肿瘤相关巨噬细胞在增强前列腺癌细胞上皮—间质转化和侵袭性的机制研究

发布时间：2018-07-26 12:30

【摘要】：研究背景前列腺癌(prostate cancer,PCa)是影响西方国家男性的最常见的癌症,在2016年,仅在美国就会有超过18万个前列腺癌新发病例,占所有新发癌症的五分之一以上。全世界每年由癌症引起的近800万死亡病例中的90%以上主要是癌症的转移扩散造成的,转移是一种复杂的多步骤过程,通过该过程癌细胞从原发性肿瘤扩散到周围组织和远端器官。在肿瘤进展期间,单核细胞和巨噬细胞募集到肿瘤周围,从而改变肿瘤微环境以加速肿瘤的进展,从肿瘤和基质细胞产生的各种微环境信号可促使巨噬细胞改变它们的功能表型。巨噬细胞可分为两种:经典M1型和M2型巨噬细胞,M1型巨噬细胞是参与炎症反应和抗肿瘤免疫的关键,相反,M2型巨噬细胞则影响抗炎效果并有促肿瘤活性。肿瘤相关巨噬细胞(tumor-associated macrophages,TAM)表现出与M2型巨噬细胞相似的功能,并且是肿瘤微环境的关键调节剂。在肿瘤晚期,TAM为肿瘤生长、存活和血管生成提供有利的微环境,产生多种促炎细胞因子,如IL-6,其参与肿瘤细胞周期凋亡和抑制重要基因的诱导。诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)是M1型巨噬细胞的特异性标记物,而精氨酸激酶-1(arginase-1,Arg-1)是TAM的特异性标记物。在肿瘤微环境中,巨噬细胞是最丰富的炎症细胞。肿瘤细胞及其周围细胞在其微环境中的相互作用对于肿瘤的恶性进展发挥至关重要的作用。因此我们假想,在微环境中,肿瘤细胞分泌的相关因子可使M1型巨噬细胞向M2型极化(TAMs),同时,TAMs可能促进了肿瘤的侵袭性与进展速度。目的探讨肿瘤相关巨噬细胞与前列腺癌细胞在微环境中相互作用对前列腺癌侵袭与进展的机制。方法1.培养人前列腺癌细胞PC-3、C4-2细胞,培养小鼠巨噬细胞RAW264.7细胞,利用共培养技术将PC-3细胞与RAW264.7细胞共同培养24h,PBS作为对照组;将C4-2细胞与RAW264.7细胞共同培养24h,PBS作为对照组。2.利用Western blot技术观察PC-3细胞对RAW264.7细胞、C4-2细胞对RAW264.7细胞影响,检测M1特异性指标iNOS、M2特异性指标Arg-1的蛋白水平表达量。3.利用Western blot技术观察PC-3细胞、C4-2细胞对巨噬RAW264.7细胞中AMPK、p-AMPK、mTor、p-mTor的蛋白水平表达量。4.利用细胞免疫荧光染色技术观察PC-3细胞、C4-2细胞对RAW264.7细胞中iNOS、Arg-1的蛋白水平表达量。5.利用Transwell迁移侵袭实验、细胞划痕实验观察RAW264.7细胞对PC-3细胞、C4-2细胞的侵袭与转移能力。6.利用Western blot技术观察RAW264.7细胞对PC-3细胞、C4-2细胞中上皮间质转化(EMT)指标(E-cadherin、N-Cadherin、Vimentin)和侵袭能力指标 Icam-1的蛋白水平表达量。结果1.小鼠巨噬细胞RAW264.7细胞与人前列腺癌细胞PC-3、C4-2细胞共培养24h后,细胞形态发生了明显变化,巨噬细胞由圆形小细胞形成多角的大细胞、长梭形细胞。2.Western blot结果显示,与PBS对照组相比,PC-3细胞和C4-2细胞对RAW264.7细胞作用24h后,iNOS的蛋白表达量下降,Arg-1的蛋白水平表达量增加。3.Western blot结果显示,与PBS对照组相比,PC-3细胞和C4-2细胞对RAW264.7细胞作用24h后,p-AMPK的蛋白表达量增加、p-mTor的蛋白水平表达量减少。4.细胞免疫荧光结果显示,与PBS对照组相比,PC-3细胞和C4-2细胞对RAW264.7细胞作用24h后,iNOS的蛋白表达量下降,Arg-1的蛋白水平表达量增加。5.Transwell结果显示,与PBS对照组相比,RAW264.7细胞对PC-3细胞和C4-2细胞作用24h后,迁移细胞数量明显增加,细胞的迁移运动能力增加;细胞划痕实验结果显示,与PBS对照组相比,RAW264.7细胞对PC-3细胞、C4-2细胞作用24h后,细胞的迁移速度明显增加。6.Westem blot结果显示,与PBS对照组相比,RAW264.7细胞对PC-3细胞、C4-2细胞作用24h后,N-Cadherin、Vimentin、Icam-1的蛋白表达量明显增加,E-cadherin的蛋白表达量明显减少。结论1.在肿瘤微环境中,前列腺癌细胞可影响巨噬细胞形态发生改变,并可通过促进AMPK磷酸化蛋白表达,抑制mTor蛋白表达,促进巨噬细胞由M1向M2极化。2.在肿瘤微环境中,极化的M2巨噬细胞(TAM)可促进前列腺癌细胞上皮间质转化过程,从而增加前列腺癌细胞的侵袭与转移能力,加快进展速度。3.TAM与前列腺癌细胞在其微环境中的相互作用对于肿瘤的恶性进展发挥重要的作用。
[Abstract]:Background prostate cancer (PCa) is the most common cancer that affects men in the West. In 2016, more than 180 thousand new cases of prostate cancer were found in the United States, accounting for more than 1/5 of all new cancers. More than 90% of the nearly 8 million deaths caused by cancer in the world are mainly cancer transfers in the world. As a result of diffusion, metastasis is a complex multistep process through which cancer cells spread from primary tumors to surrounding tissues and distal organs. During the progression of the tumor, monocytes and macrophages raise the tumor around the tumor, thereby changing the tumor microenvironment to accelerate the progression of the tumor, from the tumor and the matrix cells. Microenvironmental signals can induce macrophages to change their functional phenotype. Macrophages can be divided into two types: classic M1 type and M2 type macrophage, M1 type macrophage is the key to the inflammatory response and anti-tumor immunity. On the contrary, the M2 type macrophage affects the anti-inflammatory effect and promotes the tumor activity. Tumor related macrophages (tumor-associat Ed macrophages, TAM) shows similar functions as M2 macrophages and is a key regulator of tumor microenvironment. In the late stage of the tumor, TAM provides a favorable microenvironment for tumor growth, survival and angiogenesis, producing a variety of proinflammatory cytokines, such as IL-6, which is involved in the induction of cell cycle apoptosis and inhibition of important genes. Inducible nitric oxide synthase (iNOS) is a specific marker for M1 type macrophages, while arginine kinase -1 (arginase-1, Arg-1) is a specific marker for TAM. In the microenvironment of the tumor, macrophages are the most abundant inflammatory cells. The interaction of tumor cells and their surrounding cells in their microenvironment The malignant progression of tumor plays a vital role. Therefore, we hypothesize that in microenvironment, the related factors secreted by tumor cells can make M1 type macrophages to M2 type polarization (TAMs). Meanwhile, TAMs may promote the invasion and progression of tumor. The mechanism of action on the invasion and progression of prostate cancer. Methods 1. PC-3, C4-2 cells were cultured and cultured RAW264.7 cells of mouse macrophages. PC-3 cells were co cultured with RAW264.7 cells by co culture technique, and PBS was used as a control group. C4-2 cells and RAW264.7 cells were co cultured with 24h, PBS as a control group.2.. The effect of PC-3 cells on RAW264.7 cells, C4-2 cells on RAW264.7 cells, M1 specificity index iNOS, and M2 specificity index Arg-1 protein level expression.3. by Western cells. PC-3 cells were observed by immunofluorescence staining, and the protein level expression of iNOS, Arg-1 in the RAW264.7 cells,.5.,.5. using Transwell migration and invasion experiment. The cell scratch test was used to observe the PC-3 cells and the invasion and metastasis ability of RAW264.7 cells, and.6. using Western cells to observe the cells. The protein level of intermediate epithelial mesenchymal transformation (EMT) index (E-cadherin, N-Cadherin, Vimentin) and invasive ability index Icam-1. Results 1. mice macrophage RAW264.7 cells and human prostate cancer cells PC-3, C4-2 cells co culture 24h, the cell morphology changes obviously, macrophages form round small cells to form a large angle. .2.Western blot results showed that, compared with the PBS control group, the protein expression of iNOS was decreased after the action of PC-3 and C4-2 cells to RAW264.7 cells, and the Arg-1 protein level expression increased.3.Western blot results. The protein expression was increased and the protein level of p-mTor decreased.4. cell immunofluorescence results. Compared with the PBS control group, the protein expression of iNOS was decreased after the action of PC-3 and C4-2 cells on RAW264.7 cells, and the Arg-1 protein level expression increased.5.Transwell. The RAW264.7 cells were compared with the PBS control group. After the action of cell and C4-2 cells for 24h, the number of migratory cells increased significantly and the mobility of cells increased. The results of cell scratch test showed that, compared with the PBS control group, the migration speed of RAW264.7 cells significantly increased.6.Westem blot results after the action of 24h in the PC-3 cells and C4-2 cells, and the RAW264.7 cells were compared with the PBS control group. After the action of 24h in PC-3 cells and C4-2 cells, the protein expression of N-Cadherin, Vimentin, Icam-1 was significantly increased and the protein expression of E-cadherin decreased significantly. Conclusion 1. in the microenvironment of the tumor, the prostate cancer cells can affect the morphogenesis of macrophages, and can promote the expression of AMPK phosphorylated protein, inhibit the expression of mTor protein and promote the giant. The phagocytic cell from M1 to M2 polarization.2. in the tumor microenvironment, polarization M2 macrophage (TAM) can promote the transformation process of epithelial mesenchymal transition of prostate cancer cells, thus increasing the invasion and metastasis of prostate cancer cells, speeding up the interaction between the progress speed.3.TAM and the prostate cancer cells in its microenvironment, which plays a heavy role in the malignant progress of the tumor. The role.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.25

【相似文献】