FPR和TLR9在缺氧诱导人卵巢癌细胞耐药中的作用及机制

发布时间：2018-03-28 07:46

本文选题：人卵巢细胞　切入点：缺氧　出处：《第三军医大学》2017年硕士论文

【摘要】：研究背景卵巢癌是世界上最致命的妇科恶性肿瘤,因此病全球每年死亡人数超过125,000人,严重威胁着女性的生殖健康。尽管自铂类药物出现以来,总体上患者生存有所改善,但大多数接受治疗的妇女最终都会导致化疗耐受(耐药)。了解耐药的机制,如何逆转这种耐药,成为当前研究的热点。缺氧广泛存在于各种实体瘤,缺氧是导致癌细胞耐药的重要因素。因为95%的氧是在线粒体消耗的,因此线粒体是细胞缺氧时最受影响的细胞器。根据内共生学说的观点,线粒体是“噬氧菌”被真核祖细胞吞噬,在长期的共生过程中,逐渐演化而成的。因此,线粒体具有细菌的属性。缺氧可引起线粒体等细胞器功能不足或受损,引起部分细胞受损甚至死亡,细胞内的核酸、多肽等释放到细胞外,或被包封在细胞内的膜泡结构内。例如线粒体DNA(mt DNA)含有大量与细菌类似的未甲基化CpG序列,mt DNA激活Toll样受体-9(Toll like receptor 9,TLR9)等介导的信号途径,引起多种反应;另外,线粒体也含有与细菌类似的甲基化多肽,这些多肽可激活甲酰肽受体(formyl peptide receptor,FPR)介导的信号途径。TLR9、FPR与肿瘤多种恶性行为相关,但在缺氧诱导肿瘤耐药过程中的作用与机制,尚缺乏深入研究。研究目的探讨FPR和TLR9在缺氧诱导人卵巢癌细胞耐药中的作用及机制。研究方法1.将人卵巢癌细胞(SKOV3)置于1%O2的缺氧条件下不同时间(0h、6h、12h、24h),24h后检测顺铂对细胞的抑制率。2.将SKOV3置于常氧(21%O2)和缺氧(1%O2)培养箱内不同时间(6h、12h、24h后,收集上清。同时,用FPR特异性拮抗剂t-Boc、TLR9拮抗剂氯喹(CQ)或生理盐水(作为对照组)处理未缺氧的SKOV3细胞,然后用缺氧上清刺激这些未缺氧的SKOV3细胞,24h后检测顺铂对细胞的抑制率。3.通过WB的方法检测人卵巢癌组织和细胞中FPR的表达,通过WB、免疫荧光和RT-PCR的方法检测人卵巢癌组织和细胞中TLR9的表达。4.用FPR激动剂fMLF或者用tBoc将FPR拮抗后再加f MLF刺激SKOV3,通过趋化实验检测FPR的功能活性。用fMLF或tBoc+f MLF刺激细胞不同时间(6h、12h、24h),CCK8检测顺铂对细胞的抑制率。用TLR9激动剂ODN2006刺激细胞不同时间(6h、12h、24h),顺铂处理细胞24h后通过CCK8检测顺铂对细胞的抑制率。5.用收集的常氧上清、缺氧上清刺激经过t Boc或CQ处理的SKOV3后,用Western Blot检测多药耐药相关蛋白(MRP)、P糖蛋白(P-gp)、P53、Beclin1的表达。研究结果1.缺氧促进卵巢癌细胞耐药用“缺氧时SKOV3抑制率变化的百分比”表示“缺氧诱导的耐药性”(HICR值)。与未缺氧组比较,缺氧各组细胞对顺铂的抑制率不同程度降低,HICR值不同程度增加。缺氧6h组的HICR值增高无统计学意义(p0.05)。缺氧12h和24h组,细胞对顺铂的抑制率显著降低,HICR值显著增高(p0.01)。2.缺氧上清促进卵巢癌细胞耐药用不同时间的缺氧上清处理SKOV3,上清浓度为60%和80%时,与常氧12h和24h上清处理组相比,缺氧12h和24h上清刺激细胞后,细胞对顺铂的敏感性显著降低(p0.05),即耐药性增加;当上清浓度为100%时,缺氧6h上清刺激细胞后,也显著降低对顺铂的敏感性,显著增加肿瘤细胞的耐药性(p0.05)。3.卵巢癌组织和卵巢癌细胞中FPR和TLR9的表达FPR与TLR9在人卵巢癌组织和人卵巢癌细胞(SKOV3)中均有表达,卵巢癌组织中其表达与与MRP的表达呈显著正相关(p0.05)。FPR激动剂fMLF刺激,可引起剂量依赖性的SKOV3趋化反应(p0.05)。缺氧上调两受体的表达,其蛋白水平增加程度随缺氧时间增加而增加,缺氧12h和24h其增加具有显著性统计学意义(p0.05),TLR9的mRNA水平也呈相似的变化。另外,免疫荧光结果显示TLR9在细胞膜和细胞质中均有表达。4.激活FPR或TLR9促进SKOV3细胞耐药FPR特异性激动剂f MLF或者TLR9特异性激动剂ODN2006处理SKOV3不同时间后,显著降低顺铂对细胞的抑制率(p0.05),即细胞的耐药性增加。5.拮抗FPR或TLR9抑制缺氧上清对耐药性的诱导FPR特异性拮抗剂t-Boc显著降低HICR值,即抑制了缺氧上清对耐药性的诱导作用。t Boc不同程度地降低了HICR值。对6、12h缺氧上清,10nM的t Boc即可显著降低其HICR值,102、103n M t Boc更加明显降低HICR(p0.05);对24h缺氧上清,100nM的t Boc才能显著降其HICR值(p0.05),103nM t Boc更加明显降低其HICR(p0.05)。TLR9拮抗剂(CQ)预处理后,不同程度地降低HICR值。10μM CQ即可以显著降低缺氧各组的HICR值(p0.05);100μM CQ对缺氧6h上清的HICR没有显著影响,但可以显著降低缺氧12h和24h上清的HICR值(p0.05)。1000μM CQ对各组HICR均没有显著影响(p0.05)。6.FPR或TLR9在缺氧上清上调耐药相关蛋白变化的作用缺氧上清显著上调MRP、P-gp、P53、Beclin1表达(p0.05),FPR特异性拮抗剂t Boc显著削弱缺氧上清对这些蛋白表达的上调作用(p0.05),Beclin1的表达变化差异无统计学意义(p0.05)。除了对Beclin1外,TLR9的拮抗剂CQ也可产生类似的效应。研究结论人卵巢癌组织和人卵巢癌细胞表达FPR和TLR9两受体;缺氧不仅上调卵巢癌细胞上两受体的表达,而且引起两受体的具有生物活性的化合物(配体)释放到细胞外。FPR和TLR9的激活在缺氧诱导卵巢癌细胞获得耐药性过程中发挥重要作用,这一作用可能与FPR或TLR9被激活引起MRP、P-gp、P53、Beclin1等耐药相关蛋白表达上调有关。FPR和TLR9有望成为化疗增敏的新靶点。
[Abstract]:Background: ovarian cancer is the most lethal gynecologic malignant tumor, so the death toll disease worldwide each year more than 125000 people, a serious threat to women's reproductive health. Although since the emergence of platinum drugs since the overall survival has improved, but the majority of treated women will eventually lead to chemotherapy resistance (resistance). Drug resistance mechanisms, how to reverse the drug resistance, become the focus of current research. Hypoxia exists in a wide variety of solid tumor, hypoxia is an important factor in drug resistant cancer cells. Because 95% of the oxygen consumption in mitochondria, mitochondria is organelles hypoxia most affected. According to the endosymbiotic theory point of view, mitochondria is "bite