HMGB1在人脐血造血干细胞归巢和增殖分化中的作用及其机制研究
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摘要
中文摘要
背景与目的高迁移率族蛋白B1(high mobility group box 1,HMGB1)是一种DNA结合蛋白,组织损伤后释放至胞外,招募多种类型干细胞,参与受损组织修复。造血干细胞移植(haematopoietic stem cell transplantation ,HSCT)的预处理必然导致骨髓组织受损,释放HMGB1,推测其可能参与造血重建。本实验将首次观察预处理后骨髓基质细胞HMGB1的释放、HMGB1对人脐血造血干细胞归巢和增殖分化的影响,并对其机制予以探讨;以期为促进HSCT术后造血重建寻找新靶点。方法体外培养人骨髓基质细胞,加速器照射后,利用ELISA方法检测培养上清中HMGB1含量的变化。利用MACS系统分选人脐血CD34+细胞,HMGB1与其共培养6天,通过流式细胞术检测脐血CD34+细胞分化指标(CD13、CD14、CD11c、CD41,CD71)的变化。利用克隆形成实验观察HMGB1与对造血干细胞增殖分化的影响。应用transwell小室趋化装置观察HMGB1对人脐血CD34+细胞的迁移活性的影响。流式细胞术检测HMGB1的受体RAGE、TLR2和TLR4在人脐血CD34+细胞上的表达。利用抗-RAGE抗体、抗- TLR2抗体和抗- TLR4抗体阻断RAGE、TLR2和TLR4,重复趋化实验,体外观察RAGE、TLR2和TLR4是否参与HMGB1可能诱导的人脐血CD34+细胞迁移。结果1.经X线照射后骨髓基质细胞培养上清中HMGB1含量增高:人骨髓基质细胞,加速器照射(12Gy)后,培养上清中HMGB1含量为(4.3±0.9)ng/ml,较不照射组HMGB1含量(0.4±0.2)ng/ml升高(p<0.01)。2.HMGB1表面受体检测人脐血CD34+细胞表达HMGB1的受体RAGE(43.1±7.2)%、TLR2(36.1±6.6)%和TLR4(23.1±5.2)%。3.HMGB1促进CD34+细胞向红系和粒单系增殖分化:脐血分选富集的纯度为(98.25±0.93)%,与HMGB1体外液体共培养6天后,和对照组比较,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 vs 32.6±5.9)%、CD14(12.6±2.7 vs 25.4±4.4)%、CD11c(9.8±2.1 vs 20.3±3.9)%、、CD71(26.6±4.6 vs 47.1±7.4)%,而巨核系标记CD41(1.1±0.4% vs 1.3±0.5%)的表达无明显变化。同样,克隆形成实验示共培养14天后,红系集落、粒-巨噬细胞集落和总集落的生成较对照组明显增多(p<0.05)。4.HMGB1诱导脐血CD34+细胞的迁移:HMGB1在一定的浓度范围内随浓度递增其对人脐血CD34+细胞的迁移作用逐渐增强,当HMGB1浓度为100 ng/ml时,趋化活性最强,趋化指数为3.96±0.46,与对照组比较差异显著(p<0.01),抗-RAGE抗体可部分抑制HMGB1对脐血CD34+细胞的迁移作用。
结论照射后骨髓基质细胞培养上清HMGB1含量明显升高;HMGB1可促进人脐血CD34+细胞向粒单核及红系分化,并促进红系集落和粒-巨噬细胞集落的生成;一定浓度的HMGB1可加强脐血CD34+细胞迁移功能,此作用有可能通过RAGE介导。
Background and objective High mobility group box 1(HMGB1)is an abundant chromatin-binding protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells,can play a role in tissue regeneration. Presently, we build over this hypothesis. The fore treatment of haematopoietic stem cell transplantation(HSCT)will lead the bone marrow tissue damage, and release of HMGB1,which is expected to promote the migration of HSCs to the damaged tissue, and to play a role in reconstruction of hematopoiesis , but little is known about the mechanisms regulating these processes. Here, we elected 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 cells homing,proliferation and differentiation and their mechanisms, in order to find new target in reconstruction of hematopoiesis.
Methods MSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12Gy X-Gy irradiated was determined using a enzymelinked immunosorbent assay (ELISA). CD34+ cells were positively selected with a MACS CD34 isolation kit. Subsequently, freshly isolated CD34+ cells were cultured in the presence of HMGB1 for 6 days. Phenotype of cultured cells surface molecules (CD13、CD14、CD11c、CD41、CD71) were analyzed by flow cytometry. Also, the proliferation and differentiation ability of cord blood HSCs were characterized by colony forming cell assay. In addition, the receptors of HMGB1(RAGE、TLR2、TLR4) on cord blood CD34+ cells were detected by flow cytometry. The effects of HMGB1 on HSCs homing were evaluated by chemotaxis assay using Boyden chambers. To analyze the underlying mechanism of HMGB1-induced HSCs migration, we engaged inhibitory antibodies against its receptors, RAGE, TLR2, and TLR4.
