檢測人類子宮內膜異位症的典型鈣粘蛋白並與子宮腔內之子宮內膜作一比較; IDENTIFICATION OF THE CLASSICAL CADHERIN SUBTYPES PRESENT IN THE HUMAN ENDOMETRIOSIS AND IN COMPARISON WITH THE EUTOPIC ENDOMETRIUM
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(2) 誌謝 自從中國醫藥學院醫學系畢業，即從事於婦產科臨床醫學工作至 今。有感於臨床治療，往往是發病之後的補救措施。因此，一直希望有機 會能夠再由基礎醫學的研究來探討疾病的形成，以期由細胞或分子層面來 治療或預防疾病。終於，再前年得償夙願，能夠進入母校醫學研究所從事 研究工作。 匆匆兩年過去了，要感謝的人實在太多。首先感謝蔡長海董事長 能夠提供及鼓勵臨床醫師進修的機會。也感謝指導教授蔡鴻德主任不管在 臨床工作或是基礎研究的提攜及督促，還有共同指導教授陳澤昭博士和張 文正教務長的殷切指導。也感謝科內葉聯舜主任、林武周主任及楊東川主 任等學長提供研究的經驗，還有吳慧音同學及黃鈺媚小姐在實驗過程中的 鼎力幫助使得整個實驗得以完成。 雖然初步的研究告一段落，課程也劃上休止符，但卻是我個人從 事更深入研究的開始，藉由兩年師長所傳授的經驗，建立起自己基礎醫學 研究的磐石，相信在未來能夠使自己往前邁進，不懼艱難。 最後感謝我摯愛的妻子以及兩位嗷嗷待哺的小公主，有他們的陪 伴此論文才得以順利完成。. 2.
(3) 中文摘要 背 景 子宮內膜異位症(endometriosis)是指子宮內膜的腺體及其基質出現 在子宮腔外，在生育年齡的婦女其發生率將近 3~10%，然而在不孕症的婦 女，其發生率則高到 25~35%。子宮內膜異位症造成的骨盆腔疼痛及不孕 等症狀，是病人及醫療的極大負擔。至目前為止，其致病的機轉尚未明瞭， 最被廣為接受的理論為經血逆流所造成，然而逆流的子宮內膜組織如何附 著並侵入腹膜並得以增殖和成長，仍然是一個謎。鈣粘蛋白(cadherin)是一 種細胞沾粘分子[cell adhesion molecules (CAM)]，其功能為細胞沾粘作 用，同時在胚胎發育中主導不同組織及器官的形成，同時它們與癌細胞的 侵襲性也有關。因此我們認為鈣粘蛋白可能與子宮內膜異位症的形成、侵 入、及成長有關。本論文乃探討子宮內膜異位症中鈣粘蛋白的表現，並分 析它們與子宮腔內之子宮內膜(eutopic endometrium)的不同表現，以瞭解 子宮內膜異位形成的沾粘機轉(adhesive mechanism)。. 方法 收集九位子宮內膜異位症的病人，依其月經週期分為增殖期(n=5)及 分 泌 期 (n=4)。 首先利用 degenerate reverse transcription-polymerase chain reaction (RT-PCR) 來 檢 測 子 宮 內 膜 異 位 組 織 中 典 型 鈣 粘 蛋 白 (classical cadherin)的種類。之後，再以 semiquantitative RT-PCR 的方法， 比較同一病人其子宮內膜異位組織與子宮腔內之子宮內膜組織中，這些鈣 粘蛋白 mRNA 的表現差異。. 3.
(4) 結果 我們確認子宮內膜異位組織中有六種典型鈣粘蛋白存在，它們分別為 Epithelial-cadherin(E-cad). 、. 、. Placental-cadherin(P-cad). Neural-cadherin(N-cad)、cadherin-6、cadherin-9、和 cadherin-11。比較 這六種鈣粘蛋白在子宮腔內之子宮內膜與子宮內膜異位的表現差異時，我 們發現 P-cad mRNA 表現在子宮內膜異位組織中較子宮腔內之子宮內膜組 織有明顯增多。這種表現差異不論是增殖期或是分泌期都相同。. 結論 我們確認六種典型鈣粘蛋白在子宮內膜異位組織。其中只有 P-cad 在子宮內膜異位組織中比子宮腔內之子宮內膜高出許多。因此，我們認為 P-cad 可能在子宮內膜異位症的形成之沾粘機轉中扮演著重要的角色。. 4.
