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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 role of M2 macrophages in the invasion and metastasis of ovarian cancer and the possible mechanism of Toll-like receptor (TLRs) signaling pathway. Methods THP-1 cells were induced by 320 nmol / L phorbol ester (PMA) and M2 macrophages were identified by direct immunofluorescence. Transwell chamber was used to establish a non-contact co-culture model with M2 cells and ovarian cancer cells SKOV3. 48 h after co-culture, real-time quantitative PCR (realtime PCR) detection of SKOV3 TLR1,2,6 and matrix metalloproteinase MMP-9 levels, Western blot detection of My D88, TRAF6, P- The expressions of NF-κB and MMP-9 were evaluated by MTT assay. The changes of MMP-9 in SKOV3 treated with TLR1, 2 and 6 agonists at 6, 12 and 24 h were evaluated. Results PMA could induce THP-1 to become type M2 macrophages. After co-cultured with type 2 macrophages for 24 and 48 h, the expression of MMP-9 was significantly higher than that of control group (P <0.05) (P <0.05). After co-cultured for 24 and 48 h, the expression of My D88, TRAF6 and P-NF-κB in SKOV3 also increased synchronously (P <0.05). The expressions of MMP-9 mRNA in SKOV3 6, 12 and 24 h after stimulation with Pam3CSK4, HKLM and FSL-1 by TLR1, 2 and 6 agonists were significantly increased (P <0.05). Conclusion M2-type macrophages induced by differentiation may increase MMP-9 level and increase the invasion and metastasis of SKOV3 in ovarian cancer cells by stimulating and activating TLR1, 2 and 6 signaling pathways.