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研究生:王森稔
研究生(外文):Wang Shen-Nien
論文名稱:膽囊腺瘤及膽囊癌之黏液素基因的表現情形
論文名稱(外文):Mucin Gene Expression in Adenoma and Cancer of the Human Gallbladder
指導教授:李金德李金德引用關係
指導教授(外文):Lee King-Teh
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:74
中文關鍵詞:黏液素基因膽囊腺瘤膽囊癌
外文關鍵詞:Mucin Gene ExpressionGallbladder adenomaGallbladder cancer
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截至目前為止,已有九種以上的黏液素基因(MUC)被發現及探討。研究顯示,有許多癌症及發炎性的疾病,其細胞組織中的某些細胞表面抗原位置(cell surface epitopes)的分佈情形和正常組織的有所不同,黏液素(mucin)是其中一例。所以,有關於各種癌症(cancer)及癌症前趨病灶(precancerous lesion)之間,其各種黏液素基因的表現情形及有何差異,一直不斷地被討論和研究。在本篇研究中,我們試著去探討MUC1﹑MUC2﹑MUC3﹑MUC4﹑MUC5B﹑MUC5AC﹑及MUC6,在正常膽囊上皮組織﹑膽囊腺瘤(adenoma)和膽囊癌(cancer)中的表現情形。我們總共收集了十五個正常膽囊﹑十七個膽囊腺瘤和二十二個膽囊癌,然後利用免疫組織化學染色和反轉錄聚合酵素連鎖反應這兩種方法,來建立各種黏液素基因在這三種不同的膽囊組織中的表現情形。
膽囊腺瘤組織中的細胞,透過光學顯微鏡檢查,大致可以分為三種:胃幽門腺體細胞(pyloric gland cells)﹑腸黏膜細胞(intestinal cells)以及膽管上皮細胞(biliary cells),而這三種細胞可以不同比例同時存在。我們發現以pyloric gland cells為主要部分的膽囊腺瘤有十二個,佔71%(12/17);以intestinal cells為主要部分的膽囊腺瘤有四個,佔24%(4/17);至於biliary cells雖沒有成為主要部分,不過每個樣本中都可以發現它的存在。經過免疫染色之後可以發現,膽管上皮細胞主要是表現MUC1﹑MUC3﹑MUC5AC及MUC6 (65%﹑94%﹑76%及59%);胃幽門腺體細胞主要是表現MUC1﹑MUC3和MUC6 (100%﹑100%﹑100%);而腸黏膜細胞主要是表現MUC1﹑MUC2﹑MUC3﹑MUC5AC及MUC6(60%﹑100%﹑100%﹑90%﹑80%)。和膽管上皮細胞比較起來,在胃幽門腺體細胞中,MUC1和MUC6的表現有升高的情形,其差異呈現統計學上的意義(100% vs. 65%, P=0.021;100% vs. 59%, P=0.011);在腸黏膜細胞中,MUC2的表現有升高,其差異呈現統計學上的意義(100% vs. 29%, P=0.000)。至於MUC3則恆定地表現在這三種細胞中不受影響。
在22個患有膽囊癌的病人中,腺癌(adenocarcinoma)佔最大部分,發生率約59% (13/22);而乳突狀腺癌(papillary adenocarcinoma)佔第二位,其發生率約23% (5/22)。其它還包括:鱗狀腺癌(adenosquamous carcinoma)有二個,黏液狀腺癌(mucinous adenocarcinoma)有一個,清澈細胞癌 (clear cell carcinoma)有一個。免疫染色發現,膽囊癌組織主要是表現MUC1及MUC3這兩種核心蛋白,其陽性比率都可達到百分之百﹔而MUC2﹑MUC5AC和MUC6的表現情形較差,其陽性比率分別是41%﹑59%和36%。有些膽囊癌組織中,會合併intestinal metaplasia的發生,其發生率是36% (8/22)。結果顯示,這些合併intestinal metaplasia發生的膽囊癌組織中,MUC2﹑MUC5AC和MUC6這三種secreted mucins的表現反而有增加的趨勢。其中又以MUC2百分之百的表現情形,更能反映出此種現象。
正常膽囊上皮組織﹑膽囊腺瘤和膽囊癌這三種不同的膽囊組織,利用免疫組織化學染色及反轉錄聚合酵素連鎖反應去探討其黏液素基因的表現情形,可以發現:正常膽囊上皮組織主要是表現MUC1﹑MUC3﹑MUC5AC及MUC6(100%﹑100%﹑67%﹑93%);膽囊腺瘤組織主要是表現MUC1﹑MUC3﹑MUC5AC及MUC6(88%﹑94%﹑71%﹑88%),和正常膽囊上皮組織一樣,而且其表現的差異並沒有呈現統計學上的意義;至於膽囊癌組織主要是表現MUC1﹑MUC3及MUC5B(100%﹑100%﹑72%),和正常膽囊上皮組織的比較起來,其差異主要是在MUC5B和MUC6這兩種黏液素基因(72% vs. 29%, P=0.036;36% vs. 93%, P=0.002)。
接著,我們將13個膽囊腺癌(adenocarcinoma)的檢體,根據組織分化不同的程度,進行黏液素基因表現的分析比較。結果發現,無論分化程度為何,MUC1﹑MUC3及MUC5B表現的陽性比率都很高;至於MUC2﹑MUC4﹑MUC5AC及MUC6的表現,似乎隨著組織分化程度漸差,而有逐漸降低的情形。經過分析可以發現,其中又以MUC2﹑MUC5AC及MUC6這三種secreted mucins,其表現情形隨著組織分化程度變差而降低的情形,有呈現統計學上的意義(其P值分別是0.05﹑0.049及0.01)。
