胞外HMGB1调控SDF-1/CXCR-4轴介导脐血CD34^+细胞的迁移作用研究  

Extracellular HMGB1 Promotes the Migration of Cord Blood CD34^+ Cells via SDF-1 /CXCR-4 Axis

作　　者：杨璐璐[1] 孙自敏[1] 刘欣[1] 朱小玉[1] 王兴兵[1] 汪健[1] 

机构地区：[1]安徽医科大学附属省立医院血液科,安徽合肥230001

出　　处：《中国实验血液学杂志》2014年第5期1415-1421,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金青年基金项目(31101006)

摘　　要：本研究探究高迁移率族蛋白B1(high mobility group box1,HMGB1)和/或基质细胞衍化因子(SDF-1)对脐血CD34+细胞的迁移作用,并进一步探讨HMGB1是否通过调控SDF-1/CXCR-4轴介导脐血CD34+细胞的迁移。分离脐血单个核细胞,应用免疫磁珠分选CD34+细胞,流式细胞仪检测脐血CD34+细胞的纯度。利用趋化迁移实验(Transwell)测定HMGB1和/或SDF-1对脐血CD34+细胞的迁移作用。1.0×105细胞/孔脐血CD34+细胞加入上孔中,应用不同浓度梯度的HMGB1和/或SDF-1(0、10、25、50、100、200 ng/ml)检测HMGB1和/或SDF-1促进脐血CD34+细胞迁移的最适作用浓度。新鲜分离的脐血CD34+细胞表达SDF-1受体CXCR-4和HMGB1受体TLR2,TLR4和RAGE。为进一步探讨何种受体在HMGB1和SDF-1介导的细胞迁移中起作用,应用抗SDF-1抗体,抗CXCR-4抗体,抗RAGE抗体,抗TLR2抗体和抗TLR4抗体阻断研究HMGB1和/或SDF-1对脐血CD34+细胞的迁移作用。结果表明,磁珠分选脐血CD34+细胞纯度为97.40±1.26%。25 ng/ml的SDF-1不能介导脐血CD34+细胞的迁移,最佳作用浓度为100 ng/ml。HMGB1的最低作用浓度为100 ng/ml。通过剂量研究实验发现,细胞迁移最佳联合作用浓度为HMGB1 100 ng/ml+SDF-1 50 ng/ml。抗体阻断实验结果表明,抗SDF-1(4μg/ml)和抗CXCR-4(5μg/ml)抗体均可以阻断HMGB1单独或联合SDF-1介导的细胞迁移运动,而抗RAGE抗体,抗TLR2抗体及抗TLR4抗体并不能阻断HMGB1和/或SDF-1对脐血CD34+细胞的迁移作用。结论:HMGB1和SDF-1均可以促进脐血CD34+细胞的迁移,HMGB1可能通过CXCR-4而非RAGE和TLR受体加强SDF-1诱导的细胞迁移作用。具体的作用机制还需进一步探讨。This study was aimed to investigate the effect of high mobility group boxl （HMGB1） and/or stromal cell derived factor-1 （SDF-1） on the migration of cord blood CD34 ＋ cells,and to explore whether HMGB1 promotes cord blood CD34 ＋ cell migration via SDF-1/CXCR4 axis.Cord blood mononuclear cells were isolated by Ficoll-Paque density centrifugation,CD34 ＋ cells were collected by a positive immunoselection procedure（CD34 MicroBeads） according to the manufacturer＇s instructions,the purity of the isolated CD34 ＋ cells was detected by flow cytometry.In vitro chemotaxis assays were performed using Transwell cell chambers to detect cells migration.1 × 105cells/well cord blood CD34 ＋ cells were added into the upper chambers.Different concentrations of HMGB1 and/or SDF-1 （0,10,25,50,100,200 ng/ml） were used to detect the optimal concentrations of HMGB1 and/or SDF-1 for inducing migration of cord blood CD34 ＋ cells.Freshly isolated cord blood CD34 ＋ cells express CXCR4（SDF-1 receptor）,and HMGB1 receptor TLR-2,TLR-4 and RAGE.To explore which receptors were required for the synergy of HGMB1 and/or SDF-1 on cells migration,the anti-SDF-1,anti-CXCR4 and anti-RAGE antibodies were used to detect the effect of HGMB1 alone or with SDF-1 on cord blood CD34 ＋ cells migration.The results showed that the purity of CD34 ＋ cells isolated from cord blood mononuclear cells by magnetic cell sorting was 97.40 ± 1.26%,the 25 ng/ml SDF-1 did not induce migration of cord blood CD34 ＋ cells,whereas the optimal migration was observedat 100 ng/ml.HMGB1 alone did not induce migration up to 100 ng/ml.The dose test found that the the best synergistic concentrations for cells migration were 100 ng/ml HMGB1 combined with 50 ng/ml SDF-1.The blocking test showed that both the anti-SDF-1 （4 μg/ml） and anti-CXCR4 （5 μg/ml） antibodies could block cell migration induced by HMGB1 or combined with SDF-1,but the cord blood CD34 ＋ cells in the presence of anti-RAGE,anti-TLR-2 and anti-TLR-4 antibodies did not

关 键 词：HMGB1 迁移 脐血 SDF-1/CXCR-4轴 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]