Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20080
標題: 巨噬細胞與非小型細胞肺腺癌細胞交互作用後之基因剖繪
Expression profiles and functional characterization of genes in macrophages interacted with non-small cell lung cancer cells
作者: 許惠茹
Hsu, Hui-Ju
關鍵字: IL-8;介白質-8;tumor infiltrating macrophage;non-small cell lung cancer;腫瘤浸潤巨噬細胞;非小型肺癌細胞
出版社: 生物醫學研究所
摘要: 
在1976年 Folkman 指出血管新生是決定癌症轉移與增生的重要步驟,當癌細胞團塊增長至2-3mm3時,單純的細胞擴散已不足提供充足的養分與氧氣,這時癌細胞需要血管新生供給細胞增生機會。血管新生由多種血管增生因子調節,目前已知的血管增生因子有:靜脈內皮細胞生長因子(vascular endothelial growth factor,VEGF)、介白質(Interleukin,IL)-1、IL-8、血小板內皮細胞生長因子(platelet derived growth factor,PDGF)與腫瘤壞死因子(tumor necrosis factor,TNF)-α 等。腫瘤細胞、腫瘤間質細胞與腫瘤周圍的發炎細胞均可感應缺氧壓力而分泌血管增生因子,活化的巨噬細胞也可以分泌 IL-1、TNF-α 與 IL-8 等血管增生因子,Polverini 等人在1984年指出癌細胞周圍浸潤的巨噬細胞能夠藉由周邊分泌調節幫助(paracrine)癌細胞血管新生。前人的研究也發現在非小型細胞肺癌病人切片中,IL-8 的表現與巨噬細胞浸潤、血管新生成正比,與病人的生存率成反比。並且,將非小型細胞肺癌細胞與巨噬細胞共同培養 (coculture)後,肺癌細胞與巨噬細胞中 IL-8 mRNA 表現上升,而 IL-8 的分泌可被 TNF-α 與 IL-1 等細胞激素調控,所以巨噬細胞是非小型細胞肺癌細胞表現血管増生因子 IL-8 的重要媒介。因此學生的研究方向為,進一步探討巨噬細胞與非小型肺癌細胞交互作用後,巨噬細胞中 IL-8 mRNA 表現之變化,並利用 TNF-α 與 IL-1 蛋白研究巨噬細胞表現 IL-8 的調控機制。以及利用微陣列晶片剖繪被肺癌細胞活化的巨噬細胞基因表型的變化。學生利用 Transwell 系統或癌細胞培養液(conditioned medium,CM) 進行非小型細胞肺腺癌細胞株(CL1-5)與巨噬細胞的共同培養,並以 real-time quantitative RT-PCR 驗證巨噬細胞中基因表現變化的趨勢。此外,將 TNF-α、IL-1α 與 IL-1β 蛋白取代 CL1-5 CM,以刺激巨噬細胞,並觀察 IL-8 的表現變化。更進一步,將 IL-1 RA 及 TNF-α、IL-1α 與 IL-1β 抗體分別加入 Transwell系統,以抑制巨噬細胞與非小型細胞肺癌細胞的交互作用,藉以釐清 TNF-α、IL-1α 與 IL-1β 於癌細胞活化巨噬細胞的過程中所扮演的角色。結果發現,巨噬細胞與 CL1-5 共培養後,巨噬細胞中 IL-8 mRNA 的表現隨著時間而增加,而 IL-1α mRNA 也出現相同的情況。以 TNF-α、IL-1α 與 IL-1β 蛋白培養巨噬細胞時,IL-1α 明顯誘發巨噬細胞表現 IL-8 mRNA,TNF-α 與 IL-1β 則不明顯。而 IL-1 RA 與抗 IL-1α 抗體能夠抑制被癌細胞活化的巨噬細胞表現 IL-8 mRNA,因此 IL-1α 是誘發巨噬細胞表現 IL-8 的重要媒介,也是癌細胞與巨噬細胞交互作用的主要橋樑。而微陣列晶片結果顯示巨噬細胞被 CL1-5 CM 活化後有110個基因表現下降2倍以上,有117個基因表現上升2倍以上,出現變化的基因包括訊息傳遞因子、發炎相關因子與血管新生調節因子等,其中的 CD59、Neuropilin-1 與 IL-10 受體 β 被我們挑選出來以西方墨點法與 RT-PCR 進一步確認。CD59 能夠保護細胞免於補體毒殺,Neuropilin-1 是 VEGF 的共受體,而 IL-10 是細胞激素分泌的抑制者。這三個基因於發炎機制上均有一席之地,再加上結果顯示巨噬細胞被 CL1-5 CM 活化後三個基因表現均上升,所以這些基因對於腫瘤環境可能有重要影響,同時也深具研究價值。這些基因未來會繼續探討,期望能夠勾勒出活化的巨噬細胞細胞內訊息傳遞與巨噬細胞與癌細胞完整的作用機制。

In 1976, Folkman indicated that the supply of oxygen and nutrition by cell diffusion is not sufficient for tumor proliferation when tumor’s diameter attach 2-3mm3. Angiogenesis is required for tumor growth, progression, and metastasis. And angiogenesis is regulated by the local activity of a variety of angiogenic factors, such as interleukin (IL)-8, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PDGF) and tumor necrosis factor α (TNF-α). Before angiogenesis initiation, tumor cells, stroma cells and tumor infiltrating inflammatory cells, including macrophage could sense hypoxia and secrete angiogenic factor. Furthermore, in 1984, Polverini hypothesized that tumor infiltrating macrophage foster tumor angiogenesis by paracrine regulation. Previous studies had showed IL-8 mRNA expression correlates positively with tumor angiogenesis, macrophage infiltration and negatively with patient survival in non-small-cell lung cancer (NSCLC) In addition, coculture of NSCLC cells and macrophages showed increased expression of IL-8 mRNA in NSCLC cells and macrophage, and IL-8 expression has been published regulated by TNF-α and IL-1. Therefore, macrophages played a crucial role in initiating the expression