CXCR7在急性单核细胞白血病中的表达及对THP-1细胞功能的影响
发布时间：2018-10-20 15:12
【摘要】：目的本实验主要探讨了基质细胞衍生因子-1α(stromal cell derived factor-1α,SDF-1α)受体CXCR7(CXC-chemokine receptor 7,CXCR7)在急性单核细胞白血病(AML-M5)中的表达,及其对急性单核细胞白血病细胞系THP-1细胞增殖、凋亡和侵袭等功能的影响。方法采集10例初次诊断AML-M5患者外周血和10例正常人外周血,应用Ficoll密度梯度离心法提取外周血单个核细胞(PBMC);体外悬浮培养人外周血急性单核细胞白血病细胞系THP-1细胞。(1)采用流式细胞术检测初次诊断AML-M5患者、正常人PBMC幼稚细胞表面及THP-1细胞表面CXCR7表达;(2)采用Western Blot法检测初诊AML-M5患者PBMC、正常人PBMC及THP-1细胞CXCR7蛋白表达水平;(3)采用RT-PCR检测初诊AML-M5患者PBMC、正常人PBMC及THP-1细胞CXCR7 m RNA表达水平;(4)采用CCK8法检测SDF-1α/CXCR7趋化轴对THP-1细胞体外增殖活性的作用,CXCR7单克隆抗体11G8阻断THP-1细胞表面CXCR7表达后,观察细胞体外增殖能力的变化;(5)采用Annexin V/PI双染标记法检测CXCR7单克隆抗体对无血清培养条件下THP-1细胞凋亡的影响;(6)采用Transwell法观察SDF-1α/CXCR7趋化轴对THP-1细胞体外侵袭能力的影响,CXCR7单克隆抗体阻断细胞表面CXCR7表达后,观察细胞侵袭能力的变化。结果(1)初次诊断AML-M5患者PBMC幼稚细胞表面CXCR7表达高于正常对照(P0.05),THP-1细胞表面CXCR7也有高表达;(2)THP-1细胞、AML-M5患者PBMC中CXCR7蛋白水平明显高于正常对照(P0.05);(3)THP-1细胞、AML-M5患者PBMC中CXCR7 m RNA水平高于正常对照(P0.05);(4)SDF-1α刺激可增高THP-1细胞增殖活性,但这种增殖活性可被CXCR7单克隆抗体抑制(P0.01);(5)CXCR7单克隆抗体不影响无血清培养条件下THP-1细胞的凋亡(P0.05);(6)SDF-1α/CXCR7趋化轴促进THP-1细胞体外侵袭能力,CXCR7单克隆抗体可明显抑制SDF-1α诱导的THP-1细胞侵袭(P0.01)。结论(1)CXCR7在正常对照者中几乎不表达,但在初诊急性单核细胞白血病患者及急性单核细胞白血病细胞系THP-1细胞中均有高表达。(2)SDF-1α可增强急性单核细胞白血病细胞系THP-1细胞的增殖活性,用CXCR7单克隆抗体11G8阻断CXCR7与SDF-1α之间相互作用可有效减弱THP-1细胞增殖活性。(3)在无血清培养条件下,THP-1细胞的凋亡活性不受CXCR7单克隆抗体的影响。(4)SDF-1α/CXCR7生物轴参与急性单核细胞白血病THP-1细胞的体外侵袭能力,同时应用CXCR4和CXCR7两种阻断剂时能最大程度地降低SDF-1α诱导的THP-1细胞体外侵袭能力。
[Abstract]:Objective to investigate the expression of stromal cell derived factor-1 伪 (SDF-1 伪) receptor CXCR7 (CXC-chemokine receptor 7 CXCR7) in acute monocytic leukemia (AML-M5) and its effects on proliferation, apoptosis and invasion of THP-1 cell line. Methods Peripheral blood samples were collected from 10 patients with AML-M5 and 10 normal controls. Ficoll density gradient centrifugation was used to extract THP-1 cells from peripheral blood mononuclear cells (PBMC);) cultured in vitro. (1) flow cytometry was used to detect the initial diagnosis of AML-M5 patients. (2) the expression of CXCR7 protein in PBMC and THP-1 cells of PBMC, patients with newly diagnosed AML-M5 was detected by Western Blot assay, and (3) the PBMC and THP-1 cells CXCR7 m of PBMC and THP-1 cells of AML-M5 patients were detected by RT-PCR. (4) the effect of SDF-1 伪 / CXCR7 chemotactic axis on the proliferation of THP-1 cells in vitro was detected by CCK8 assay, and the CXCR7 expression on THP-1 cells was blocked by CXCR7 monoclonal antibody 11G8. (5) to detect the effect of CXCR7 monoclonal antibody on apoptosis of THP-1 cells in serum-free culture by Annexin V/PI double staining method, and (6) to observe the invasion ability of SDF-1 伪 / CXCR7 chemotaxis axis on THP-1 cells in vitro by Transwell assay. After CXCR7 monoclonal antibody blocked the expression of CXCR7 on cell surface, The changes of cell invasion ability were observed. Results (1) the expression of CXCR7 on the surface of immature PBMC cells in AML-M5 patients was higher than that of normal controls (P0.05), and the expression of CXCR7 on the surface of THP-1 cells was also high. (2) CXCR7 protein level in PBMC of AML-M5 patients was significantly higher than that of normal controls (P0.05); (3) THP-1 cells, CXCR7 m RNA water in AML-M5 patients PBMC. The proliferation activity of THP-1 cells was increased by SDF-1 伪 stimulation compared with normal control (P0.05); (4). But the proliferation activity was inhibited by CXCR7 monoclonal antibody (P0.01); (5) CXCR7 monoclonal antibody did not affect the apoptosis of THP-1 cells (P0.05); (6) SDF-1 伪 / CXCR7 chemotactic axis promoted the invasion ability of THP-1 cells in vitro. CXCR7 monoclonal antibody could significantly inhibit SDF-1 伪 -induced invasion of THP-1 cells (P0.01). Conclusion (1) CXCR7 was almost not expressed in normal controls. However, there was high expression of SDF-1 伪 in both newly diagnosed patients with acute monocytic leukemia and in THP-1 