高迁移率族蛋白B1对人脐血造血干细胞增殖分化的影响

Effects of HMGB1 on human cord blood CD34^＋ hematopoietic stem cells proliferation and differentiation in vitro

目的观察辐射后骨髓基质细胞（MSC）高迁移率族蛋白B1（high mobility group box 1，HMGB1）的释放，研究HMGB1对人脐血造血干细胞（HSC）增殖分化的影响。方法体外培养人骨髓MSC，利用ELISA方法检测经12Gyγ射线照射后培养上清中HMGB1含量的变化。HMGB1与CD34^＋的人脐血HSC体外液体共培养6d，通过流式细胞术检测CD34^＋细胞分化指标（CD13、CD14、CD11c、CD41、CD71）的变化。集落形成实验观察HMGB1对HSC增殖分化的影响。结果辐射后，骨髓MSC培养上清中HMGB1含量为（4．3±0．9）ng／ml，较对照组HMGB1含量[（0．4±0．2）ng／m1]明显升高（P〈0．01）。脐血CD34^＋细胞表面表达HMGBl受体RAGE、TLR2和TLR4。HSC与HMGBI共培养6d后，与对照组比较，红系（CD71）和粒单系（CD13、CD14、CD11c）标记的表达明显增强，分别为CD13（18．4±3．8）％和（32．6±5．9）％、CD14（12．6±2．7）％和（25．4±4．4）％、CD11c（9．8±2．1）％和（20．3±3．9）％、CD71（26．6±4．6）％和（47．1±7．4）％，而巨核系标记CD41的表达[（1．1±0．4）％和（1．3±0．5）％]无明显变化。集落形成实验示共培养14d后，红系集落、粒一巨噬细胞集落和总集落的生成较对照组明显增多（P〈0．05），联用抗-TLR2和抗-TLR4抗体可部分抑制这一作用。结论辐射促进骨髓MSC释放HMGB1，胞外HMGB1可以促进HSC的增殖分化。辐射后，骨髓MSC释放的HMGB1与造血恢复或造血重建的关系值得进一步研究。

Objective To study both the release of HMGB1 from irradiation-treated mesenchymal stem cells （MSCs） and the effects of HMGB1 on human cord blood CD34^＋ hematopoietic progenitor cell proliferation and differentiation. Methods MSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12 Gy γ-ray irradiation was determined by enzyme linked immunosorbent assay （ELISA）. CD34 ^＋ cells were positively selected with a MACS CD34 isolation kit. The freshly isolated CD34^＋ cells were cultured in the presence of HMGBI for 6 days. Pheuotype of cultured cells surface molecules （CD13, CD14, CD11 c, CD41 and CD71 ） were analyzed by flow eytometry. The proliferation and differentiation capacities of cord blood HSCs were assayed by colony forming cell assay. The receptors of HMGB1 （RAGE, TLR2 and TLR4） on cord blood CD34^＋ cells were detected by flow cytometry. Results HMGB1 level in the supernatant [ （4.3± 0.9 ）ng/ml ] of the irradiated MSC was significantly higher than that in control [ （0.4±0.2） ng/ml] （ P 〈0.01 ）. Human cord blood CD34^＋ cells expressed the HMGB1 receptors RAGE, TLR2 and TLR4. The HMGBl-treated CD34^＋ cells contained higher proportions of CD13 ^＋ [ （ 32.6 ± 5.9）% vs （18.4 ±3.8）%1, CD14^＋[（25.4±4.4）% vs （12.6±2.7）%1, CD11c^＋[（20.3±3.9）% vs （9.8 ±2.1）% ], CD71^＋ [ （47.1±7.4）% vs （26.6 ±4.6）% l cells compared with control group did. But HMGB1 did not induce the generation of CD41 ^＋ cells [ （ 1.3±0. 5 ） % vs （ 1.1 ± 0.4） % ]. Furthermore, HMGB1 profoundly induced the growth of BFU-E, CFU-GM and total CFU in a dose-dependent manner, and this effect was partially inhibited by TLR2 and TLR4 antibodies. Conclusion Human MSC treated with γ- ray irradiation can release HMGB1, which can induce the proliferation and differentiation of human cord CD34^＋ cells.