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目的 :研究Toll样受体4(TLR4)在人类乳腺癌MCF-7细胞株中的表达及在肿瘤增殖和转移中的作用。方法 :脂多糖(LPS)刺激MCF-7细胞株,RT-PCR、Real-time PCR、FCS和Western blotting检测TLR4在m RNA和蛋白水平表达变化;MTT检测细胞增殖;RT-PCR和real-time PCR检测基质金属蛋白酶(MMP-2)、MMP-9和血管内皮生长因子(VEGF)的m RNA表达并用ELISA检测培养上清中其蛋白表达水平;Western blot检测TLR4信号传导通路下游蛋白My D88的表达;CBA测定细胞培养上清的炎性细胞因子;划痕实验检测癌细胞的生物学侵袭力。结果:实验表明,LPS刺激MCF-7细胞株能明显上调TLR4 m RNA和蛋白表达(P<0.05)。MTT结果显示,LPS对癌细胞增殖没有影响。LPS激活TLR4后,MMP-2、MMP-9和VEGF在m RNA和蛋白水平的表达均明显上调(P<0.05)。划痕实验表明,LPS能显著增强MCF-7细胞株的划痕愈合能力。此外,LPS能诱导TLR4信号通路下游My D88蛋白表达的显著上调(P<0.05),IL-6的分泌增多(P<0.05)。结论:LPS能明显增强MCF-7细胞株TLR4的表达,增强癌细胞的侵袭力。
Objective: To investigate the expression of TLR4 in human breast cancer MCF-7 cell line and its role in tumor proliferation and metastasis. Methods: The expression of TLR4 at m RNA and protein levels was detected by RT-PCR, Real-time PCR, FCS and Western blotting respectively. The proliferation of MCF-7 cells was detected by RT-PCR and real-time PCR was used to detect the mRNA expression of MMP-2, MMP-9 and VEGF in the culture supernatant, Western blot was used to detect the expression of My D88 in the downstream of TLR4 signal transduction pathway Expression; CBA determination of cell culture supernatant of inflammatory cytokines; scratch test to detect the biological invasiveness of cancer cells. Results: The results showed that LPS stimulation of MCF-7 cells significantly upregulated the expression of TLR4 mRNA and protein (P <0.05). MTT results showed that LPS had no effect on the proliferation of cancer cells. LPS activated TLR4, MMP-2, MMP-9 and VEGF in the m RNA and protein levels were significantly increased (P <0.05). Scratch experiments showed that LPS can significantly enhance the scratch healing ability of MCF-7 cell line. In addition, LPS induced a significant up-regulation of My D88 protein expression (P <0.05) and IL-6 secretion downstream of TLR4 signaling pathway (P <0.05). Conclusion: LPS can significantly enhance the expression of TLR4 in MCF-7 cells and enhance the invasiveness of cancer cells.