探討CCRL2和CCL5的相互作用在攝護腺癌細胞爬行上的影響性

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題名:	探討CCRL2和CCL5的相互作用在攝護腺癌細胞爬行上的影響性 Analysis of CCRL2 – CCL5 interaction in prostate cancer cell migration
作者:	李蕙汝;Li, Hui-Ju
貢獻者:	癌症生物學研究所碩士班
關鍵詞:	CCRL2;CCR5;攝護腺癌;CCRL2;CCR5;prostate cancer
日期:	2012-07-27
上傳時間:	2012-08-31 16:34:40 (UTC+8)
出版者:	中國醫藥大學
摘要:	在先前的研究中發現到，在攝護腺癌病患血清中的CCL5濃度高低和病情的惡化程度有正相關的情況。CCL5在目前被發現到相對的趨化素受體有CCR1、CCR3和CCR5，而最近研究指出，CCRL2是一種非典型的趨化素受體，在趨化素CCL5所影響的免應反應中具有潛在的影響性。在我們的初步的實驗結果上發現到，將前列腺癌病患的腫瘤組織切片，利用免疫組織染色結果上看到CCRL2在較高期數病患的組織中相對於低期數的病患有較高的表達情形。因此在這邊，我們推測在CCL5所造成攝護腺癌的惡化中，CCRL2扮演一個具有影響性的角色。為了進一步的了解CCRL2在攝護腺癌中的表達情況，利用即時定量聚合酶連鎖反應來探測CCRL2在不同攝護腺癌細胞株鐘的表達量。結果上發現到，在高侵入性PC3細胞株中有高量的CCRL2表現，相較於較為良性的LNCaP和C4-2細胞株CCRL2則相對的表現量較低。利用短暫性在細胞中大量表達CCR5和CCRL2，或是單獨的大量表達再藉由接上了生物素CCL5來觀察，不管在哪一種情況下，CCL5都會和CCRL2和CCR5相互接觸，不會只有專一一種受體。而在PC3中利用小髮夾RNA來降低細胞的CCRL2表現量之後，在CCL5所引誘的爬行上可以看到降低的趨勢。而在C4-2細胞中大量的表達CCRL2的情況下，可以看到對於CCL5所誘導的細胞侵入上會有上升的情況。進而在免疫螢光染色的結果上觀察到，利用CCL5去刺激後，降低CCRL2在細胞中的表現會降低CCR5分布在爬行前緣的情況。總和來說，CCRL2可說是領導CCL5刺激時CCR5的分布位置。為了想要進一步的了解CCRL2是否和CCR5下游反映是否有關連，觀察CCR5下游p38和JNK的磷酸化情況。結果觀察到，降低了CCRL2的表現時，CCR5的下游磷酸化並不會也同時下降，反而還上升。進一步的想要了解CCL5-CCRL2是否有特別的下游反應機制，利用磷酸化蛋白上的觀察找到在增加細胞CCRL2的表現量情況下，刺激CCL5之後，GSK3-α/β, Akt和β-catenin觀察到會有上升的情況。最後結論上可以得到，CCRL2在攝護腺癌上可以影響CCL5所引誘的CCR5分布位置的極性，進一步的影響癌細胞的爬行，未來的觀察上想要知道CCRL2是否影響癌細胞侵入的器官專一性。 Previously, we have showed the increased CCL5 level in serum of prostate cancer patients with a correlation with stage of disease. Other than CCR1, CCR3 and CCR5 that are known receptor of CCL5, a recent study in B cell indentified CCRL2 as a novel non-classical chemokine receptor that potentially involved in CCL5-mediated immunomodulatory function. In addition, our preliminary data show the revealed elevated CCRL2 expression in T4 staged prostate cancer tissues compared to lower staged specimens. We therefore interest to know whether CCL5 involved in prostate cancer progression, and this effect may through the expression of CCRL2. To verify CCRL2 expression in prostate cancer, quantitative RT-PCR was performed to determine the expression of CCRL2 in prostate cancer cell lines. We found that a significantly increased expression of CCRL2 in highly invasive prostate cancer PC3 cells compared to low tumorigenic LNCaP and C4-2 cells. Overexpression of CCR5 and CCRL2 either alone or together increased the binding of biotinylated CCL5 on the cell surface of C4-2 cells, suggesting that CCL5 may independently bind to both CCR5 and CCRL2. Knockdown of CCRL2 in PC3 cells with shCCRL2 decreased CCL5-induced directional migration. Conversely, overexpression of CCRL2 in C4-2 cells enhanced chemotactic migration of cells to CCL5. Immunofluorescence result revealed a strong staining of CCR5 located on the leading edge of cells toward CCL5, and this staining was scattering through the whole cell upon the reduction of CCRL2. Taken together, these data suggest a role of CCRL2 in guiding CCR5 to CCL5. To further verify this suppressed CCL5 chemoattraction by shCCRL2 is through the influence of CCR5 signaling pathway, the phosphorylation status of p38 and JNK was compared between parental and shCCRL2 transfected PC3 cells which express endogenous CCR5 in response to CCL5 stimulation. The result demonstrated that shCCRL2 increased rather than decreased CCR5 downstream signal activities, suggesting that knockdown CCRL2 may increase the binding of CCL5 to CCR5. To investigate CCL5-CCRL2 downstream signal, phospho-protein array study was carry out and result found that GSK3-α/β, Akt and β-catenin was increased in CCRL2 overexpression cells under CCL5 stimulation. In conclusion, our study indicates overexpression of CCRL2 in prostate cancer might play important role in regulation of CCL5 induced CCR5 polarization, which result in increased prostate cancer cell migration. Further evaluation whether CCRL2 also involve in organ specific metastasis was current ongoing.
顯示於類別:	[癌症生物學研究所] 博碩士論文

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