cell line. (2) SDF-1 伪 could enhance the proliferation activity of THP-1 cell line. Blocking the interaction between CXCR7 and SDF-1 伪 with CXCR7 monoclonal antibody 11G8 could effectively reduce the proliferation activity of THP-1 cells. (3) in serum-free culture, the apoptotic activity of THP-1 cells was not affected by CXCR7 monoclonal antibody. (4) SDF-1 伪 / CXCR7 biological axis was involved in acute mononuclear cells. The invasiveness of THP-1 cells in vitro, At the same time, CXCR4 and CXCR7 could reduce the invasiveness of THP-1 cells induced by SDF-1 伪 to the maximum extent.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
本文编号：2283541
[Abstract]:Objective to investigate the expression of stromal cell derived factor-1 伪 (SDF-1 伪) receptor CXCR7 (CXC-chemokine receptor 7 CXCR7) in acute monocytic leukemia (AML-M5) and its effects on proliferation, apoptosis and invasion of THP-1 cell line. Methods Peripheral blood samples were collected from 10 patients with AML-M5 and 10 normal controls. Ficoll density gradient centrifugation was used to extract THP-1 cells from peripheral blood mononuclear cells (PBMC);) cultured in vitro. (1) flow cytometry was used to detect the initial diagnosis of AML-M5 patients. (2) the expression of CXCR7 protein in PBMC and THP-1 cells of PBMC, patients with newly diagnosed AML-M5 was detected by Western Blot assay, and (3) the PBMC and THP-1 cells CXCR7 m of PBMC and THP-1 cells of AML-M5 patients were detected by RT-PCR. (4) the effect of SDF-1 伪 / CXCR7 chemotactic axis on the proliferation of THP-1 cells in vitro was detected by CCK8 assay, and the CXCR7 expression on THP-1 cells was blocked by CXCR7 monoclonal antibody 11G8. (5) to detect the effect of CXCR7 monoclonal antibody on apoptosis of THP-1 cells in serum-free culture by Annexin V/PI double staining method, and (6) to observe the invasion ability of SDF-1 伪 / CXCR7 chemotaxis axis on THP-1 cells in vitro by Transwell assay. After CXCR7 monoclonal antibody blocked the expression of CXCR7 on cell surface, The changes of cell invasion ability were observed. Results (1) the expression of CXCR7 on the surface of immature PBMC cells in AML-M5 patients was higher than that of normal controls (P0.05), and the expression of CXCR7 on the surface of THP-1 cells was also high. (2) CXCR7 protein level in PBMC of AML-M5 patients was significantly higher than that of normal controls (P0.05); (3) THP-1 cells, CXCR7 m RNA water in AML-M5 patients PBMC. The proliferation activity of THP-1 cells was increased by SDF-1 伪 stimulation compared with normal control (P0.05); (4). But the proliferation activity was inhibited by CXCR7 monoclonal antibody (P0.01); (5) CXCR7 monoclonal antibody did not affect the apoptosis of THP-1 cells (P0.05); (6) SDF-1 伪 / CXCR7 chemotactic axis promoted the invasion ability of THP-1 cells in vitro. CXCR7 monoclonal antibody could significantly inhibit SDF-1 伪 -induced invasion of THP-1 cells (P0.01). Conclusion (1) CXCR7 was almost not expressed in normal controls. However, there was high expression of SDF-1 伪 in both newly diagnosed patients with acute monocytic leukemia and in THP-1 cell line. (2) SDF-1 伪 could enhance the proliferation activity of THP-1 cell line. Blocking the interaction between CXCR7 and SDF-1 伪 with CXCR7 monoclonal antibody 11G8 could effectively reduce the proliferation activity of THP-1 cells. (3) in serum-free culture, the apoptotic activity of THP-1 cells was not affected by CXCR7 monoclonal antibody. (4) SDF-1 伪 / CXCR7 biological axis was involved in acute mononuclear cells. The invasiveness of THP-1 cells in vitro, At the same time, CXCR4 and CXCR7 could reduce the invasiveness of THP-1 cells induced by SDF-1 伪 to the maximum extent.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.71
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