【作者】 刘萍；

【导师】 倪虹；

【作者基本信息】 天津医科大学 ， 肿瘤学（专业学位）， 2016， 硕士

【摘要】 目的:卵巢癌在妇科恶性肿瘤中的病死率很高。全面的细胞减灭术和术后辅以铂类加紫杉醇为基础的联合化疗是其标准治疗方案。虽然在治疗上有了很大的进步,但最终仍会出现疾病的复发或转移。究其根源,主要是因为化疗耐药的产生。如何预测卵巢癌耐药,指导卵巢癌患者的治疗,在卵巢癌的治疗研究中成为了一个热点问题。晚期卵巢癌患者常伴有恶性腹水的产生,并且腹水中的细胞因子与患者的无进展生存和预后密切相关。此外,卵巢腹水可促进肿瘤细胞的增殖,并可抑制细胞的凋亡。由此我们推测,卵巢癌腹水可能促进卵巢癌耐药的发生。因此,在本研究中我们首先检测卵巢癌腹水对卵巢癌细胞生物学行为的影响,然后探究卵巢癌腹水与卵巢癌化疗耐药的相关性。方法:1.搜集23例卵巢癌腹水上清,用不同的腹水培养卵巢癌SKOV3细胞,并分为实验组(腹水培养组)和对照组。2.采用MTT实验、细胞划痕修复实验和Transwell侵袭实验检测卵巢癌腹水对SKOV3细胞的增殖、迁移和侵袭能力的影响。3.对实验组和对照组的细胞行不同浓度化疗药物的处理,并采用MTT实验检测各组肿瘤细胞对紫杉醇敏感性的变化。4.化疗药物处理24h后,采用流式细胞仪检测不同组内SKOV3细胞的凋亡情况。5.采用Western Blot检测耐药蛋白在对照组、腹水处理组和腹水肿瘤细胞组的表达水平。结果:1.MTT实验和细胞划痕修复实验结果显示,腹水可促进卵巢癌SKOV3细胞的增殖,并可促使细胞向划痕区域明显迁移(P<0.05)。Transwell侵袭实验结果显示,腹水培养组穿膜细胞数与SKOV3空白对照组相比,差异无统计学意义(P>0.05),腹水没有增加SKOV3细胞的侵袭能力。2.对实验组(腹水肿瘤细胞组与腹水培养组)和对照组的细胞行不同浓度化疗药物处理48h后,采用MTT实验检测各组细胞对紫杉醇的敏感性。结果显示,在相同药物浓度下,腹水肿瘤细胞组和腹水组的细胞抑制率明显低于对照组,而腹水肿瘤细胞组又明显低于腹水组。经腹水处理后SKOV3细胞对紫杉醇的敏感性显著下降(P<0.05),与对照组相比,下降了约2.3倍。3.流式细胞仪检测各组细胞的凋亡,结果显示,腹水培养组与对照组相比,细胞凋亡率无明显变化(P>0.05);紫杉醇作用24h后,腹水培养组的凋亡率明显低于对照组(P<0.05)。4.卵巢癌腹水可以增加肿瘤细胞内耐药蛋白P-gp和MRP1的表达。结论:1.卵巢癌腹水可促进卵巢癌SKOV3细胞的增殖和迁移。2.卵巢癌腹水可增加卵巢癌SKOV3细胞对紫杉醇的耐药性,减少细胞凋亡。3.卵巢癌腹水可增加肿瘤细胞耐药蛋白的表达。更多还原

【Abstract】 Objectives: Ovarian cancer mortality is highest in gynecologic malignancies. The standard treatment of ovarian cancer is ideal cytoreductive surgery and neoadjuvant chemotherapy based on platinum and paclitaxel after surgery. Although there has great progress in the treatment, the relapse or metastasis of the disease will eventually happen. The main reason for this lies in chemo-resistance. How to predict drug resistance and guide treatment in ovarian cancer has become a hot issue. Advanced ovarian cancer patients are often accompanied by the malignant ascites, and the cytokine in ascites is closely related to progression-free survival and prognosis of cancer patients. In addition, ovarian ascites can promote tumor cell proliferation and inhibit apoptosis. Thus, we hypothesized that ovarian cancer ascites may promote the occurrence of ovarian cancer drug resistance. Therefore, in this study, we firstly detect the ascites how to influent the biological behavior of ovarian cancer cells, and then explore the correlation between ascites and chemoresistance.Methods: 1. To collect 23 cases of ovarian cancer ascites supernatant, and use them to culture ovarian cancer SKOV3 cells. The cells were divided into experimental groups(ascites culture group) and control group according to the culture conditions. 2. MTT assay, wound scratch assay and transwell were used to investigate the effect of ascites on the ability of proliferation, migration and invasion of SKOV3. 3. Treatment of different concentrations of chemotherapeutic drugs in the experimental group and the control group. MTT method was used to describe ovarian cancer SKOV3 cells, cells treated with ascites and ascites tumor cells sensitivity of paclitaxel. 4. Flow cytometry was used to detect the apoptosis of SKOV3 cells in different groups after chemotherapy drugs treatment. 5. Western Blot was used to detect the expression levels of resistance protein in control group, cells treated with ascites group and ascites tumor cells group.Results: 1. MTT assay and wound healing assay showed that ascites can promote the proliferation and migration of ovarian cancer SKOV3 cells(P<0.05). Transwell assay showed that, compared with SKOV3 blank control group, there was no significant difference in the cell number in cells treated with ascites group(P>0.05), and ascites did not increase the invasion ability of SKOV3 cells. 2. MTT method was used to detect the sensitivity of the cells in the ascites tumor cell group, the ascites culture group and the control group to the chemotherapy drug paclitaxel. The results showed that, in the same drug concentration, the cell inhibition rate of ascites tumor cells and ascites group was significantly lower than that of the control group, while the ascites tumor cell group was significantly lower than that of the ascites group. After treatment of ascites, the sensitivity of SKOV3 cells to paclitaxel was significantly decreased(P<0.05), which decreased by about 2.3 times compared with the control group. 3. Flow cytometry assay showed that, compared with the control group, cell apoptosis rate of the ascites culture group had no significant change(P > 0.05); After the effect of paclitaxel on 24 h, the apoptosis rate of the ascites culture group was significantly lower than that of the control group(P<0.05). 4. Ovarian cancer ascites can increase the expression of multidrug resistance protein of SKOV3 cells and tumor cells in ascites.Conclusions: 1. Ovarian cancer ascites can promote the proliferation and migration of ovarian cancer SKOV3 cells. 2. Ovarian cancer ascites increased the resistance of ovarian cancer SKOV3 cells to paclitaxel and reduced cell apoptosis. 3. Ovarian cancer ascites can increase the expression of drug resistance protein in tumor cells.更多还原