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目的:评价病毒巨噬细胞炎性蛋白ⅡN端肽(NT21MP)是否通过干扰SDF-1α/CXCR4信号抑制人乳腺癌细胞株SKBR3细胞的趋化作用。方法:以RT-PCR和免疫组化检测SKBR3和MCF-7两种人乳腺癌细胞中CXCR4的表达;用细胞转移实验检测在NT21MP存在或缺乏的情况下SDF-1α诱导SKBR3细胞的趋化作用;以Fluo3/AM为细胞内游离钙离子的荧光指示剂,用激光扫描共聚焦显微镜测定NT21MP对SDF-1α诱导SKBR3细胞内游离钙浓度的影响;Western blot分析ERK1/2和FAK蛋白的磷酸化水平变化。结果:相对于MCF-7细胞,SKBR3细胞中CXCR4蛋白表达水平较高;经SDF-1α处理后,SKBR3的迁移能力提高,CXCR4抑制剂AMD3100可有效抑制SKBR3细胞的迁移,细胞经NT21MP预处理后,可剂量依赖性地抑制SKBR3细胞的迁移(P<0.05);NT21MP也可抑制由SDF-1α诱导的细胞内Ca2+峰值(P<0.05),而钙离子浓度升高是SKBR3细胞迁移的重要信号之一;另外,相对于阴性对照组,NT21MP也可下调SDF-1α诱导的SKBR3中信号蛋白ERK1/2和FAK的磷酸化水平(P<0.05)。结论:NT21MP可抑制SDF-1α诱导的SK-BR3细胞的迁移,可能与其上游钙离子释放和ERK1/2及FAK磷酸化阻断信号有关。
AIM: To evaluate whether chemotactic effect of virus macrophage inflammatory protein Ⅱ N-terminal peptide (NT21MP) on human breast cancer cell line SKBR3 by interfering with SDF-1α / CXCR4 signaling. Methods: The expression of CXCR4 in SKBR3 and MCF-7 human breast cancer cells was detected by RT-PCR and immunohistochemistry. The chemotaxis of SDF-1α-induced SKBR3 cells in the presence or absence of NT21MP was detected by cell metastasis The effect of NT21MP on SDF-1αinduced the intracellular free calcium concentration in SKBR3 cells was measured by Fluo3 / AM as fluorescent indicator of intracellular free calcium ion. The phosphorylation of ERK1 / 2 and FAK protein was analyzed by Western blot Horizontal changes. Results: The expression of CXCR4 protein in SKBR3 cells was higher than that in MCF-7 cells. The migration ability of SKBR3 was enhanced after SDF-1α treatment. The CXCR4 inhibitor AMD3100 could effectively inhibit the migration of SKBR3 cells. After NT21MP pretreatment , Inhibited the migration of SKBR3 cells in a dose-dependent manner (P <0.05). NT21MP also inhibited the intracellular Ca2 + peak induced by SDF-1α (P <0.05), while the increase of calcium concentration was an important signal of SKBR3 cell migration In addition, NT21MP also down-regulated SDF-1α-induced phosphorylation of ERK1 / 2 and FAK in SKBR3 (P <0.05) relative to the negative control group. CONCLUSION: NT21MP can inhibit SDF-1α-induced migration of SK-BR3 cells, which may be related to the release of calcium ions and the blockade of ERK1 / 2 and FAK phosphorylation.