aerobic" eukaryotic progenitor cell phagocytosis, in the long process of symbiosis, and gradually evolved. Therefore, the attribute of mitochondria are bacteria. Hypoxia can cause mitochondrial fine etc. Organelle insufficiency or damaged, causing part of the cell damage and even death. The nucleic acid in cell, peptide released outside the cell, or encapsulated within the cell membrane vesicle structure. Such as mitochondrial DNA (MT DNA) contains a lot of bacteria like unmethylated CpG sequences, MT DNA activation of Toll like receptor -9 (Toll like receptor 9, TLR9) mediated signaling pathways, causing a variety of reactions; in addition, mitochondria also contain methylated peptides with similar bacteria, these peptides can activate the formyl peptide receptor (formyl peptide, receptor, FPR) signal transduction mediated by.TLR9 FPR was associated with tumor size, but a variety of malignant behavior. Induction effect and mechanism of tumor resistance during hypoxia, is still a lack of in-depth research. Objective to investigate the effect of FPR and TLR9 in hypoxia induced effect and mechanism of drug resistant human ovarian carcinoma cells. Methods 1. human ovarian cancer cells (SKOV3) under hypoxia 1%O2 Under the condition of different time (0h, 6h, 12h, 24h, 24h) after inhibition of cisplatin on cell detection rate of.2. SKOV3 (21%O2) in normoxia and hypoxia (1%O2) incubator at different time (6h, 12h, 24h, the supernatant was collected. At the same time, with the FPR antagonist t-Boc and TLR9 antagonist agent chloroquine (CQ) or saline (as control group) without hypoxia in SKOV3 cells, and then use these hypoxia stimulation hypoxia supernatant of SKOV3 cells, the expression of 24h after inhibition of cisplatin on cell detection rate of.3. detected by WB in human ovarian cancer cells and tissues of FPR, through WB, the expression of.4. TLR9 method and immunofluorescence detection of RT-PCR human ovarian cancer cells and tissues with FPR agonist fMLF or tBoc antagonist FPR plus f stimulation of MLF SKOV3, through the functional activity of chemotaxis assay. FPR with fMLF or tBoc+f stimulation of MLF cells at different times (6h, 12h, 24h, CCK8) detection cisplatin on cells The inhibition rate of cell stimulation. Different time with TLR9 agonist ODN2006 (6h, 12h, 24h), cisplatin treated cells was detected by CCK8 24h after cisplatin on cell growth rate of.5. in normoxic supernatant were collected, hypoxia was stimulated after t Boc or CQ SKOV3, with Western Blot detection of multidrug resistance related protein (MRP), P (P-gp), P53, protein expression of Beclin1. Results: 1. hypoxia promotes ovarian cancer cells resistant to hypoxia SKOV3 inhibition percentage of medicinal "rate change" resistance to hypoxia induced "(HICR). And the hypoxia group, hypoxia group inhibition rate of different cells to cisplatin decreased and HICR value increased in different degrees. In hypoxia 6h group elevated HICR value was not statistically significant (P0.05). 