CXCL12/CXCR4通路在人肺腺癌细胞诱导破骨细胞前体细胞向破骨细胞分化中的作用

Role of CXCL12/CXCR4 pathway in human lung adenocarcinoma cells-induced differentiation of osteo⁃clast precursor cells into osteoclasts

目的探讨趋化因子12(CXCL12)/趋化因子受体4(CXCR4)通路在人肺腺癌细胞诱导破骨细胞前体细胞向破骨细胞分化中的作用及其可能的分子机制。方法以小鼠单核巨噬细胞白血病细胞RAW264.7为破骨细胞前体细胞,随机分为干预组和对照组。两组均在Transwell小室的下室接种RAW264.7细胞,上室接种人肺腺癌细胞A549;干预组在小室内分别加入含12.5、25.0、50.0、100.0µg/mL CXCR4竞争性拮抗剂AMD3100的培养基,对照组仅加入培养基;培养7 d后取两组细胞,行抗酒石酸碱性磷酸酶(TRAP)染色并计数分化成的破骨细胞数量,qPCR检测Transwell下室破骨细胞标志基因组织蛋白酶K(CTSK)、降钙素受体(CTR)、抗酒石酸酸性磷酸酶(TRAP)、破骨细胞分化因子受体(RANK)mRNA,ELISA法检测小室上清液CXCL12、白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)。结果干预组在12.5、25.0、50.0、100.0µg/mL AMD3100处理后,RAW264.7细胞向破骨细胞分化数量分别为(81±3)、(49±3)、(34±3)、(11±2)个,均低于对照组的(99±5)个(P均<0.05);且随着AMD3100处理浓度的增加,RAW264.7细胞向破骨细胞分化数量逐渐减少(P均<0.05)。与对照组比较,干预组在不同浓度AMD3100干预后破骨细胞标志基因CTSK、CTR、TRAP、RANK mRNA表达减少,小室上清液CXCL12、IL-6、TNF-α水平降低(P均<0.05);且随着AMD3100处理浓度的增加,破骨细胞各标志物表达逐渐减少,小室上清液CXCL12、IL-6、TNF-α水平逐渐降低(P均<0.05)。结论抑制CXCL12/CXCR4通路可减少人肺腺癌细胞诱导破骨细胞前体细胞向破骨细胞分化,该作用可能是通过减少IL-6、TNF-α表达实现的。

Objective To explore the role of the chemokine 12(CXCL12)/chemokine receptor 4(CXCR4)path⁃way in human lung adenocarcinoma cells-induced differentiation of osteoclast precursor cells into osteoclasts and the possible molecular mechanism.Methods The Abelson leukemia virus-transformed murine monocyte/macrophage-like RAW 264.7 cells acted as osteoclast precursor cells,and were randomly divided into the treatment group and control group.Both groups were inoculated with RAW 264.7 cells in the lower Transwell chamber and A549 cells in the upper Transwell chamber.The treatment group was separately treated with a culture medium containing the competitive CXCR4 antagonist AMD3100 at 12.5,25.0,50.0,and 100.0μg/mL in the Transwell chamber,and the control group was only given a culture medium.After 7 days of culture,cells in both groups received tartrate-resistant alkaline phosphatase(TRAP)staining to count the number of RAW 264.7 cells differentiating into osteoclasts.Quantitative PCR was used to measure the mRNA levels of the following osteoclast marker genes in the lower Transwell chamber:cathepsin K(CTSK),calcitonin receptor(CTR),TRAP,and receptor activator of nuclear factor-κB(RANK).ELISA was used to determine the levels of CXCL12,interleukin 6(IL-6),and tumor necrosis factor-α(TNF-α)in the supernatant in the chamber.Results In the treatment group,the numbers of RAW 264.7 cells differentiating into osteoclasts were 81±3,49±3,34±3,and 11±2 after 12.5,25.0,50.0,and 100.0µg/mL AMD3100 treatment,all of which were significantly lower than that in the con⁃trol group(99±5),and the number of RAW 264.7 cells differentiating into osteoclasts decreased significantly with the increased concentrations of AMD3100(all P<0.05).Compared with the control group,the treatment group showed significantly reduced mRNA expression of the osteoclast marker genes CTSK,CTR,TRAP,and RANK and significantly de⁃creased levels of CXCL12,IL-6,and TNF-αin the cell supernatant after treatment with various concentrations of AMD3100(all P<0.05).As the concentration of AMD3100 increased,the expression of the osteoclast marker genes and the levels of CXCL12,IL-6,and TNF-αgradually decreased(all P<0.05).Conclusion Inhibiting the CXCL12/CX⁃CR4 pathway can reduce human lung adenocarcinoma cells-induced differentiation of osteoclast precursor cells into osteoclasts,which may be achieved by reducing the expression of IL-6 and TNF-α.