抑制卵巢癌细胞COX-2对内皮祖细胞迁移趋化能力影响的体外研究

发布时间：2018-02-05 05:44

本文关键词： 内皮祖细胞舒林酸环氧化酶2 迁移趋化能力　出处：《第三军医大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景：卵巢癌是女性生殖系统常见恶性肿瘤。因其发病隐匿，很容易转移，且极易复发，死亡率高居女性生殖系统肿瘤首位。加之现阶段预防和治疗手段的不足，5年生存率低下，严重威胁女性生活质量，给家庭社会带来巨大负担。随着对该病认识的发展，已证实卵巢癌发生发展的重要病理基础是其活跃的血管生成，它影响着肿瘤的侵袭转移以及疾病转归。近期研究证明，内皮祖细胞(endothelial progenitor cells，EPCs)在血管生成中居于关键环节，血管内皮细胞可能由骨髓来源的循环EPCs掺入并分化而来。基于此本课题组提出假设：卵巢癌中肿瘤细胞与EPCs共处局部微环境，肿瘤细胞通过分泌多种细胞因子或表达相关活性酶类与EPCs作用，促进肿瘤的血管生成。本研究拟在体外通过抑制卵巢癌细胞环氧化酶2(cyclooxygenase-2，COX-2)的表达，通过非接触共培养的方式观察其对EPCs迁移趋化能力的影响。目的：通过环氧化酶抑制剂舒林酸来抑制卵巢癌SKOV-3细胞COX-2的表达，然后与EPCs共培养观察其迁移趋化能力的变化，并初探可能的机制。方法： 1.运用密度梯度离心法分离脐血中的单核类细胞，联合应用生长因子促进目的细胞生长和贴壁筛选法纯化细胞，，后经双荧光染色法和免疫荧光技术鉴定该脐血来源的细胞； 2.采用阶梯式浓度的舒林酸作用于卵巢癌SKOV-3细胞不同时间，经RT-PCR和WB检测COX-2表达情况，并做多因素方差分析，找出抑制作用最明显的组合； 3.在体外共培养卵巢癌COX-2受抑SKOV-3细胞和EPCs，采用对比观察法比较EPCs迁移趋化能力的变化，并检查细胞中HPA表达量和培养液中VEGF含量；关联分析COX-2、HPA、VEGF与EPCs迁移数量的关系。结果： 1.EPCs初期以圆形细胞为主；第3天后开始做贴壁生长，略呈梭状；第7天开始逐渐形成细胞集落，第14天开始出现典型“铺路石”形态。对EPCs进行FITC-UEA-1和DiI-Ac-LDL摄取功能鉴定，呈双阳性。免疫荧光检测EPCs表面标志CD34和CD133均呈阳性反应。 2.采用环氧化酶抑制剂舒林酸处理卵巢癌SKOV-3细胞，RT-PCR结果显示药物与抑制效率间存在量效-时效关系。然后选取抑制作用较强的3个组合再次作用与卵巢癌SKOV-3细胞，行WB检测COX-2蛋白表达情况，结果显示:当舒林酸为2mmol/L处理48h时，COX-2表达下降最为显著。 3.体外观察到卵巢癌COX-2受抑细胞可使EPCs迁移趋化能力明显降低，COX-2、HPA、VEGF分别与EPCs迁移数量相关；且COX-2表达量分别与HPA、VEGF表达量相关。结论： 1.经过分离培养鉴定脐血来源的单核细胞，成功获得EPCs； 2.经过舒林酸处理卵巢癌SKOV-3细胞，确能使COX-2表达受抑制，成功构建卵巢癌细胞COX-2的抑制模型； 3.最后在体外观察到卵巢癌COX-2受抑细胞可使EPCs迁移趋化能力明显降低，且此作用可能与体系中的HAP、VEGF含量降低有关。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

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