我們的結論是,不論膽囊癌﹑膽囊腺瘤或正常膽囊上皮組織,它們都個別表現出其獨特且專一性的黏液素基因,同時也都或多或少表現出secreted form及membrane-bound form這兩類黏液素。當膽囊上皮組織在惡性轉變過程中,會影響secreted mucins的表現;至於membrane-bound mucins,則恆定地表現於這三種組織中不受影響。其中,當MUC5B mRNA的表現增加時,或者是MUC6 apomucin的表現減少時,將可以反映出膽囊組織可能發生了惡性轉變。同時,MUC2﹑MUC5AC及MUC6這三種secreted mucins的表現減少時,將可以反映出膽囊腺癌組織的分化程度較差。

To date, more than nine mucin genes have been characterized and their expression in malignant and premalignant lesions is under evaluation. In this study, we examined the expression of MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC6 in adenoma and cancer of the human gallbladder. We retrospectively collected 15 normal gallbladders, 17 gallbladder adenomas, and 22 gallbladder cancers, and characterized the expression profiles of mucin genes by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR).
Histologically, gallbladder adenomas are classified as: pyloric gland type, intestinal type, and biliary type. Tubular adenomas of pyloric gland type were more common in our group and its incidence was 71%( 12/17). The biliary type mainly expressed MUC1, MUC3, MUC5AC, and MUC6 apomucins (65%, 94%, 76%, and 59%, respectively). MUC1 and MUC6 apomucins were more frequently expressed in the pyloric gland type (100% and 100%, respectively), whereas MUC2 and MUC 5AC apomucins were more frequently expressed in the intestinal type (100% and 90%, respectively). MUC3 apomucin was constantly expressed in these three types.
Of 22 patients with GB cancers, adenocarcinoma was the most common in 59% (13/22), and followed by papillary adenocarcinoma,23% (5/22). The GB cancers constantly and wholly showed MUC1 and MUC3 apomucins in 100% cases. MUC2, MUC 5AC, and MUC6 apomucins were infrequently expressed in 41%, 59%, and 36%, respectively. The incidence of intestinal metaplasia was 36% (8/22) in GB cancer. All of the cancer specimens with intestinal metaplasia expressed MUC2 apomucin.
Considering the mucin genes expression in these three groups, MUC1, MUC2, MUC3, MUC4, and MUC5AC were constantly expressed. MUC5B mRNA was more frequently detected in the cancer group (72%), compared with the normal group (29%)(P=0.014). MUC6 apomucin was less frequently expressed in the cancer group (36%), compared with the normal group (93%)(P=0.001). Regardless of differentiation, the patients with adenocarcinoma constantly and highly frequently expressed MUC1, MUC3, and MUC5B. However, the poorer the differentiation became, the less frequently MUC2, MUC4, MUC5AC, and MUC6 expressed (especially MUC6, P<0.05). These results suggest that the expression of the secreted mucins is related to malignant transformation of the gallbladder. And, increased expression of MUC5B mRNA or decreased expression of MUC6 apomucin could reflect the poor differentiation.

