臺灣博碩士論文加值系統

口腔癌目前是台灣癌症致死率較高的癌症之一，與食用檳榔、抽菸以及微生物感染等口腔危險因子有密切關係。纖維母細胞(Fibroblasts)為結締組織的主要細胞，可產生與代謝膠原纖維蛋白，在傷口癒合上扮演重要角色，不正常激活的纖維母細胞可表現較高的膠原蛋白(collagen)、阿法平滑肌肌動蛋白 (α-smooth muscle actin, α-SMA)以及CXCL12，可能與癌化有關。巨噬細胞極化分兩大類，M1 type的標誌蛋白為CD86、IL-12以及TNF-α等，可以促進發炎反應;而M2 type的標誌蛋白為精氨酸酶(Arginase-1)、分化群163 (CD163)、IL-10以及TGF-β等，會抑制發炎反應並可能會促進腫瘤細胞的生成。大腸直腸癌研究發現M2巨噬細胞可能會促使纖維母細胞活化表現出α-SMA等因子，可能與癌化有關，然而，在口腔癌中巨噬細胞極化的情形對口腔纖維母細胞的影響仍未明確。實驗室先前研究發現檳榔萃取物可能使巨噬細胞往M2 type極化，因此本研究想進一步探討此M2-like極化之巨噬細胞對於口腔纖維母細胞活化的影響，也探討口腔癌組織中巨噬細胞以及纖維母細胞的表現。本實驗所使用的巨噬細胞為THP-1細胞(人類單核球細胞株)以phorbol 12-myrsitate 13-acetate (PMA) 分化而成，再以Porphyromonas gingivalis 脂多醣(LPS)、尼古丁(Nicotine, NT)及/或檳榔萃取物(ANE)處理24小時，以西方墨點法和酵素連結免疫吸附法發現，M1 type巨噬細胞的標誌: CD86及細胞激素IL-12在所有組別間沒有太大差異; M2 type巨噬細胞標誌: Arginase-1及CD163的表現，在所有實驗組中都比對照組高，尤其是有加ANE的組別; M2細胞激素TGF-β則是對照組與各實驗組產量都差不多;有趣的是，LPS會使TNF-α及IL-10產量提高，但NT及ANE則不會。接下來以不同處理之巨噬細胞上清液作為條件培養液(CM-Mac)來處理口腔纖維母細胞，發現有ANE處理過的CM-Mac可使口腔纖維母細胞collagen及IL-6有明顯上升，α-SMA也有上升的趨勢;而有LPS處理的條件培養液也會使口腔纖維母細胞IL-6明顯上升;意外發現經LPS或ANE處理的CM-Mac使CXCL12明顯降低。若以ANE、LPS及/或NT直接處理口腔纖維母細胞，發現所有實驗組別與對照組相比其collagen與α-SMA的表現都比較高。臨床檢體方面:口腔癌旁正常組織、口腔癌症組織和口腔正常組織是經台北榮民總醫院IRB通過和受試者同意，從牙科臨床所取得的，以免疫組織化學染色方法發現，在結締組織中，CD68、CD163以及Arginase-1的表現在口腔癌組織高於口腔癌旁正常組織(成對樣本分析)，CD86表現僅在癌旁正常組織有微弱表現;此外，結締組織中α-SMA的表現亦在口腔癌組織較高。以上結果顯示: M2-like巨噬細胞和活化的口腔纖維母細胞在口腔癌結締組織中表現較高;另一方面，在細胞實驗中，除了ANE、LPS及/或NT等之直接促進纖維母細胞活化效應外，無論有無其他危險因子，ANE都可能會促使巨噬細胞往M2方向極化(M2-like)，而這種M2-like巨噬細胞條件培養液會使口腔纖維母細胞產生較多的膠原蛋白及IL-6，但不會提高 CXCL12表現。結論:在長期嚼食檳榔者口腔微環境中，除了ANE等口腔危險因子之直接效應外，被危險因子影響的免疫細胞例如M2-like的巨噬細胞，也會繼而影響結構細胞之功能，譬如造成纖維母細胞產生較多的膠原蛋白，此可能在口腔癌化中扮演重要角色。

