本文關(guān)鍵詞： 內(nèi)皮祖細(xì)胞 舒林酸 環(huán)氧化酶2 遷移趨化能力　出處：《第三軍醫(yī)大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：背景： 卵巢癌是女性生殖系統(tǒng)常見惡性腫瘤。因其發(fā)病隱匿，很容易轉(zhuǎn)移，且極易復(fù)發(fā)，死亡率高居女性生殖系統(tǒng)腫瘤首位。加之現(xiàn)階段預(yù)防和治療手段的不足，5年生存率低下，嚴(yán)重威脅女性生活質(zhì)量，給家庭社會(huì)帶來巨大負(fù)擔(dān)。隨著對(duì)該病認(rèn)識(shí)的發(fā)展，已證實(shí)卵巢癌發(fā)生發(fā)展的重要病理基礎(chǔ)是其活躍的血管生成，它影響著腫瘤的侵襲轉(zhuǎn)移以及疾病轉(zhuǎn)歸。近期研究證明，內(nèi)皮祖細(xì)胞(endothelial progenitor cells，EPCs)在血管生成中居于關(guān)鍵環(huán)節(jié)，血管內(nèi)皮細(xì)胞可能由骨髓來源的循環(huán)EPCs摻入并分化而來。基于此本課題組提出假設(shè)：卵巢癌中腫瘤細(xì)胞與EPCs共處局部微環(huán)境，腫瘤細(xì)胞通過分泌多種細(xì)胞因子或表達(dá)相關(guān)活性酶類與EPCs作用，促進(jìn)腫瘤的血管生成。本研究擬在體外通過抑制卵巢癌細(xì)胞環(huán)氧化酶2(cyclooxygenase-2，COX-2)的表達(dá)，通過非接觸共培養(yǎng)的方式觀察其對(duì)EPCs遷移趨化能力的影響。 目的： 通過環(huán)氧化酶抑制劑舒林酸來抑制卵巢癌SKOV-3細(xì)胞COX-2的表達(dá)，然后與EPCs共培養(yǎng)觀察其遷移趨化能力的變化，并初探可能的機(jī)制。 方法： 1.運(yùn)用密度梯度離心法分離臍血中的單核類細(xì)胞，聯(lián)合應(yīng)用生長因子促進(jìn)目的細(xì)胞生長和貼壁篩選法純化細(xì)胞，，后經(jīng)雙熒光染色法和免疫熒光技術(shù)鑒定該臍血來源的細(xì)胞； 2.采用階梯式濃度的舒林酸作用于卵巢癌SKOV-3細(xì)胞不同時(shí)間，經(jīng)RT-PCR和WB檢測(cè)COX-2表達(dá)情況，并做多因素方差分析，找出抑制作用最明顯的組合； 3.在體外共培養(yǎng)卵巢癌COX-2受抑SKOV-3細(xì)胞和EPCs，采用對(duì)比觀察法比較EPCs遷移趨化能力的變化，并檢查細(xì)胞中HPA表達(dá)量和培養(yǎng)液中VEGF含量；關(guān)聯(lián)分析COX-2、HPA、VEGF與EPCs遷移數(shù)量的關(guān)系。 結(jié)果： 1.EPCs初期以圓形細(xì)胞為主；第3天后開始做貼壁生長，略呈梭狀；第7天開始逐漸形成細(xì)胞集落，第14天開始出現(xiàn)典型“鋪路石”形態(tài)。對(duì)EPCs進(jìn)行FITC-UEA-1和DiI-Ac-LDL攝取功能鑒定，呈雙陽性。免疫熒光檢測(cè)EPCs表面標(biāo)志CD34和CD133均呈陽性反應(yīng)。 2.采用環(huán)氧化酶抑制劑舒林酸處理卵巢癌SKOV-3細(xì)胞，RT-PCR結(jié)果顯示藥物與抑制效率間存在量效-時(shí)效關(guān)系。然后選取抑制作用較強(qiáng)的3個(gè)組合再次作用與卵巢癌SKOV-3細(xì)胞，行WB檢測(cè)COX-2蛋白表達(dá)情況，結(jié)果顯示:當(dāng)舒林酸為2mmol/L處理48h時(shí)，COX-2表達(dá)下降最為顯著。 3.體外觀察到卵巢癌COX-2受抑細(xì)胞可使EPCs遷移趨化能力明顯降低，COX-2、HPA、VEGF分別與EPCs遷移數(shù)量相關(guān)；且COX-2表達(dá)量分別與HPA、VEGF表達(dá)量相關(guān)。 結(jié)論： 1.經(jīng)過分離培養(yǎng)鑒定臍血來源的單核細(xì)胞，成功獲得EPCs； 2.經(jīng)過舒林酸處理卵巢癌SKOV-3細(xì)胞，確能使COX-2表達(dá)受抑制，成功構(gòu)建卵巢癌細(xì)胞COX-2的抑制模型； 3.最后在體外觀察到卵巢癌COX-2受抑細(xì)胞可使EPCs遷移趨化能力明顯降低，且此作用可能與體系中的HAP、VEGF含量降低有關(guān)。
[Abstract]:Background: Ovarian cancer is a common malignant tumor in female reproductive system. Because of its hidden incidence, easy metastasis, and easy to relapse, the mortality rate of female reproductive system tumor is the first. In addition, the current prevention and treatment methods are insufficient. The 5-year survival rate is low, which seriously threatens the quality of life of women and brings a huge burden to the family and society. With the development of understanding of the disease, it has been proved that the important pathological basis for the occurrence and development of ovarian cancer is its active angiogenesis. It affects the invasion and metastasis of tumor and the outcome of disease. Recent studies have proved that endothelial progenitor cells is endothelial progenitor cells. EPCs play a key role in angiogenesis. Vascular endothelial cells may be derived from bone marrow-derived circulating EPCs incorporation and differentiation. Based on this study, we proposed the hypothesis that tumor cells co-exist with EPCs in local microenvironment in ovarian