Results 1. HMGB1 is released from MSC treated with X-Gy irradiated. Level of HMGB1in the supernatant(4.3±0.9)ng/ml for MSC treated with 12Gy X-Gy irradiated was significantly higher when compared to control group(0.4±0.2)(p<0.01). 2. Expression of HMGB1 receptors in human cord blood CD34+ cells. Human cord blood CD34+ cells expressed the HMGB1 receptors, RAGE (43.1%±7.2%), TLR2(36.1%±6.6%) and TLR4 (23.1%±5.2%) . 3. HMGB1 induces the proliferation and differentiation of human cord blood CD34+ cells in vitro. The HMGB1-treated CD34+ cells possessed the higher proportion of CD13(32.6±5.9% vs 18.4±3.8%),CD14(25.4±4.4% vs 12.6±2.7%),CD11c(20.3±3.9% vs 9.8±2.1%),CD71(47.1±7.4% vs 26.6±4.6%) compared to control group. But, HMGB1 did not induce the generation of CD41+ cells (1.3±0.5% vs 1.1±0.4%).Colony forming cell assay indicate the growth of BFU-E、CFU-GM and total CFU upregulated. 4. HMGB1 stimulate migration of cord blood CD34+ cells HMGB1 stimulated migration of CD34+ cells in a concentration-dependent manner, when the concentration was 100ng/ml, the strong chemotaxis was shown(p<0.01). A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of CD34+, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated CD34+ migration.
Conclusion Treatment of human MSC with X-Gy irradiation can cause release of HMGB1. Human cord blood CD34+ progenitor cells constitutively express functional HMGB1 receptors, RAGE, TLR2 and TLR4. HMGB1 can induce the differentiation of human cord CD34+ cells along the granulo-monocytic and erythroid system. A certain concentration of HMGB1 was able to induce the chemotaxis in vitro of CD34+ cells, and the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE.
背景与目的高迁移率族蛋白B1(high mobility group box 1,HMGB1)是一种DNA结合蛋白,组织损伤后释放至胞外,招募多种类型干细胞,参与受损组织修复。造血干细胞移植(haematopoietic stem cell transplantation ,HSCT)的预处理必然导致骨髓组织受损,释放HMGB1,推测其可能参与造血重建。本实验将首次观察预处理后骨髓基质细胞HMGB1的释放、HMGB1对人脐血造血干细胞归巢和增殖分化的影响,并对其机制予以探讨;以期为促进HSCT术后造血重建寻找新靶点。方法体外培养人骨髓基质细胞,加速器照射后,利用ELISA方法检测培养上清中HMGB1含量的变化。利用MACS系统分选人脐血CD34+细胞,HMGB1与其共培养6天,通过流式细胞术检测脐血CD34+细胞分化指标(CD13、CD14、CD11c、CD41,CD71)的变化。利用克隆形成实验观察HMGB1与对造血干细胞增殖分化的影响。应用transwell小室趋化装置观察HMGB1对人脐血CD34+细胞的迁移活性的影响。流式细胞术检测HMGB1的受体RAGE、TLR2和TLR4在人脐血CD34+细胞上的表达。利用抗-RAGE抗体、抗- TLR2抗体和抗- TLR4抗体阻断RAGE、TLR2和TLR4,重复趋化实验,体外观察RAGE、TLR2和TLR4是否参与HMGB1可能诱导的人脐血CD34+细胞迁移。结果1.经X线照射后骨髓基质细胞培养上清中HMGB1含量增高:人骨髓基质细胞,加速器照射(12Gy)后,培养上清中HMGB1含量为(4.3±0.9)ng/ml,较不照射组HMGB1含量(0.4±0.2)ng/ml升高(p<0.01)。2.HMGB1表面受体检测人脐血CD34+细胞表达HMGB1的受体RAGE(43.1±7.2)%、TLR2(36.1±6.6)%和TLR4(23.1±5.2)%。3.HMGB1促进CD34+细胞向红系和粒单系增殖分化:脐血分选富集的纯度为(98.25±0.93)%,与HMGB1体外液体共培养6天后,和对照组比较,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 vs 32.6±5.9)%、CD14(12.6±2.7 vs 25.4±4.4)%、CD11c(9.8±2.1 vs 20.3±3.9)%、、CD71(26.6±4.6 vs 47.1±7.4)%,而巨核系标记CD41(1.1±0.4% vs 1.3±0.5%)的表达无明显变化。同样,克隆形成实验示共培养14天后,红系集落、粒-巨噬细胞集落和总集落的生成较对照组明显增多(p<0.05)。4.HMGB1诱导脐血CD34+细胞的迁移:HMGB1在一定的浓度范围内随浓度递增其对人脐血CD34+细胞的迁移作用逐渐增强,当HMGB1浓度为100 ng/ml时,趋化活性最强,趋化指数为3.96±0.46,与对照组比较差异显著(p<0.01),抗-RAGE抗体可部分抑制HMGB1对脐血CD34+细胞的迁移作用。
结论照射后骨髓基质细胞培养上清HMGB1含量明显升高;HMGB1可促进人脐血CD34+细胞向粒单核及红系分化,并促进红系集落和粒-巨噬细胞集落的生成;一定浓度的HMGB1可加强脐血CD34+细胞迁移功能,此作用有可能通过RAGE介导。
