肿瘤相关巨噬细胞的异质性与卵巢癌异质性的关联

发布时间：2018-06-03 04:41

本文选题：肿瘤相关巨噬细胞 + 卵巢癌　；参考：《北京协和医学院》2017年博士论文

【摘要】：研究背景及目的巨噬细胞根据其功能与表达细胞因子的不同,可以分为两个极化方向:能够杀死病原体与肿瘤的M1,以及促进肿瘤细胞的生长、迁移甚至侵袭能力的M2。后者在免疫组化或流式细胞检测中常用CD68+CD206或CD68+CD163进行M2细胞的特异标记以进行识别与分选。此外,M2细胞还可以细分为若干亚群,这些亚群之间在细胞因子及细胞表面分子的表达上均有所差异。近来多个实验证实,肿瘤组织的微环境内有大量巨噬细胞,我们称之为肿瘤相关巨噬细胞(TAM),TAM在细胞表型上可能与M2更为相似;此外,肿瘤细胞也可以诱导巨噬细胞向M2方向极化,IL-6、LIF、IFN-γ等细胞因子可能在这一诱导过程中起了重要作用。另一方面,关于肿瘤细胞异质性的研究一直是肿瘤学的重点,除了肿瘤细胞本身的异质性,肿瘤相关巨噬细胞是否也具有异质性即是本实验所讨论的重点。通过不同类型的卵巢癌细胞系与单一来源的巨噬细胞进行相互作用,以期找到其中的差异性及其背后的原因。研究方法1.利用聚酯透明膜transwell使卵巢癌细胞与巨噬细胞系进行非接触性共培养,诱导巨噬细胞极化;2.利用流式细胞术对诱导后的巨噬细胞进行分群分析,计算CD68+CD206双阳性细胞(即M2细胞)占全部CD68阳性细胞(即巨噬细胞)的比例,比较不同诱导条件下M2细胞极化比例的差异,及其随时间的变化;3.收集经不同条件诱导极化的巨噬细胞的上清,利用ELISA对细胞上清中的细胞因子浓度进行测定与分析;4.将经过不同诱导条件极化的巨噬细胞与A2780反向共培养一定时间后,测定不同组A2780生长速率的变化以及细胞因子表达上的差异;研究结果1.不同卵巢癌诱导巨噬细胞向M2极化的能力不一样,且群体中M2比例随时间变化的规律也不同。2.不同卵巢癌细胞诱导出的M2细胞分泌的细胞因子、对肿瘤细胞的生长促进作用也具有异质性,即不同肿瘤细胞诱导出的M2细胞本身也具备异质性。3.反向诱导72h后,平均IL-10浓度与相应肿瘤细胞生长速率呈现显著正相关关系,提示IL-10可能通过巨噬细胞-卵巢癌的相互关系影响卵巢癌的增殖与发展。4.永生化卵巢上皮细胞在诱导巨噬细胞向M2极化方面表现出了与卵巢癌相似的特性,若比较永生化卵巢上皮细胞基因结构与正常卵巢上皮细胞之间的差异,或许可提示巨噬细胞极化过程中有重要作用的基因。结论卵巢癌细胞在诱导巨噬细胞向M2极化方面具有异质性,此种差异不仅存在于透明细胞癌与腺癌这样不同组织类型的卵巢癌之间,也存在于同样是腺癌但是来源不同的肿瘤之间;卵巢癌诱导出的M2在细胞因子的表达、对肿瘤细胞生长的促进作用方面均具有异质性,这种异质性本身即是肿瘤异质性的体现。
[Abstract]:Background and objective macrophages can be divided into two polarized directions according to their function and expression of cytokines: M1, which can kill pathogens and tumors, and M2, which can promote the growth, migration and even invasion of tumor cells. The latter used CD68 CD206 or CD68 CD163 as specific markers to identify and select M2 cells in immunohistochemistry or flow cytometry. In addition, M 2 cells can be subdivided into several subsets, which have different expression of cytokines and molecules on cell surface. A number of recent experiments have confirmed that there are a large number of macrophages in the microenvironment of tumor tissues, which we call tumor-associated macrophages Tam Tam TAM may be more similar to M2 in cell phenotype; in addition, Tumor cells can also induce macrophages to polarize to M2 direction. Cytokines such as IL-6, LIFN- 纬 and IFN- 纬 may play an important role in this induction. On the other hand, the study of tumor cell heterogeneity has always been the focus of oncology. Besides the heterogeneity of tumor cells, whether tumor related macrophages have heterogeneity is the focus of this study. Different types of ovarian cancer cell lines interact with macrophages from a single source in order to find out the differences and the underlying causes. Method 1. The ovarian cancer cell line and macrophage cell line were co-cultured by polyester hyaline membrane (transwell), and the macrophage polarization was induced. The percentage of CD68 CD206 double positive cells (M2 cells) in all CD68 positive cells (i.e. macrophages) was calculated by flow cytometry, and the polarization ratio of M2 cells under different induction conditions was compared. And its change with time. The supernatants of macrophages induced by different conditions were collected and the concentration of cytokines in the supernatants was determined and analyzed by ELISA. After the macrophages with different induced conditions were co-cultured with A2780 for a certain time, the growth rate of A2780 and the difference of cytokine expression in different groups were measured. The ability of different ovarian cancer to induce macrophage polarization to M2 is different, and the rule of M2 ratio changing with time is also different. The cytokines secreted by M2 cells induced by different ovarian cancer cells have heterogeneity in promoting the growth of tumor cells, that is, M2 cells induced by different tumor cells have heterogeneity. 3. After 72 hours of reverse induction, the average concentration of IL-10 was positively correlated with the growth rate of tumor cells, suggesting that IL-10 may influence the proliferation and development of ovarian cancer through the interaction of macrophages and ovarian cancer. Immortalized ovarian epithelial cells showed similar characteristics to ovarian cancer in inducing macrophage polarization to M2. The difference between gene structure of immortalized ovarian epithelial cells and normal ovarian epithelial cells was compared. It may suggest that macrophages play an important role in the process of polarization of genes. Conclusion Ovarian cancer cells have heterogeneity in inducing macrophage polarization to M2. This difference exists not only between ovarian carcinoma with clear cell carcinoma and adenocarcinoma, but also with ovarian carcinoma. The expression of M2 induced by ovarian cancer has heterogeneity in promoting the growth of tumor cells, and this heterogeneity is the manifestation of heterogeneity of tumor.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.31

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