of angiogenic factor- IL-8 in lung cancer cells during tumor angiogenesis. In this study, we evaluate the expression of IL-8 in macrophages after interacting with NSCLC cells and the regulation by TNF-α and IL-1. Furthermore, microarray analysis was used to find out the difference of gene expression between naïve and sensitized macrophages. By this way, we can further figure out the regulation involved in this interaction and profile gene expression and function in sensitized macrophages. We measured macrophage IL-8 mRNA expression (by real-time quantitative RT-PCR), and a cancer cell/macrophage coculture system was used to evaluate the interaction between macrophages (cell line THP-1) and human lung adenocarcinoma cell line, CL1-5. To determine the effect of IL-1 and TNF- on IL-8 mRNA levels, macrophages were incubated for 24 hours with various concentrations of recombinant human IL-1, IL-1β and TNF- in the absence of CL1-5 cells, then total RNA were isolated to measure IL-8 mRNA expression. In addition, various concentrations of recombinant human IL-1 receptor antagonist (IL-1 RA) were added to the CL1-5/macrophage cocultures for 24 hours to investigate the suppression on IL-8 expression, and the regulation of TNF-、IL-1 and IL-1 was also detected by antibody neutralization. In CL1-5/macrophage cocultures, IL-8 mRNA expression in macrophages increased with time, so as IL-1 mRNA expression. In the stimulation by recombinant IL-1, IL-1, and TNF-, a significant increase in IL-8 mRNA expression was seen in the macrophages treated by IL-1. In addition, the results of antibody neutralization showed that anti-IL-1 antibody could significantly suppress IL-8 mRNA expression in macrophages. Similarly, IL-8 mRNA expression was suppressed by IL-1 RA treatment. Above these data, IL-1 seems to be the most important inducer for IL-8 expression in macrophage, and in this study sensitized macrophages have more ability to promote angiogenesis than naïve macrophages. In microarray analysis, there were 110 genes that have a 2 or more fold decrease after 24 hours coculture by conditioned medium, and 117 genes that have 2 or more fold increase after 24 hours coculture. There were dozens of genes involved in the interaction including signal transduction, inflammatory, angiogenesis, and so on. We chose certain genes that have obvious variation after coculture including CD59、neuropilin-1 and IL-10 receptor  from microarray results for further investigation. The result of western blot showed that the expression of CD59 and neuropilin-1 was increased in macrophage after coculture, and in the results of RT-PCR, neuropilin-1 and IL-10 receptor  mRNA were also up-regulated in sensitized macrophage. IL-10 inhibits cytokine secretion by inflammatory cells. CD59 can protect cell from complement cytotoxicity, and neuropilin-1 is VEGF coreceptor. These genes may play important role in angiogenesis. There must be complicated pathways participated in CL1-5/macrophage interaction. Further characterization of genes identified in this study will be in progress in the future.
URI: http://hdl.handle.net/11455/20080
Appears in Collections:生物醫學研究所

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