSC增殖最快的时间点为共培养的第7天。将细胞分为六组,HSC组、MSC组单细胞培养,MSC+BMP-2组、HSC+BMP-2组分别加入100 ng/mL BMP-2共培养,HSC+MSC组两种细胞共培养,HSC+MSC+BMP-2组两种细胞与100 ng/mL BMP-2共培养。在培养第7天时,采用细胞计数板计数HSC数量,采用实时荧光定量qP CR法检测增殖指数Ki67及促血管形成相关因子血管生成素受体(Tie2)、血管内皮生长因子(VEGF)、血管生成素1(Ang1)mRNA表达。结果 HSC组、HSC+BMP-2组、HSC+MSC组、HSC+MSC+BMP-2组HSC数量分别为(15.37±0.42)、(37.81±1.07)、(117.00±2.53)、(129.06±5.13)×10~4个,组间比较P<0.05或<0.01。HSC组、HSC+BMP-2组、HSC+MSC组、HSC+MSC+BMP-2组Ki67及Tie2 mRNA相对表达量逐渐升高,MSC组、MSC+BMP-2组、HSC+MSC组、HSC+MSC+BMP-2组VEGF、Ang1 mRNA相对表达量逐渐升高,组间比较P<0.05或<0.01。结论 BMP-2可协同MSC促进HSC增殖,其机制可能与BMP-2促进促血管形成相关因子VEGF、Ang1和Tie2表达有关。

Objective To investigate the role of human bone morphogenetic protein 2(BMP-2)in mesenchymal stem cells(MSCs)promoting the proliferation of hematopoietic stem cells(HSCs)and its possible mechanism.Methods The whole bone marrow adherent culture method was used to expand MSC from primary to the third passage,MSCs were identified by flow cytometry with a purity of 52.4%;the human bone marrow CD34+cells(that is HSC)were sorted by mini MACS magnetic bead separator from adults′bone marrow,and the purity of the HSCs was 81.9%identified by flow cytometry.The HSCs were non-contact co-cultured with the third passage of MSCs by Transwell.On day 3,7,and 10,we counted the number of HSCs.The fastest proliferation time of HSCs was the seventh day of co-culture.In the next experiment,the cells were divided into six groups:the HSC group and MSC group(we used the single cell culture),HSC+BMP-2 group and MSC+BMP-2 group(which were added with 100 ng/mL BMP-2),HSC+MSC group(the two groups of cells were co-cultured),and the HSC+MSC+BMP-2 group(HSCs were co-cultured with MSCs and then were added with 100 ng/mL BMP-2).On day 7 of culture,the number of HSCs was counted by using cell counting plate,and the proliferation index Ki67 and the expression of angiogenesis-related factors Tie2,VEGF,and Ang1 mRNA was detected by real-time fluorescent quantitative qRT-PCR.Results The number of HSCs in the HSC group,HSC+BMP-2 group,HSC+MSC group,HSC+MSC+BMP-2 group was respectively(15.37±0.42)×10 4,(37.81±1.07)×10 4,(117.00±2.53)×10 4,and(129.06±5.13)×10 4,and significant difference was found between groups(P<0.05 or P<0.01).The relative expression of Ki67 and Tie2 mRNA of HSCs in the HSC group,HSC+BMP-2 group,HSC+MSC group,and HSC+MSC+BMP-2 group increased gradually.Similarly,the VEGF and Ang1 mRNA relative expression of MSCs in the MSC group,MSC+BMP-2 group,MSC+HSC group,and MSC+HSC+BMP-2 group increased gradually,and significant difference was found between groups(P<0.05 or P<0.01).Conclusion BMP-2 could promote the proliferation effect of MSC on HSC by enhancing the expression of angiogenesis-related factors VEGF,Ang1,and Tie2.