(5) 目錄 頁數 誌謝-----------------------------------------------------------------------------------------1 中文摘要-----------------------------------------------------------------------------------2 目錄-----------------------------------------------------------------------------------------4 圖目錄--------------------------------------------------------------------------------------5 表目錄--------------------------------------------------------------------------------------6 符號與縮寫--------------------------------------------------------------------------------7 第一章 前言------------------------------------------------------------------------------9 第二章 檢測子宮內膜異位組織中的典型鈣粘蛋白種類-----------------------21 第三章 子宮內膜異位組織與子宮腔內之子宮內膜鈣粘蛋白之比較--------40 第四章 討論、總結與未來研究方向-----------------------------------------------52 第五章 參考文獻-----------------------------------------------------------------------56 英文摘要---------------------------------------------------------------------------------81 作者簡歷---------------------------------------------------------------------------------82. 5.
(6) 圖目錄 Figure 1.. Schematic cadherin structure model------------------------------14. Figure 2.. Schematic representation of classical cadherin structure----15. Figure 3.. Schematic representation of PCR-II vector----------------------31. Figure 4.. Agar plate for transformed bacteria selection-------------------33. Figure 5.. Photomicrographs. plasmid. DNA. and. cloned. PCR. products-38 Figure 6.. Validation of semiquantitative RT-PCR for GAPDH and E-cad------------------------------------------------------------------------43. Figure 7.. Validation of semiquantitative RT-PCR for P-cad and N-cad--------------------------------------------------------------------------------44. Figure 8.. Validation of semiquantitative RT-PCR for cad-6 and cad-9---------------------------------------------------------------------------------45. Figure 9.. Validation of semiquantitative RT-PCR for cad-11-------------46. Figure 10. Comparison of the cadherin mRNA levels present in eutopic endometrium. or. endometriosis. 6. obtained. during. the.
(7) proliferative phase------------------------------------------------------48 Figure 11. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the secretary phase----------------------------------------------------------------------50. 7.
(8) 表目錄 Table 1.. The primers used in degenerate or semiquantitative RT-PCR------------------------------------------------------------------------------27. Table 2.. The PCR conditions---------------------------------------------------28. Table 3.. Analysis of cadherin cDNA generated from endometriotic lesions---------------------------------------------------------------------39. 8.
(9) 符號與縮寫 Ala ANOVA Bp BSA °C CaCl2 Cad CAM CAR CDNA DDT DNA Dsc Dsg EB EC E-cad ECM ER IL G GAPDH His Kb KCl MgCl2 Min Ml MM MMLV MMPs MRNA µl µM N-cad PBS P-cad Pcdh. Alanine Analysis of variance Base pair Bovine serum albumin Degrees centigrade Calcium chloride Cadherin Cell adhesion molecule Cell adhesion recognition Complementary DNA Dithiothreatol Deoxyribonucleic acid Desmocollins Desmogleins Ethidium bromide Extracellular Epithelial cadherin Extracellular matrix Estrogen receptor Interlukin Grams Glyceraldehyde-3-phosphate dehydrogenase Histidine Kiobases Potassium chloride Maginum chloride Minutes Milliter Millimolar Moloney Murine Leukemia Virus Matrix metalloproteinases Messenger RNA Microliter Micromolar Neural cadherin Phosphate-buffered saline Placental cadherin Protocadherin 9.
(10) PCR PR RNA RNAsin RT Tris-HCl Val VEGF. Polymerase chain reaction Progesterone receptor Ribonucleic acid Ribonuclease inhibitor Reverse transcriptase Tris(hydroxymethyl)-aminomethane-hydrochloric acid Valine Vascular endothelial growth factor. 10.
