【摘要】：研究背景前列腺癌(prostate cancer,PCa)是影響西方國家男性的最常見的癌癥,在2016年,僅在美國就會有超過18萬個(gè)前列腺癌新發(fā)病例,占所有新發(fā)癌癥的五分之一以上。全世界每年由癌癥引起的近800萬死亡病例中的90%以上主要是癌癥的轉(zhuǎn)移擴(kuò)散造成的,轉(zhuǎn)移是一種復(fù)雜的多步驟過程,通過該過程癌細(xì)胞從原發(fā)性腫瘤擴(kuò)散到周圍組織和遠(yuǎn)端器官。在腫瘤進(jìn)展期間,單核細(xì)胞和巨噬細(xì)胞募集到腫瘤周圍,從而改變腫瘤微環(huán)境以加速腫瘤的進(jìn)展,從腫瘤和基質(zhì)細(xì)胞產(chǎn)生的各種微環(huán)境信號可促使巨噬細(xì)胞改變它們的功能表型。巨噬細(xì)胞可分為兩種:經(jīng)典M1型和M2型巨噬細(xì)胞,M1型巨噬細(xì)胞是參與炎癥反應(yīng)和抗腫瘤免疫的關(guān)鍵,相反,M2型巨噬細(xì)胞則影響抗炎效果并有促腫瘤活性。腫瘤相關(guān)巨噬細(xì)胞(tumor-associated macrophages,TAM)表現(xiàn)出與M2型巨噬細(xì)胞相似的功能,并且是腫瘤微環(huán)境的關(guān)鍵調(diào)節(jié)劑。在腫瘤晚期,TAM為腫瘤生長、存活和血管生成提供有利的微環(huán)境,產(chǎn)生多種促炎細(xì)胞因子,如IL-6,其參與腫瘤細(xì)胞周期凋亡和抑制重要基因的誘導(dǎo)。誘導(dǎo)型一氧化氮合酶(inducible nitric oxide synthase,iNOS)是M1型巨噬細(xì)胞的特異性標(biāo)記物,而精氨酸激酶-1(arginase-1,Arg-1)是TAM的特異性標(biāo)記物。在腫瘤微環(huán)境中,巨噬細(xì)胞是最豐富的炎癥細(xì)胞。腫瘤細(xì)胞及其周圍細(xì)胞在其微環(huán)境中的相互作用對于腫瘤的惡性進(jìn)展發(fā)揮至關(guān)重要的作用。因此我們假想,在微環(huán)境中,腫瘤細(xì)胞分泌的相關(guān)因子可使M1型巨噬細(xì)胞向M2型極化(TAMs),同時(shí),TAMs可能促進(jìn)了腫瘤的侵襲性與進(jìn)展速度。目的探討腫瘤相關(guān)巨噬細(xì)胞與前列腺癌細(xì)胞在微環(huán)境中相互作用對前列腺癌侵襲與進(jìn)展的機(jī)制。方法1.培養(yǎng)人前列腺癌細(xì)胞PC-3、C4-2細(xì)胞,培養(yǎng)小鼠巨噬細(xì)胞RAW264.7細(xì)胞,利用共培養(yǎng)技術(shù)將PC-3細(xì)胞與RAW264.7細(xì)胞共同培養(yǎng)24h,PBS作為對照組;將C4-2細(xì)胞與RAW264.7細(xì)胞共同培養(yǎng)24h,PBS作為對照組。2.利用Western blot技術(shù)觀察PC-3細(xì)胞對RAW264.7細(xì)胞、C4-2細(xì)胞對RAW264.7細(xì)胞影響,檢測M1特異性指標(biāo)iNOS、M2特異性指標(biāo)Arg-1的蛋白水平表達(dá)量。3.利用Western blot技術(shù)觀察PC-3細(xì)胞、C4-2細(xì)胞對巨噬RAW264.7細(xì)胞中AMPK、p-AMPK、mTor、p-mTor的蛋白水平表達(dá)量。4.利用細(xì)胞免疫熒光染色技術(shù)觀察PC-3細(xì)胞、C4-2細(xì)胞對RAW264.7細(xì)胞中iNOS、Arg-1的蛋白水平表達(dá)量。