结直肠癌微环境中PKM2促进巨噬细胞募集的作用和机制研究

发布时间：2018-02-08 08:46

本文关键词： PKM2 结直肠癌 CCL2 巨噬细胞　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：一、前言结直肠癌是消化道中常见的恶性肿瘤之一,其发病率和病死率仅次于胃癌、食管癌和原发性肝癌。因此,深入研究结肠直肠癌分子机制及其关键信号通路分子的鉴定,对进一步提高结肠直肠癌的治疗水平具有重要意义。研究证实结肠直肠癌的发生进展是一个众多因素相互共同作用的过程。肿瘤微环境是肿瘤生长的内环境。而巨噬细胞具有促肿瘤血管新生和重塑肿瘤微环境等功能作用,但其如何募集到肿瘤部位及其发挥相关功能作用机理至今尚不完全清楚。丙酮酸激酶M2(PKM2)是糖酵解过程中一个关键限制酶,同时也是一个重要的转录辅助因子,其PKM2在癌症发生发展过程中的非糖酵解功能和作用日益得到人们的关注。在结直肠癌微环境中,PKM2是如何塑造肿瘤微环境,进而促进结直肠癌的生长和存活,其机制尚不清楚。二、研究目的探讨结直肠癌细胞中PKM2在结直肠癌微环境中对巨噬细胞募集的作用及其机制。三、研究方法本研究主要通过siRNA干扰等方法探讨PKM2对巨噬细胞募集的相关趋化因子表达的影响;通过实时荧光定量PCR等方法证实丙酮酸激酶对巨噬细胞募集相关趋化因子表达和活性影响的分子机制,进而逆造肿瘤炎症微环境,进而促进结直肠癌发生发展进程。研究结果1.临床组织标本免疫组化的结果显示:相对于正常的对照组而言,在结直肠癌组织中丙酮酸激酶M2型表达量明显增多,同时巨噬细胞的数量和分布也集中在肿瘤腺体周边,同时,蛋白质印迹的结果也表明丙酮酸激酶M2在结直肠癌细胞中的表达量相对于正常肠上皮细胞的含量明显增多2.条件培养基对巨噬细胞的趋化实验结果显示,与对照组相比,利用特异性siRNA敲低PKM2的表达,明显降低巨噬细胞的趋化和迁移。为了进一步避免脱靶,我们利用已证实具有干扰效果的shPKM2慢病毒感染细胞并收集其条件培养基,随后进行趋化实验,其结果与上述的一致。3.Real-time PCR实验结果显示:相对于对照组而言,干扰结直肠癌细胞中PKM2的表达能够明显降低CCL2在mRNA水平上的表达,同时酶联免疫吸附实验结果也进一步表明,PKM2能够影响CCL2的表达和分泌4.肠上皮细胞中内源性的PKM2与IKKβ相互作用,然而,敲低PKM2的表能够明显抑制p65(Ser276)的磷酸化活性。5.p65的过表达可拯救敲低PKM2而引起的结直肠癌细胞CCL2的转录活性的降低,且可恢复PKM2敲低引起的细胞侵袭能力的降低巨噬细胞趋化作用五、结论1.证实了结直肠癌组织中PKM2的表达量与巨噬细胞的数目和分布成正相关。2.PKM2调节结直肠癌细胞CCL2的表达和分泌促进巨噬细胞的趋化作用3.PKM2与p65相互作用,并调节p65 Ser276位点的磷酸化。4.敲掉结直肠癌细胞的PKM2的表达明显抑制p65结合到CCL2的启动子区域进而降低其转录活性
[Abstract]:1. Colorectal cancer is one of the most common malignant tumors in the digestive tract. Its morbidity and mortality are second only to gastric cancer, esophageal cancer and primary liver cancer. It is important to further improve the treatment level of colorectal cancer. It is confirmed that the progression of colorectal cancer is a process of interaction of many factors. The tumor microenvironment is the internal environment of tumor growth. Macrophages have the function of promoting tumor angiogenesis and remodeling tumor microenvironment. However, it is still unclear how it can recruit the tumor site and how it functions. Pyruvate kinase M2PKM2 is a key restriction enzyme in glycolysis, and it is also an important transcriptional cofactor. The non-glycolytic function and role of PKM2 in carcinogenesis and development have attracted more and more attention. In the microenvironment of colorectal cancer, PKM2 is responsible for shaping the tumor microenvironment and promoting the growth and survival of colorectal cancer. The mechanism is not clear. Second, the purpose of this study was to investigate the role of PKM2 in the microenvironment of colorectal cancer and its mechanism of macrophage recruitment. Methods siRNA interference was used to investigate the effect of PKM2 on the expression of chemokines in macrophage recruitment. The effects of pyruvate kinase on the expression and activity of chemokines associated with macrophage recruitment were confirmed by real-time fluorescence quantitative PCR, and then the inflammatory microenvironment of tumor was reversed. The immunohistochemical results of clinical tissues showed that the expression of pyruvate kinase M2 was significantly increased in colorectal cancer tissues compared with the normal control group. 2. At the same time, the number and distribution of macrophages are also concentrated around the tumor glands, at the same time, The results of Western blot also showed that the expression of pyruvate kinase M2 in colorectal cancer cells was significantly higher than that in normal intestinal epithelial cells 2. The chemotactic effect of conditioned medium on macrophages showed that the expression of pyruvate kinase M2 in colorectal cancer cells was significantly higher than that in control group. The expression of specific siRNA knockout PKM2 was used to significantly reduce the chemotaxis and migration of macrophages. In order to further avoid miss, we used shPKM2 lentivirus, which has been proved to have interference effect, to infect cells and collect its conditioned medium. The chemotaxis experiment was carried out, and the results were consistent with the results of .3. Real-time PCR experiment. The results showed that interfering the expression of PKM2 in colorectal cancer cells could significantly reduce the expression of CCL2 at the mRNA level compared with the control group. At the same time, the results of enzyme-linked immunosorbent assay further showed that PKM2 could affect the expression and secretion of CCL2. 4. Endogenous PKM2 interacts with IKK 尾 in intestinal epithelial cells. Knockout of PKM2 could significantly inhibit the phosphorylation activity of p65 ser276. 5. P65 could save the decrease of CCL2 transcription in colorectal cancer cells induced by knock down PKM2. And can restore the PKM2 knockdown induced cell invasion ability to reduce macrophage chemotaxis. It is confirmed that the expression of PKM2 is positively correlated with the number and distribution of macrophages. 2. PKM2 regulates the expression and secretion of CCL2 in colorectal cancer cells and promotes the chemotaxis of macrophages 3.PKM2 and p65 interact with each other. The phosphorylation of p65 Ser276 site was regulated. 4. The expression of PKM2 in knock out colorectal cancer cells significantly inhibited p65 binding to the promoter region of CCL2 and decreased its transcriptional activity.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

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