(11) 第一章 前言. 1-1： 導論(Introduction)： 子宮內膜異位症(endometriosis)在生育年齡的婦女是十分常見的 疾 病 ， 其發生率將近 3~10%，然而在不孕症的婦女其發生率將高到 25~35%[1,2]。由於子宮內膜異位症會引起疼痛症狀及不孕症問題，因此常 造成婦女很大的困擾。但由於子宮內膜異位症的發病原理及致病機轉至今 尚並不完全明瞭，因此也導致治療上的困難。遠在 1860 年，Rokitansky 即提及子宮內膜異位症[3]，但直到 1921 年 Sampson 才描述，具有病理構 造及功能的子宮黏膜出現在子宮外，稱為『子宮內膜異位症』[4]。子宮內 膜異位症最常見的症狀是經痛(dysmenorrhea)其次為不孕症(infertility)、性 交疼痛(dyspareunia)、下腹痛、下背痛。子宮內膜異位症可發生在身體任 何器官[5]，疾病若發生在肺部則月經來潮時會有咳血或是氣胸[6-8]，若發 生在泌尿道則會有血尿或是小便疼痛甚至導致輸尿管阻塞[9-11]，若發生在 胃腸道則會有血便、大便疼痛，甚至會引起腸阻塞[12-16]。. 11.
(12) 1-2： 子宮內膜異位症之病理機轉： 自 1927 年 Sampson 對於子宮內膜異位症的病理機轉提出假說至 今，共有幾項，(一)、植入學說(Implantation theory)：子宮內膜組織經由輸 卵管逆流至腹腔，附著、增殖、成長於腹膜中，進而形成子宮內膜異位症 [17-22]，(二)、胚胎化生學說(Coelomic metaplasia theory)：腹腔的胚腔上 皮細胞(coelomic epithelium)經由內分泌或發炎反應的刺激，而化生成為子 宮內膜細胞[23-26]。(三)、直接擴散學說(Direct extension theory)：子宮內 膜細胞直接侵入子宮肌肉層，形成子宮腺肌症，甚至侵入膀胱、輸尿管、 尿道或腸子[27]。(四)、淋巴管和血管轉移學說(Lymphatic and vascular metastasis theory)：子宮內膜組織經由淋巴液和血液，轉移至遠方的器官 [5-16,28-33]。(五)、機械式植入學說(Mechanical transplantation theory)： 例如開刀或生產而產生子宮內膜異位症[34]。(六)、複合式學說 (Composite theory)：認為是由以上機轉所共同形成，非單一學說可解釋所有疾病[35]。 以上以植入學說較被接受且有動物實驗証明。 然而報告指出婦女經血逆流幾乎都會發生，但是為何只有 3~10% 的婦女產生子宮內膜異位症，因此推測可能有某些因素促使子宮內膜異位 症的產生。例如，(一)、遺傳基因(Genetic factors)：有些學者認為子宮內 膜異位症可能是遺傳疾病，因而導致免疫系統改變使得異位的子宮內膜細 胞得以成長[36,37]。(二)、免疫系統因素(Immune system factors)：子宮內. 12.
(13) 膜 異 位 症 的 病 人 ， 腹 腔 體 液 (peritoneal fluid) 中 發 現 巨 噬 細 胞 (macrophage)、輔助性 T 細胞(helper T-cell )及自然殺手細胞(natural killer cell, [NK cell])增加情形，而這些免疫細胞會產生細胞激素(cytokines)，包 括 interleukin-1(IL-1)、tumor necrosis factor-á(TNF-á)、IL-6、IL-8。而這 些細胞激素與免疫補體細胞(immunocompetent cells)的成長及分化，以及 嗜中性白血球(neutrophils)和血管生成(angiogenic)有關[38-45]。(三)、血管 生成因素(Angiogenic factors)：血管內皮細胞成長因素 vascular endothelial growth factor(VEGF)是一種血管生成因素，可刺激血管內皮細胞成長及分 化。有些學者發現血管內皮細胞成長因素可能是由被活化的巨噬細胞所分 泌，它可以使腹膜上植入的子宮內膜細胞形成新生血管 (neovascularization)，而且腹腔體液中血管內皮細胞成長因素的濃度與子 宮 內 膜 異 位 症 的 嚴 重 程 度 有 關 [46-49]。 (四 )、 類 固 醇 接 受 器 (Steroid receptors)：子宮內膜異位症的病人其植入腹膜的子宮內膜細胞上發現有 estrogen. receptor(ER),. progesterone. receptor. (PR),. androgen. receptor(AR)，且發現 P-450 aromatase mRMA 的轉錄(transcripts)，這可 能代表子宮內膜細胞可局部製造雌激素促進子宮內膜細胞的成長及增殖 [50-53]。(五)、附著沾黏及侵入(Adhesion and invasion)：腹腔的子宮內膜 細胞藉 CAM 如 intergins、鈣粘蛋白及 maxtrix metalloproteinases(MMPs) 等，與腹膜的細胞外基質(extracellular matrix)相結合促進子宮內膜細胞侵. 13.