5.利用Transwell遷移侵襲實(shí)驗(yàn)、細(xì)胞劃痕實(shí)驗(yàn)觀察RAW264.7細(xì)胞對PC-3細(xì)胞、C4-2細(xì)胞的侵襲與轉(zhuǎn)移能力。6.利用Western blot技術(shù)觀察RAW264.7細(xì)胞對PC-3細(xì)胞、C4-2細(xì)胞中上皮間質(zhì)轉(zhuǎn)化(EMT)指標(biāo)(E-cadherin、N-Cadherin、Vimentin)和侵襲能力指標(biāo) Icam-1的蛋白水平表達(dá)量。結(jié)果1.小鼠巨噬細(xì)胞RAW264.7細(xì)胞與人前列腺癌細(xì)胞PC-3、C4-2細(xì)胞共培養(yǎng)24h后,細(xì)胞形態(tài)發(fā)生了明顯變化,巨噬細(xì)胞由圓形小細(xì)胞形成多角的大細(xì)胞、長梭形細(xì)胞。2.Western blot結(jié)果顯示,與PBS對照組相比,PC-3細(xì)胞和C4-2細(xì)胞對RAW264.7細(xì)胞作用24h后,iNOS的蛋白表達(dá)量下降,Arg-1的蛋白水平表達(dá)量增加。3.Western blot結(jié)果顯示,與PBS對照組相比,PC-3細(xì)胞和C4-2細(xì)胞對RAW264.7細(xì)胞作用24h后,p-AMPK的蛋白表達(dá)量增加、p-mTor的蛋白水平表達(dá)量減少。4.細(xì)胞免疫熒光結(jié)果顯示,與PBS對照組相比,PC-3細(xì)胞和C4-2細(xì)胞對RAW264.7細(xì)胞作用24h后,iNOS的蛋白表達(dá)量下降,Arg-1的蛋白水平表達(dá)量增加。5.Transwell結(jié)果顯示,與PBS對照組相比,RAW264.7細(xì)胞對PC-3細(xì)胞和C4-2細(xì)胞作用24h后,遷移細(xì)胞數(shù)量明顯增加,細(xì)胞的遷移運(yùn)動(dòng)能力增加;細(xì)胞劃痕實(shí)驗(yàn)結(jié)果顯示,與PBS對照組相比,RAW264.7細(xì)胞對PC-3細(xì)胞、C4-2細(xì)胞作用24h后,細(xì)胞的遷移速度明顯增加。6.Westem blot結(jié)果顯示,與PBS對照組相比,RAW264.7細(xì)胞對PC-3細(xì)胞、C4-2細(xì)胞作用24h后,N-Cadherin、Vimentin、Icam-1的蛋白表達(dá)量明顯增加,E-cadherin的蛋白表達(dá)量明顯減少。結(jié)論1.在腫瘤微環(huán)境中,前列腺癌細(xì)胞可影響巨噬細(xì)胞形態(tài)發(fā)生改變,并可通過促進(jìn)AMPK磷酸化蛋白表達(dá),抑制mTor蛋白表達(dá),促進(jìn)巨噬細(xì)胞由M1向M2極化。2.在腫瘤微環(huán)境中,極化的M2巨噬細(xì)胞(TAM)可促進(jìn)前列腺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)化過程,從而增加前列腺癌細(xì)胞的侵襲與轉(zhuǎn)移能力,加快進(jìn)展速度。3.TAM與前列腺癌細(xì)胞在其微環(huán)境中的相互作用對于腫瘤的惡性進(jìn)展發(fā)揮重要的作用。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.25

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