卵巢癌原位—转移模型的建立及高转移潜能细胞株和相关差异基因筛选

发布时间：2018-03-06 20:41

本文选题：上皮性卵巢癌　切入点：卵巢癌原位-转移模型　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：背景：卵巢癌是死亡率最高的妇科恶性肿瘤。其中,又以上皮性卵巢癌为常见。卵巢癌的难治性主要有两方面,早期不容易发现和转移复发。大多数卵巢癌病人在诊断时常伴有转移。与原发灶相比,转移灶中的肿瘤细胞大多对常规治疗手段并不敏感,容易引起卵巢癌进一步发展。因此较好的控制转移病灶的癌细胞防止复发和进一步转移,我们才有可能改变当前卵巢癌治疗效果差的局面。目的：建立一个理想的卵巢癌生长和转移小鼠模型,基本重现卵巢原位肿瘤形成及腹腔和其他远隔器官转移的整个经过：利用该模型筛选得到具有稳定遗传性的高转移潜能亚细胞株,最终通过芯片分析,获取卵巢癌器官特异性转移相关基因,为临床治疗转移提供参考依据。方法：利用慢病毒转染构建稳定表达两种报告基因的卵巢癌细胞系SKOV3-Luciferase/RFP;经过流式分选筛选纯化肿瘤细胞：利用活体成像系统跟踪观察小鼠体内肿瘤生长转移情况；利用基因芯片检测不同转移特性亚细胞株基因表达情况,筛选出差异基因；实时定量PCR, western blot和免疫组化验证差异基因表达。结果：通过小鼠卵巢癌原位-转移模型可以很好地观察肿瘤细胞在小鼠体内从卵巢原位扩散到其他部位的整个过程：利用原位-转移模型经过三次筛选,成功获得分别具有腹腔和肺部高转移能力的S系和L系细胞株；经动物实验验证S系和L系细胞展现出不同程度的转移能力增强以及转移部位选择性；基因芯片检测结果显示S系细胞表达升高的基因在L系细胞中表达水平一般,功能主要包括调控细胞增殖,迁移,血管生成和脂肪代谢等；L系细胞内表达水平升高的基因功能主要集中于维持干细胞特性和调节细胞免疫等方面。结论：构建的卵巢癌原位-转移模型有较好的稳定性和可靠性；不同的选择性转移能力是因为亚细胞株携带有不同的转移相关基因,这些基因可以帮助肿瘤细胞适应不同的组织微环境。靶向这些转移相关基因的治疗,有可能通过抑制转移发生和清除已经转移的卵巢癌细胞帮助提高治疗效果,改善病人预后。
[Abstract]:Background: ovarian cancer is the highest mortality gynecologic malignant tumor. Among them, epithelial ovarian cancer is also common. There are two main aspects of ovarian cancer's refractory. Most ovarian cancer patients are often diagnosed with metastasis. Compared with the primary tumor, most of the tumor cells in the metastatic tumor are not sensitive to conventional treatment. It is easy to cause further development of ovarian cancer. Therefore, cancer cells that have better control of metastatic lesions prevent recurrence and further metastasis. It's possible to change the current poor treatment of ovarian cancer. Objective: to establish an ideal model of ovarian cancer growth and metastasis in mice. The whole process of ovarian in situ tumor formation and metastasis of abdominal cavity and other distant organs was basically reappeared. A stable and hereditary subcell line with high metastatic potential was obtained by using this model, and finally microarray analysis was used. To obtain genes associated with organ specific metastasis of ovarian cancer, Methods: using lentivirus transfection to construct ovarian cancer cell line SKOV3-Luciferase / RFP which stably expressed two kinds of reporter genes. Tumor growth and metastasis in mice were observed. The gene expression of subcell lines with different transfer characteristics was detected by gene microarray, and the differentially expressed genes were screened out. Real-time quantitative PCR, western blot and immunohistochemistry were used to verify the differential gene expression. Results: the in situ metastasis model of mouse ovarian cancer could be used to observe the whole process of tumor cells spreading from ovary in situ to other parts in mice. Process: three times screening using in situ transfer model, The S and L cell lines with high metastatic ability in abdominal cavity and lung were successfully obtained, and the enhancement of metastasis ability and the selectivity of metastasis site were demonstrated by animal experiments. The results of gene microarray analysis showed that the expression level of the genes with high expression in S cell line was normal in L cell line, and its function mainly included regulating cell proliferation and migration. The gene functions of increased expression level in L cell lines such as angiogenesis and fat metabolism were mainly focused on maintaining stem cell characteristics and regulating cellular immunity. Conclusion: the constructed ovarian cancer in situ metastasis model has good stability and reliability. The different ability of selective transfer is due to the fact that subcell lines carry different genes associated with metastasis, which can help tumor cells adapt to different tissue microenvironments and target the treatment of these metastasis-associated genes. It is possible to improve the therapeutic effect and prognosis by inhibiting metastasis and removing metastatic ovarian cancer cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.31;R-332

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