Oral cancer, one of the leading causes of cancer death in Taiwan, is closely related to risk factors such as areca nut chewing habit, smoking and microbial infection. Activation of fibroblasts showing higher expression of collagen, α-smooth muscle actin (α-SMA) and CXCL12 is observed in carcinogenesis. Macrophage polarization can be classified into two types: M1 and M2. M1 type markers like CD86, IL-12 and TNF- may enhance inflammation, while M2 type markers like arginase-1, CD163, IL-10 and TGF- may promote tumor growth. M2 macrophages could promote the stroma expression of -SMA in colorectal cancer, however, little is known about the influence of macrophage polarization on fibroblast activation during oral carcinogenesis. Previous studies showed that areca nut extract (ANE) may polarize macrophages into M2-like phenotype. The study further examined the effects of these M2-like macrophages on the activation of oral fibroblasts. Phenotypes of macrophages and fibroblasts in oral cancer specimens were also evaluated. Specific protein expression was detected by western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). Macrophages differentiated from THP-1 cells by phorbol 12-myrsitate 13-acetate (PMA) were further treated with Porphyromonas gingivalis LPS, nicotine (NT) and/or areca nut extract (ANE) for 24 hours. As compared to control group, expression of M1 markers: CD86 and IL-12 showed no significant change, while expression of M2 markers: Arginase-1 and CD163 was higher in macrophages treated with ANE with/without LPS and NT. However, the expression of TGF-β showed no significant change between groups. Production of IL-10 and TNF-α was significantly increased by LPS, but not by NT and ANE. The supernatants collected from treated macrophages were used as conditioned media (CM-Mac) for the treatment of fibroblasts. Significantly increased expression of collagen and IL-6 by fibroblasts was found in cells treated with CM-Mac from ANE-related groups, and α-SMA was also increased in ANE-related groups though without statistically significance. CM-Mac from LPS-related groups also increased the production of IL-6 by fibroblasts. However, both CM-Mac from LPS-related and ANE-related groups decreased the production of CXCL12 by fibroblasts. On the other hand, oral fibroblasts directly treated with ANE, NT and/or LPS, also increased the production of collagen and α-SMA. Tissue specimens were obtained from routine dental surgery at the department of stomatology in Taipei Veterans General Hospital after IRB approval and informed consent. Using IHC, levels of positive staining for CD68、CD163、arginase-1 and α-SMA, but not CD86, in stromal connective tissues were significantly higher in oral cancer than in adjacent normal tissues. Taken together, the results showed that the expression of M2 macrophages and activated fibroblasts were higher in connective tissue stroma of OSCC. In in vitro cell experiments, ANE with/without NT and LPS might polarize macrophages into M2-like phenotype. CM-Mac from these ANE-related groups in turn could increase collagen production and IL-6 secretion, but not CXCL12 secretion, by fibroblasts. In conclusion, besides the direct effects of oral risk factors, the immune cells altered by risk factors, the M2 or M2-like macrophages, could in turn modify the resident cell function resulting in abnormal collagen production by fibroblasts, which might play an important role in oral carcinogenesis.