12h and 24h hypoxia group, the inhibition of cisplatin was significantly reduced, HICR value increased significantly (P0.01).2. hypoxia supernatant promote resistant ovarian cancer cells do not at the same time The hypoxia supernatant SKOV3, supernatant concentration was 60% and 80%, compared with normoxia 12h and 24h supernatant group, hypoxia 12h and 24h supernatant stimulated cells, cellular sensitivity to cisplatin was significantly decreased (P0.05), the resistance increases; when the supernatant concentration was 100%, 6h was stimulated cells after hypoxia. Also significantly decreased the sensitivity to cisplatin, significantly increased the resistance of tumor cells (P0.05) and FPR TLR9.3. cells in ovarian cancer and ovarian cancer in the expression of FPR and TLR9 in human ovarian carcinoma tissues and human ovarian cancer cells (SKOV3) were expressed in ovarian cancer tissue and its expression with the expression of MRP was significantly positive correlation (P0.05).FPR agonist fMLF stimulation caused a dose-dependent chemotactic response of SKOV3 (P0.05). The expression of two receptor is upregulated by hypoxia, the protein level increases with the increase of 12h and hypoxia, hypoxia significantly increased the 24h system Statistically significant (P0.05), TLR9 mRNA levels showed similar changes. In addition, immunofluorescence showed that TLR9 was in the cytoplasm and cell membrane expression of.4. activated FPR or TLR9 promote SKOV3 cell drug resistance FPR specific agonist f or MLF specific TLR9 agonist ODN2006 SKOV3 at different time after inhibition significantly decreased cisplatin on cell rate (P0.05), namely cell drug resistance increased.5. antagonist of FPR or TLR9 inhibited the hypoxia supernatant on drug resistance induced by FPR specific antagonist t-Boc significantly reduce the value of HICR, which inhibited the hypoxia supernatant on drug resistance induced by.T Boc reduced HICR value. The 6,12h hypoxia supernatant, 10nM t Boc can significantly reduce the value of the HICR, 102103n M t Boc HICR (P0.05) significantly lower; on 24h 100nM t Boc hypoxia supernatant can significantly reduce the value of HICR (P0.05), 103nM t Boc more significantly lower HICR (P0.05).TL R9 antagonist (CQ) after pretreatment, reduce the HICR value of.10 M CQ can significantly reduce hypoxia HICR value (P0.05); 100 M CQ 6h HICR on the hypoxia supernatant had no significant effect, but can significantly reduce hypoxia 12h and 24h supernatant values of HICR (P0.05).1000. M CQ had no significant influence on the HICR (P0.05).6.FPR or TLR9 related protein changes in hypoxia resistant effect of hypoxia supernatant supernatant increased significantly up-regulated the expression of MRP, P-gp, P53, the expression of Beclin1 (P0.05), FPR specific antagonist t Boc significantly weaken the effect of hypoxia on the expression of the supernatant up-regulated protein (P0.05), no statistically significant differences in the expression of Beclin1 (P0.05). With the exception of the Beclin1, the TLR9 antagonist CQ can also have a similar effect. The conclusion of the study of human ovarian carcinoma and human ovarian cancer cell FPR and the expression of TLR9 two receptor; expression of hypoxia not only two receptor was up-regulated in ovarian cancer cells. Moreover, due to biologically active compounds (two receptor ligands) released to activation of extracellular.FPR and TLR9 in hypoxia induced ovarian cancer cells play an important role in the process of acquiring drug resistance, may be related to FPR or TLR9 is activated by MRP, P-gp, P53, Beclin1 expression of drug resistance related protein upregulation of.FPR and TLR9 is expected to become a new target for chemotherapy.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.31

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