中文摘要………………………………………………………….1
英文摘要………………………………………………………….5
誌謝……………………………………………………………….8
第一章 前言…………………………………………………….9
第二章 文獻回顧………………………………………………13
第一節 黏液素研究的歷史沿革…………………………………...13
第二節 黏液素的基本結構………………………………………...14
第三節 黏液素在臨床上的應用…………………………………...15
第四節 膽囊癌及膽囊腺瘤的定義和分類………………………...17
第五節 WHO組織學分類…………………………………………19
第三章 材料與方法……………………………………………22
第一節 研究對象…………………………………………………...22
第二節 免疫組織化學染色………………………………………...22
I. 免疫染色抗體的種類……………………………………...22
II. 免疫染色抗體一覽表……………………………………...23
III. 免疫染色的流程…………………………………………...23
IV. 免疫染色結果判讀標準…………………………………...24
第三節 反轉錄聚合酵素連鎖反應………………………………...25
I. 流程………………………………………………………...25
II. 條件………………………………………………………...26
III. 判讀標準…………………………………………………...26
第四節 統計分析方法……………………………………………...26
第四章 結果……………………………………………………27
第一節 病患資料…………………………………………………...27
第二節 膽囊腺瘤的組織分類及分布情形………………………...27
第三節 三種膽囊腺瘤組織其黏液醣蛋白的表現情形…………...28
第四節 膽囊癌的種類及分布情形………………………………...31
第五節 不同膽囊癌其黏液醣蛋白的表現情形…………………...31
第六節 不同膽囊癌MUC4和MUC5B其mRNA的表現情形…34
第七節 不同膽囊組織其黏液素基因的表現情形………………...35
第八節 膽囊腺癌其黏液素基因的表現情形……………………...38
第五章 討論……………………………………………………40
第六章 結論……………………………………………………48
第七章 參考文獻………………………………………………49
附圖
圖2-1: adenoma………………………………….…………………………………55
圖2-2: tubular adenoma of pyloric-gland type…...………………………………55
圖2-3: tubular adenoma of pyloric-gland type...…………………………………55
圖2-4: tubular adenoma of intestinal type..………………………………………56
圖2-5: tubular adenoma of intestinal type….……..………………………………56
圖2-6: biliary cystadenoma…………….…..……………………………………....56
圖2-7: adenocarcinoma…….………………………………………………………57
圖2-8: papillary adenocarcinoma…….……………………………………………57
圖2-9: mucinous adenocarcinoma…………………………………………………57
圖2-10: adenosquamous carcinoma….……………………………………………58
圖2-11: clear cell adenocarcinoma………………………………………...………58
圖4-1: 膽囊腺瘤組織的biliary cells中, 五種黏液醣蛋白的表現情形………....59
圖4-2: 膽囊腺瘤組織中, intestinal type的細胞其黏液素的表現情形…...……...60
圖4-3: 膽囊腺瘤組織中, pyloric gland type的細胞其黏液素的表現情形…..….61
圖4-4: Cystadenoma組織中, 其黏液素的表現情形……………...……..……….62
圖4-5: 膽囊腺癌組織其免疫染色的情形…………………………………………63
圖4-6: 合併intestinal metaplasia的膽囊腺癌組織其免疫染色的情形….……..64
圖4-7: 膽囊乳突狀腺癌組織其免疫染色的情形…………………………………65
圖4-8: 膽囊鱗狀腺癌組織其免疫染色的情形……………………………………66
圖4-9: 膽囊黏液狀腺癌組織其免疫染色的情形…………………………………67
圖4-10: 膽囊清澈細胞癌組織其免疫染色的情形………………………………..68
附錄
附錄一: 本研究所收錄病人之基本資料…………………………………………..69
附錄二: 膽囊腺瘤組織中,不同細胞型態其免疫染色結果一覽表……………..71
附錄三: 不同膽囊癌組織中,其免疫染色結果一覽表…………………………..73

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