本文選題：人卵巢細胞　切入點：缺氧　出處：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景卵巢癌是世界上最致命的婦科惡性腫瘤,因此病全球每年死亡人數(shù)超過125,000人,嚴(yán)重威脅著女性的生殖健康。盡管自鉑類藥物出現(xiàn)以來,總體上患者生存有所改善,但大多數(shù)接受治療的婦女最終都會導(dǎo)致化療耐受(耐藥)。了解耐藥的機制,如何逆轉(zhuǎn)這種耐藥,成為當(dāng)前研究的熱點。缺氧廣泛存在于各種實體瘤,缺氧是導(dǎo)致癌細胞耐藥的重要因素。因為95%的氧是在線粒體消耗的,因此線粒體是細胞缺氧時最受影響的細胞器。根據(jù)內(nèi)共生學(xué)說的觀點,線粒體是“噬氧菌”被真核祖細胞吞噬,在長期的共生過程中,逐漸演化而成的。因此,線粒體具有細菌的屬性。缺氧可引起線粒體等細胞器功能不足或受損,引起部分細胞受損甚至死亡,細胞內(nèi)的核酸、多肽等釋放到細胞外,或被包封在細胞內(nèi)的膜泡結(jié)構(gòu)內(nèi)。例如線粒體DNA(mt DNA)含有大量與細菌類似的未甲基化CpG序列,mt DNA激活Toll樣受體-9(Toll like receptor 9,TLR9)等介導(dǎo)的信號途徑,引起多種反應(yīng);另外,線粒體也含有與細菌類似的甲基化多肽,這些多肽可激活甲酰肽受體(formyl peptide receptor,FPR)介導(dǎo)的信號途徑。TLR9、FPR與腫瘤多種惡性行為相關(guān),但在缺氧誘導(dǎo)腫瘤耐藥過程中的作用與機制,尚缺乏深入研究。研究目的探討FPR和TLR9在缺氧誘導(dǎo)人卵巢癌細胞耐藥中的作用及機制。研究方法1.將人卵巢癌細胞(SKOV3)置于1%O2的缺氧條件下不同時間(0h、6h、12h、24h),24h后檢測順鉑對細胞的抑制率。2.將SKOV3置于常氧(21%O2)和缺氧(1%O2)培養(yǎng)箱內(nèi)不同時間(6h、12h、24h后,收集上清。同時,用FPR特異性拮抗劑t-Boc、TLR9拮抗劑氯喹(CQ)或生理鹽水(作為對照組)處理未缺氧的SKOV3細胞,然后用缺氧上清刺激這些未缺氧的SKOV3細胞,24h后檢測順鉑對細胞的抑制率。3.通過WB的方法檢測人卵巢癌組織和細胞中FPR的表達,通過WB、免疫熒光和RT-PCR的方法檢測人卵巢癌組織和細胞中TLR9的表達。4.用FPR激動劑fMLF或者用tBoc將FPR拮抗后再加f MLF刺激SKOV3,通過趨化實驗檢測FPR的功能活性。用fMLF或tBoc+f MLF刺激細胞不同時間(6h、12h、24h),CCK8檢測順鉑對細胞的抑制率。用TLR9激動劑ODN2006刺激細胞不同時間(6h、12h、24h),順鉑處理細胞24h后通過CCK8檢測順鉑對細胞的抑制率。5.用收集的常氧上清、缺氧上清刺激經(jīng)過t Boc或CQ處理的SKOV3后,用Western Blot檢測多藥耐藥相關(guān)蛋白(MRP)、P糖蛋白(P-gp)、P53、Beclin1的表達。研究結(jié)果1.缺氧促進卵巢癌細胞耐藥用“缺氧時SKOV3抑制率變化的百分比”表示“缺氧誘導(dǎo)的耐藥性”(HICR值)。與未缺氧組比較,缺氧各組細胞對順鉑的抑制率不同程度降低,HICR值不同程度增加。缺氧6h組的HICR值增高無統(tǒng)計學(xué)意義(p0.05)。缺氧12h和24h組,細胞對順鉑的抑制率顯著降低,HICR值顯著增高(p0.01)。