2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658539摘要：肺癌是台灣及其他工業化國家最常見之癌症死亡原因之一（第二位）。肺癌之預後極差，在目前之治療模式下，其五年存活率低於15%。因此找尋其他有效之輔助治療方法極為重要。最近研究提供了一個重要之觀念，那就是發炎是腫瘤形成與發展之重要步驟。(Yuan,A et al. Am J Respir Crit Care Med 2000、Int. J. Cancer 2005、Clin. Cancer Res.2003、J. Clin. Oncol, 2005, 23:953). 巨噬細胞是腫瘤微環境中重要的發炎細胞成員。腫瘤巨噬細胞(TAM)可具有抗腫瘤或促腫瘤不同之作用(Yuan A et al. ClinCancer Res 2003, Am J Respir Cell Mol Biol 2005, J Clin Oncol 2005, Int J.Cancer 2005)，而其作用決定於不同之刺激因子。證據顯示巨噬細胞在不同刺激下，可分化成不同表現型之 M1(第一型，典型活化巨噬細胞，具抗菌及抑癌作用)及M2 (第二型，另類活化巨噬細胞，又包括M2a, M2b 及M2c，具促癌作用) 巨噬細胞，並可能擁有不同之功能。我們之前針對M1 或M2 (a, c)型巨噬細胞對於肺癌細胞之腫瘤形成、侵略轉移等行為之影響，以及對肺癌細胞基因表現之調節功能進行研究，結果發現不同表現型之腫瘤巨噬細胞(M1 vs M2a/M2c)在實驗室內及活體內對調節肺腺癌細胞之增生、移行、侵略、血管新生、腫瘤生成及藥物抗藥性上具有不同及反相之功能。不同表現型之腫瘤巨噬細胞亦能活化或抑制肺腺癌細胞內不同之訊息傳遞路徑，而這些活化或抑制之基因產物可做為預測肺癌病患之臨床存活預後之有意義指標。因此腫瘤巨噬細胞偏極分化的調控最近便成為熱門之研究題目。微小核醣核酸(microRNA)是一種不轉譯蛋白的核醣核酸家族，最近被發現是一種可調控各種生物現象之強力轉錄後調控因子。越來越多之證據顯示免疫細胞之功能特別是可被微小核醣核酸所調控。最近之研究顯示，微小核醣核酸之異常表現對發炎過程及發炎細胞之功能調控扮演極重要之角色。在我們先前之研究中，我們利用微小核醣核酸微陣列生物晶片來評估分析不同亞型巨噬細胞(M1與M2a/c)中微小核醣核酸之差異表現。結果顯示約有十數個微小核醣核酸在M1與M2巨噬細胞中有顯著之差異表現。其中微小核醣核酸miR146a 在M1與M2巨噬細胞之差異表現高達500倍。最近之證據顯示，miR146a 在人類發炎疾病如風濕性關節炎中，有高度之表現。另外miR146a常與另一個微小核醣核酸miR 155， 在發炎過程中一起作用。MiR-155 可經由對CTLA-4之抑制來增加T淋巴球之增生反應，可調控巨噬細胞上Toll-like receptor 之活化及功能，也在人類慢性發炎疾病中有高度表現。然而關於這些微小核醣核酸對腫瘤微環境中巨噬細胞偏極化成為不同表現型巨噬細胞之影響及調控，目前仍不清楚。這是一個一年期之計畫。在此計畫中，我們將用先前建立之模式方法來將活化單核細胞偏極分化成不同亞型之巨噬細胞，並利用及時定量反轉錄多聚合脢反應來確定微小核醣核酸miR 146a and miR 155在不同亞型之巨噬細胞之表現狀態。然後我們將把miR 146a and miR 155 之模擬性微小核醣核酸(mimics)或抑制性寡核酸(anti-miRs)轉染至活化單核細胞中，來評估miR 146a and miR 155對巨噬細胞偏極分化之影響。我們也將評估miR 146a and miR 155對已偏極分化成M1或M2之巨噬細胞，是否有反向偏極分化之效果。這些研究結果，將對我們未來發展利用微小核醣核酸調2控腫瘤發炎微環境做為治療標靶之肺癌治療新方法，提供重要之基礎及訊息。<br> Abstract: Inflammation is a critical component of tumor initiation and progression. (Yuan, A et al. Am JRespir Crit Care Med 2000、Int. J. Cancer 2005、Clin. Cancer Res. 2003、J. Clin. Oncol, 2005,23:953). Macrophages constitute a large proportion of the inflammatory cell infiltrate in tumormicroenvironments. Tumor associated macrophage (TMA) has either anti-tumor effect or tumorprogression effect (Yuan A et al. Clin Cancer Res 2003, Am J Respir Cell Mol Biol 2005, J ClinOncol 2005, Int J. Cancer 2005) dependant on different stimuli which result in their differentiationinto different phenotype macrophages.Recent evidence also showed TAMs can differentiate into different subsets macrophage:classically (M1, with antibacterial and anticancer effect) or alternatively (M2) activatedmacrophages (including M2a, M2b and M2c, with cancer promoting effects. We have previouslyshowed that different phenotype macrophages (M1vs M2a/M2c) have different and opposite invitro and in vivo effects on regulation of lung cancer cell behaviors, such as