Background and objective High mobility group box 1(HMGB1)is an abundant chromatin-binding protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells,can play a role in tissue regeneration. Presently, we build over this hypothesis. The fore treatment of haematopoietic stem cell transplantation(HSCT)will lead the bone marrow tissue damage, and release of HMGB1,which is expected to promote the migration of HSCs to the damaged tissue, and to play a role in reconstruction of hematopoiesis , but little is known about the mechanisms regulating these processes. Here, we elected 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 cells homing,proliferation and differentiation and their mechanisms, in order to find new target in reconstruction of hematopoiesis.
Methods MSCs were obtained from human bone marrow. HMGB1 released by the MSCs after treatment with 12Gy X-Gy irradiated was determined using a enzymelinked immunosorbent assay (ELISA). CD34+ cells were positively selected with a MACS CD34 isolation kit. Subsequently, freshly isolated CD34+ cells were cultured in the presence of HMGB1 for 6 days. Phenotype of cultured cells surface molecules (CD13、CD14、CD11c、CD41、CD71) were analyzed by flow cytometry. Also, the proliferation and differentiation ability of cord blood HSCs were characterized by colony forming cell assay. In addition, the receptors of HMGB1(RAGE、TLR2、TLR4) on cord blood CD34+ cells were detected by flow cytometry. The effects of HMGB1 on HSCs homing were evaluated by chemotaxis assay using Boyden chambers. To analyze the underlying mechanism of HMGB1-induced HSCs migration, we engaged inhibitory antibodies against its receptors, RAGE, TLR2, and TLR4.
Results 1. HMGB1 is released from MSC treated with X-Gy irradiated. Level of HMGB1in the supernatant(4.3±0.9)ng/ml for MSC treated with 12Gy X-Gy irradiated was significantly higher when compared to control group(0.4±0.2)(p<0.01). 2. Expression of HMGB1 receptors in human cord blood CD34+ cells. Human cord blood CD34+ cells expressed the HMGB1 receptors, RAGE (43.1%±7.2%), TLR2(36.1%±6.6%) and TLR4 (23.1%±5.2%) . 3. HMGB1 induces the proliferation and differentiation of human cord blood CD34+ cells in vitro. The HMGB1-treated CD34+ cells possessed the higher proportion of CD13(32.6±5.9% vs 18.4±3.8%),CD14(25.4±4.4% vs 12.6±2.7%),CD11c(20.3±3.9% vs 9.8±2.1%),CD71(47.1±7.4% vs 26.6±4.6%) compared to control group. But, HMGB1 did not induce the generation of CD41+ cells (1.3±0.5% vs 1.1±0.4%).Colony forming cell assay indicate the growth of BFU-E、CFU-GM and total CFU upregulated. 4. HMGB1 stimulate migration of cord blood CD34+ cells HMGB1 stimulated migration of CD34+ cells in a concentration-dependent manner, when the concentration was 100ng/ml, the strong chemotaxis was shown(p<0.01). A neutralizing anti-RAGE antibody significantly blocked the HMGB1-induced migration of CD34+, whereas neutralizing TLR2 and TLR4 antibodies did not significantly influence HMGB1-stimulated CD34+ migration.
Conclusion Treatment of human MSC with X-Gy irradiation can cause release of HMGB1. Human cord blood CD34+ progenitor cells constitutively express functional HMGB1 receptors, RAGE, TLR2 and TLR4. HMGB1 can induce the differentiation of human cord CD34+ cells along the granulo-monocytic and erythroid system. A certain concentration of HMGB1 was able to induce the chemotaxis in vitro of CD34+ cells, and the migratory effect of HMGB1 on human HSCs is predominantly mediated by RAGE.
引文
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