2.缺氧上清促進卵巢癌細胞耐藥用不同時間的缺氧上清處理SKOV3,上清濃度為60%和80%時,與常氧12h和24h上清處理組相比,缺氧12h和24h上清刺激細胞后,細胞對順鉑的敏感性顯著降低(p0.05),即耐藥性增加;當(dāng)上清濃度為100%時,缺氧6h上清刺激細胞后,也顯著降低對順鉑的敏感性,顯著增加腫瘤細胞的耐藥性(p0.05)。3.卵巢癌組織和卵巢癌細胞中FPR和TLR9的表達FPR與TLR9在人卵巢癌組織和人卵巢癌細胞(SKOV3)中均有表達,卵巢癌組織中其表達與與MRP的表達呈顯著正相關(guān)(p0.05)。FPR激動劑fMLF刺激,可引起劑量依賴性的SKOV3趨化反應(yīng)(p0.05)。缺氧上調(diào)兩受體的表達,其蛋白水平增加程度隨缺氧時間增加而增加,缺氧12h和24h其增加具有顯著性統(tǒng)計學(xué)意義(p0.05),TLR9的mRNA水平也呈相似的變化。另外,免疫熒光結(jié)果顯示TLR9在細胞膜和細胞質(zhì)中均有表達。4.激活FPR或TLR9促進SKOV3細胞耐藥FPR特異性激動劑f MLF或者TLR9特異性激動劑ODN2006處理SKOV3不同時間后,顯著降低順鉑對細胞的抑制率(p0.05),即細胞的耐藥性增加。5.拮抗FPR或TLR9抑制缺氧上清對耐藥性的誘導(dǎo)FPR特異性拮抗劑t-Boc顯著降低HICR值,即抑制了缺氧上清對耐藥性的誘導(dǎo)作用。t Boc不同程度地降低了HICR值。對6、12h缺氧上清,10nM的t Boc即可顯著降低其HICR值,102、103n M t Boc更加明顯降低HICR(p0.05);對24h缺氧上清,100nM的t Boc才能顯著降其HICR值(p0.05),103nM t Boc更加明顯降低其HICR(p0.05)。TLR9拮抗劑(CQ)預(yù)處理后,不同程度地降低HICR值。10μM CQ即可以顯著降低缺氧各組的HICR值(p0.05);100μM CQ對缺氧6h上清的HICR沒有顯著影響,但可以顯著降低缺氧12h和24h上清的HICR值(p0.05)。1000μM CQ對各組HICR均沒有顯著影響(p0.05)。6.FPR或TLR9在缺氧上清上調(diào)耐藥相關(guān)蛋白變化的作用缺氧上清顯著上調(diào)MRP、P-gp、P53、Beclin1表達(p0.05),FPR特異性拮抗劑t Boc顯著削弱缺氧上清對這些蛋白表達的上調(diào)作用(p0.05),Beclin1的表達變化差異無統(tǒng)計學(xué)意義(p0.05)。除了對Beclin1外,TLR9的拮抗劑CQ也可產(chǎn)生類似的效應(yīng)。研究結(jié)論人卵巢癌組織和人卵巢癌細胞表達FPR和TLR9兩受體;缺氧不僅上調(diào)卵巢癌細胞上兩受體的表達,而且引起兩受體的具有生物活性的化合物(配體)釋放到細胞外。FPR和TLR9的激活在缺氧誘導(dǎo)卵巢癌細胞獲得耐藥性過程中發(fā)揮重要作用,這一作用可能與FPR或TLR9被激活引起MRP、P-gp、P53、Beclin1等耐藥相關(guān)蛋白表達上調(diào)有關(guān)。FPR和TLR9有望成為化療增敏的新靶點。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.31

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