Effects of recombinant human granulocyte colony-stimulating factor on CXC chemokine receptor 4 expression and chemotaxis in bone marrow microenvironment

doi:10.3969/j.issn.2095-4344.0648

Abstract

Abstract:

BACKGROUND: The bone marrow microenvironment can mediate drug resistance and immune escape of leukemia cells through cell adhesion molecules. These adhesion molecules as new targets can be expected to break the drug resistance mechanism of leukemia cells by reducing their expression levels and functions.
OBJECTIVE: To observe the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on CXC chemokine receptor 4 (CXCR4) expression and chemotaxis in acute leukemia cells, as well as the level of stromal cell-derived factor-1 (SDF-1) in the bone marrow and peripheral blood CD4⁺CD25⁺Foxp3⁺ Treg cells.
METHODS: (1) WEHI-3 cells were co-cultured with rhG-CSF for 6, 12, 18 and 24 hours, and the expression of CXCR4 was detected by flow cytometry. The cell migration was checked by Transwell assay. Healthy Balb/C mice were subcutaneously injected with rhG-CSF. The proportion of Treg cells in the peripheral blood and spleen was detected also by flow cytometry. ELISA was used to detect the level of SDF-1 in the bone marrow.
RESULTS AND CONCLUSION: rhG-CSF significantly reduced the CXCR4 expression on the WEHI-3 cell surface, as well as the chemotactic ability of SDF-1 (P < 0.05). The level of SDF-1 in the bone marrow of healthy mice treated with rhG-CSF was significantly lower than that of the control group (P < 0.05), and the proportion of Treg cells in the peripheral blood and spleen was significantly increased (P < 0.05). In conclusion, rhG-CSF can down-regulate CXCR4 expression on the surface of leukemia cells, reduce SDF-1 level in the bone marrow and mobilize the Treg cells into the peripheral blood. These changes might ultimately break the immune escape and drug resistance under the bone marrow microenvironment, thereby improving the effect of immunotherapy against leukemia.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Granulocyte Colony-Stimulating Factor, Chemokine CXCL12, Receptors, Chemokine, T-Lymphocytes, Regulatory, Tissue Engineering

CLC Number:

R394.2

Wang Jin, Niu Ben, Ma Xiao-rong, Zhang Wang-gang. Effects of recombinant human granulocyte colony-stimulating factor on CXC chemokine receptor 4 expression and chemotaxis in bone marrow microenvironment[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5268-5273.

Figures/Tables 1

2.1 rhG-CSF对WEHI-3细胞表面CXCR4表达的影响二者共同孵育6 h时，实验组WEHI-3细胞CXCR4的表达率为(45.27±3.27)%，对照组为(49.13±3.20)%，差异有显著性意义(P < 0.05)。共同孵育12 h时，实验组WEHI-3细胞CXCR4的表达率为(37.82±4.51)%，对照组为(50.81±2.24)%，差异有显著性意义(P < 0.05)。共同孵育18 h时，实验组WEHI-3细胞的CXCR4表达量为(24.11±2.90)%，而对照组为(48.83±3.85)%，差异有显著性意义(P < 0.05)。共同孵育24 h时，实验组CXCR4的表达率为(23.10± 3.73)%，对照组为(47.04±3.81)%，差异有显著性意义(P < 0.05)。对照组WEHI-3细胞培养前后CXCR4表达无明显变化(P > 0.05)。如图1所示，当WEHI-3细胞与rhG-CSF共同孵育6，12，18，24 h时，与同时间点对照组相比，CXCR4的表达量均明显下降(P < 0.05)。随孵育时间的延长呈下降趋势，但是共同培养18 h与24 h相比二者CXCR4表达量差异无显著性意义(P > 0.05)，CXCR4表达量基本稳定，不再明显下降。 2.2 rhG-CSF对WEHI-3细胞趋化能力的影响对迁移至下室的WEHI-3细胞行结晶紫染色，如图2A-C所示，对照组未经rhG-CSF处理的WEHI-3细胞可在SDF-1α趋化因子的驱动下正常迁移至下室，空白组细胞在无SDF-1α作用下只有极少量细胞迁移至下室，而实验组经rhG-CSF处理后迁移至下室的WEHI-3细胞较对照组明显减少。倒置显微镜下细胞计数结果如图2D所示，实验组WEHI-3细胞迁移至下室细胞数显著低于对照组，二者差异有显著性意义(P < 0.05)，提示WEHI-3细胞与rhG-CSF共培养18 h后对SDF-1α趋化迁移能力显著下降(P < 0.05)。 2.3 rhG-CSF对小鼠外周血Treg细胞比例的影响如图3所示，rhG-CSF组Balb/C小鼠经过rhG-CSF处理后，外周血CD4+CD25+FOXP3+细胞占CD4+细胞比例为(6.64±1.60)%，而生理盐水组小鼠外周血中CD4+CD25+FOXP3+细胞占CD4+细胞比例为(2.96±0.29)%。rhG-CSF组小鼠外周血Treg细胞比例显著高于生理盐水组(P < 0.05)。 rhG-CSF组小鼠经过rhG-CSF处理后，脾脏CD4+CD25+FOXP3+细胞占CD4+细胞比例为(14.21±0.86)%，而生理盐水组小鼠脾脏CD4+CD25+FOXP3+细胞占CD4+细胞比例为(6.28±1.13)%。rhG-CSF组小鼠脾脏Treg细胞比例显著高于生理盐水组(P < 0.05)。 2.4 rhG-CSF对小鼠骨髓SDF-1α水平的影响如图4所示，rhG-CSF组小鼠骨髓腔冲洗液离心后，上清液中SDF-1α水平为(3.47±1.43) μg/L，而生理盐水组SDF-1α水平为(8.87±0.24) μg/L，两组间差异有显著性意义(P < 0.05)，rhG-CSF组较生理盐水组显著降低。而rhG-CSF组小鼠外周血血清SDF-1α水平与生理盐水组相比，二者间差异无显著性意义[rhG-CSF组(2.60±1.60) μg/L；生理盐水组(2.48±1.01) μg/L，P > 0.05]，提示rhG-CSF处理可导致健康Balb/C小鼠骨髓中SDF-1α水平显著下降，而对外周血SDF-1α水平无明显影响。"

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