目錄 I
表目錄 III
圖目錄 IV
中文摘要 V
Abstract VIII
緒論 1
一、口腔癌 1
二、檳榔 2
三、尼古丁(Nicotine) 3
四、脂多醣(Lipopolysaccharides, LPS) 3
五、纖維母細胞 4
六、巨噬細胞 7
七、巨噬細胞與纖維母細胞之關係 8
研究動機與假說 9
材料與方法 10
檳榔萃取物製備 10
Lipopolysaccharides、Nicotine製備 10
THP-1細胞培養 11
巨噬細胞分化 12
巨噬細胞之處理 12
巨噬細胞條件培養液製備 13
口腔組織檢體收集 13
口腔黏膜纖維母細胞培養 13
口腔纖維母細胞之處理 14
蛋白質萃取物製備及定量 14
西方墨點法(Western blotting assay) 16
酵素結合免疫吸附定量法(Enzyme-linked immunosorbent assay, ELISA) 19
口腔組織玻片製備 21
免疫組織化學染色法(Immunohistochemistry, IHC) 21
口腔組織染色結果影像擷取及定量 24
統計方法(Statistical analysis) 24
結果 26
一、PMA誘導之人類單核球細胞(THP-1)分化成巨噬細胞 26
二、LPS、NT及/或ANE對巨噬細胞極化之影響 26
巨噬細胞之M1型態極化標誌CD86以及M2型態極化標誌arginase-1與CD163之表現 26
巨噬細胞細胞激素之分泌 28
各口腔危險因子與其對照組之兩組樣本分析 29
三、巨噬細胞條件培養液對口腔纖維母細胞活化的影響 31
口腔纖維母細胞collagen之表現 31
口腔纖維母細胞α-SMA之表現 31
口腔纖維母細胞細胞激素之分泌 31
不同巨噬細胞條件培養液與其對照組之兩組樣本分析 33
四、口腔危險因子對於口腔纖維母細胞collagen及α-SMA表現之直接作用 34
各口腔危險因子與其對照組之兩組樣本分析 35
五、口腔癌症組織(OSCC)、口腔癌旁正常組織(ANT)以及口腔正常組織(Normal)其巨噬細胞標誌蛋白及口腔纖維母細胞活化標誌蛋白的表現 36
巨噬細胞分化標誌CD14和CD68的表現 36
M1巨噬細胞極化相關標誌CD86的表現 37
M2巨噬細胞極化相關標誌Arginase-1及CD163的表現 37
纖維母細胞活化標誌α-SMA的表現 38
口腔癌症組織與口腔癌旁正常組織之配對分析 38
討論 40
結論 45
表 46
圖 50
附圖 67
附表 75
附表一、一級抗體 75
附表二、西方墨點法相關溶液配製 75
參考文獻 80


表目錄
表一、 ANE或LPS對巨噬細胞M2極化之分析 46
表二、經ANE或LPS處理之巨噬細胞條件培養液對於口腔纖維母細胞活化之分析 47
表三、ANE或LPS處理對於口腔纖維母細胞活化之分析 48
表四、M1、M2 巨噬細胞標誌蛋白與活化的纖維母細胞標誌蛋白α-SMA在口腔癌結締組織中表現之分析 49


圖目錄
圖一、THP-1細胞以不同濃度之PMA誘導成巨噬細胞之型態 50
圖二、口腔危險因子對於M1巨噬細胞極化標誌CD86的影響 51
圖三、口腔危險因子對於M2巨噬細胞極化標誌arginase-1的影響 52
圖四、口腔危險因子對於M2巨噬細胞極化標誌CD163的影響 53
圖五、口腔危險因子對於巨噬細胞分泌細胞激素之影響 54
圖六、巨噬細胞條件培養液對於口腔纖維母細胞collagen表現的影響 55
圖七、巨噬細胞條件培養液對於口腔纖維母細胞α-SMA表現的影響 56
圖八、巨噬細胞條件培養液對於口腔纖維母細胞分泌 CXCL12、TGF-β以IL-6 之影響 58
圖九、口腔危險因子對於口腔纖維母細胞collagen表現的影響 59
圖十、口腔危險因子對於口腔纖維母細胞α-SMA表現的影響 60
圖十一、口腔癌症組織、癌旁正常組織以及正常組織中CD14表現 61
圖十二、口腔癌症組織、癌旁正常組織以及正常組織中CD68表現 62
圖十三、口腔癌症組織、癌旁正常組織以及正常組織中CD86表現 63
圖十四、口腔癌症組織、癌旁正常組織以及正常組織中Arginase-1表現 64
圖十五、口腔癌症組織、癌旁正常組織以及正常組織中CD163表現 65
圖十六、口腔癌症組織、癌旁正常組織以及正常組織中α-SMA 表現 66

附圖一、陽性細胞表現強度標準 67
附圖二、口腔危險因子對於M1巨噬細胞極化標誌CD86的影響 69
附圖三、口腔危險因子對於M2巨噬細胞極化標誌arginase-1的影響 71
附圖四、M1、M2 巨噬細胞標誌蛋白與活化的纖維母細胞標誌蛋白α-SMA在口腔癌結締組織中表現之分析 73
附圖五、口腔癌症組織、癌旁正常組織以及正常組織中CXCL12 表現 74

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