cancer. Tumor cells interact with EPCs by secreting a variety of cytokines or expressing related active enzymes. The aim of this study was to inhibit the expression of cyclooxygenase-2 (COX-2) in ovarian cancer cells in vitro. The effect of non-contact co-culture on the migration and chemotaxis of EPCs was observed. Objective: Cyclooxygenase inhibitor sulindac was used to inhibit the expression of COX-2 in ovarian cancer SKOV-3 cells, and then co-cultured with EPCs to observe the changes of migration and chemotaxis, and to explore the possible mechanism. Methods: 1. Mononuclear cells were isolated from cord blood by density gradient centrifugation. Growth factor was used to promote the growth of the target cells and the adherent screening method was used to purify the cells. The cord blood cells were identified by double fluorescence staining and immunofluorescence technique. 2. The expression of COX-2 in ovarian cancer SKOV-3 cells was detected by RT-PCR and WB, and multivariate analysis of variance was made. Find out the most obvious combination of inhibition; 3. COX-2 cells and EPCs cells were co-cultured in vitro to compare the changes of migration and chemotaxis of EPCs. The expression of HPA and the content of VEGF in culture medium were examined. The relationship between VEGF and EPCs migration was analyzed. Results: 1. At the beginning of EPCs, round cells were dominant; After the third day, the adherent growth began to be slightly fusiform; On the 7th day, the colony began to form gradually, and on the 14th day, the typical "paving stone" appeared. The FITC-UEA-1 and DiI-Ac-LDL uptake function of EPCs were identified. The EPCs surface markers CD34 and CD133 were positive by immunofluorescence. 2.Ovarian cancer SKOV-3 cells were treated with cyclooxygenase inhibitor sulindac. The results of RT-PCR showed that there was a dose-effect relationship between drug and inhibition efficiency, and then three combinations with strong inhibitory effect were selected to react with ovarian cancer SKOV-3 cells. The expression of COX-2 protein was detected by WB. The results showed that the expression of COX-2 decreased most significantly when sulinic acid was 2 mmol / L for 48 h. 3. In vitro, the inhibitory effect of COX-2 on the migration and chemotaxis of EPCs was found to be related to the number of EPCs migration. The expression of COX-2 was correlated with the expression of COX-2. Conclusion: 1. EPCs were successfully obtained by isolation and culture of monocytes derived from cord blood. 2.Ovarian cancer SKOV-3 cells treated with sulinic acid could inhibit the expression of COX-2, and successfully construct the inhibition model of COX-2 in ovarian cancer cells. 3. Finally, it was found that the inhibitory effect of COX-2 on ovarian cancer cells significantly decreased the migration and chemotaxis of EPCs in vitro, and this effect may be related to the decrease of EPCs content in the system.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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