proliferation, migration,invasion, angiogenesis, tumorigenesis and drug resistance. Therefore, the regulation ofpolarization of TAMs into M1 or M2 subtype macrophage become an interesting topic underintensive investigation.MicroRNAs (miRNAs), a family of small noncoding RNAs, have recently emerged as powerfulposttranscriptional regulators of various biological processes. A growing body of evidencesuggests that the development and function of cells in the immune system is particularly subject toregulation by miRNAs. Recent evidences also revealed that microRNAs play an important role inregulation of inflammation process and the functions of inflammatory cells. In our previous study,we use microRNAs microarray gene chip to investigate the expression profile of microRNAs indifferent phenotype macrophage (M1 vs M2a/c), and we found that there is a significantdifferential expression of several miRNAs between M1 and M2a/M2c. Among them,mirRNA hsa-miR-146a expressed more than 500 fold higher in M1 as compared toM2a/M2c. Recent evidences showed that miR-146 is primarily involved in the regulation ofinflammation and other process that function in the innate immune system. MiR-146a has alsobeen reported to be highly upregulated in inflammatory disease such as rhematoid arthritis. Inaddition, miR-146 is thought to be a mediator of inflammation along with another microRNA,mir-155. MiR-155 increasing the proliferative response of T(h) cells through the down-regulation ofCTLA-4, and was highly expressed in chronic inflammatory diseases. However, the effects ofmiR-146a and miR-155 on regulation of polarization of macrophage into different phenotypemacrophage are still unknown.This is a one-year project. In this project, we will use previously established model to polarize2macrophages to different phenotype macrophages, and use real-tiime RT-PCR to confirm miR 146aand miR 155 expression status in different phenotype macrophage. Then we will transfectmiR-146a and miR-155 mimics, or miR-146a and miR 155 inhibitors (anti-miRs) into activatedTHP-1 (M0) cells, and evaluate their effects on regulation of polarization of macrophage into M1 orM2 macrophage. We will also evaluate the effects of miR-146a and miR 155 on reverse polarizationof re-polarization of differentiated M1 to M2 or differentiated M2 to M1 macrophages. The effects ofmiR-146a and miR 155 on polarization of macrophage will provide important basis and informationfor developing new therapy by using microRNs as therapeutic target in modifying the inflammationprocess in tumor microenvironment in lung cancer in the future.微小核醣核酸miR 146a and miR 155巨噬細胞不同表現型巨噬細胞偏極化發炎非小細胞肺癌miR 146a and miR155polarizationMacrophagesphenotypeinflammationnon-small cell lung cancers