摘要
目的探讨M2型巨噬细胞在卵巢癌侵袭转移中的作用及其可能涉及的Toll样受体(TLRs)信号通路机制。方法用320 nmol/L佛波醇酯(PMA)诱导THP-1细胞,直接免疫荧光技术鉴定M2型巨噬细胞;Transwell小室建立M2细胞与卵巢癌细胞SKOV3体外非接触式共培养模型;24和48 h共培养后,实时荧光定量聚合酶链式反应(realtime PCR)检测SKOV3内TLR1、2、6和基质金属蛋白酶MMP-9水平,蛋白免疫印记(Western blot)检测My D88、TRAF6、P-NF-κB和MMP-9表达;TLR1、2和6激动剂分别作用SKOV3 6、12和24 h后评价MMP-9的变化。结果PMA可诱导THP-1成为M2型巨噬细胞;M2型巨噬细胞与SKOV3共培养24和48 h后,MMP-9的表达水平显著高于对照组(P<0.05);TLR1、2、和6于共培养24和48 h后在SKOV3有较高表达(P<0.05);共培养24和48 h后,TLRs信号通路蛋白My D88、TRAF6和P-NF-κB在SKOV3也同步表达增高(P<0.05);TLR1、2和6激动剂Pam3CSK4、HKLM和FSL-1分别刺激SKOV3 6、12和24 h后MMP-9 mRNA水平有显著高表达(P<0.05)。结论分化诱导后的M2型巨噬细胞,可能通过刺激和活化TLR1、2、6信号途径,引起卵巢癌细胞SKOV3内基质金属蛋白酶MMP-9水平增加,增强肿瘤的侵袭转移能力。
Objective To investigate the influence of M2-type macrophage on the expression of matrix metalloproteinase 9 （MMP-9） in ovarian cancer cells,and to analyze the underlying mechanism about Toll-like receptors signaling pathway.Methods 320 nmol/L PMA was added to induce THP-1 cell into M2-type macrophage.M2-type macrophage and SKOV3 non-contacting co-culture model was established by Transwell method.After the co-culture process of 12 and 24 h,TLR1,2,6 and MMP-9 mRNA were detected by real-time PCR.MyD88,TRAF6,P-NF-κB and MMP-9 expression level were evaluated by Western blot.Testing the expression of MMP-9 after SKOV3 being stimulated by TLR1,2 and 6 agonist for the time of 6,12 and 24 h respectively.Results Type M2 macrophages were induced for differentiation by PMA.After the co-culture process of 24 and 48 h,MMP-9 mRNA and protein relative expressions in SKOV3 cells were significantly higher （P ＜ 0.05）.The content of TLR1,2 and 6 mRNA were highly expressed in SKOV3 （P ＜ 0.05）.Co-cultured after 24 and 48 h,TLRs signaling pathway proteins MyD88,TRAF6,and P-NF-κB have synchronous increased expression in SKOV3 （P ＜ 0.05）.TLR1,2 and 6 agonist respectively stimulated SKOV3 for 6,12,24 h,MMP-9 mRNA level showed a significantly high expression （P ＜ 0.05）.Conclusions Polarization-type tumor-associated macrophage may promote MMP-9 expression in ovarian cancer cells,and its mechanism is possibly explained by stimulating and activation of TLRs signaling pathway.
出处
《基础医学与临床》
CSCD
北大核心
2014年第10期1315-1320,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(81272324
81371894)
江苏省实验诊断学重点实验室基金(XK 201114)