(14) 入[54-73]。 基於子宮內膜異位症的起源，逆流的子宮內膜細胞必須先和腹膜 細胞沾粘，進而互動(interaction) 才能形成子宮內膜異位症病灶。因此我們 著手研究細胞沾粘物質在子宮內膜異位症的發生和生長所扮演的角色。尤 其是細胞與細胞間的沾粘物質-鈣粘蛋白。. 1-3： 鈣粘蛋白： 鈣粘蛋白是一群位於細胞膜上的穿膜醣蛋白，至今至少有四十多 種鈣粘蛋白被發現，它們主導同類細胞之間的粘附作用，在鈣離子的環境 下(calcium-dependent)藉由同種親和的方式(homophilic manner)相結合 [74-77]。(見 Figure 1) 一 般 來 說 ， 不同的鈣粘蛋白仍然有類似的初級結構(primary structure) 。 其 構 造 可 分 為 三 部 份 ： N- 端 的 細 胞 外 部 分 (N-terminal extracellular domain)、穿細胞膜部分(transmembrane domain)及 C-端細胞 質部分(C-terminal cytoplasmic domain)[74-77]。而鈣粘蛋白其胺基酸的保 留性(conserved)大約為 43~58%，而最高保存性的區域(conserved region) 為 C-端細胞質部分，次高保留性的區域為靠近 N-端的細胞外部分。在細胞 外部分，是由幾組大約 110 個胺基酸重覆序列(cadherin-repeat sequence) 所構成，而鈣離子則附著於胺基酸重覆序列間。不同的鈣粘蛋白其胺基酸. 14.
(15) 重覆序列亦不同，同時也表現不同的特性及生物功能。 鈣 粘 蛋 白 可 規 納 為 四 大 類： ( 一 )、 典 型 鈣 粘 蛋 白 (classical cadherins)： 其中包括 type 1 及 type 2[74-77]， (二 )、 Desmosomal cadherins (三)、Protocadherins[78]，(四)、Cadherin-related proteins： 其中包括 truncated cadherins 及 unclassified family members。. 1-3-1： Type 1 典型鈣粘蛋白(見 Figure 2)： Type 1 典型鈣粘蛋白：包括 E-cadherin (E-cad)、N-cadherin (N-cad)、 P-cadherin (P-cad) 及 R-cadherin (R-cad)。 主 要 由 五 組 細 胞 外 部 分 (domain)、一組穿細胞膜部分及二組細胞質部分所組成[79]，五組細胞外部 分中間有四個鈣離子連接區域(calcium binding regions)。此外，在靠近 N 端的細胞外部分含有 cell adhesion recognition (CAR) sequence，它是由 三個胺基酸 His、Ala、Val (HAV)所構成[80]。藉由 CAR，相同的鈣粘蛋白 彼此接合形成拉鍊狀(zipper-like)的構造[81]。細胞質部分能夠與至少四種蛋 白 相 互 作 用 ， 此 四 種 蛋 白 為 α-catenin 、 β-catenin 、 γ-catenin 及 p120cas [82-84]。靠近 C-端細胞質部分與β-catenin 或γ-catenin 接合。而 α-catenin 則一端與β-catenin 或γ-catenin 接合，而另一端與細胞內骨架 (actin cytoskeleton)接合，像這種 cadherin-catenin complexes 才能在細胞 接合、分化及發育中發揮功能。. 15.
(16) Figure 1. From:. Schematic cadherin structure model Wijnhoven: Lancet, Volume 354(9176). July 31, 1999.356-357. 16.
(17) Figure 2.. Schematic representation of classical cadherin structure. 17.
(18) Type1 典 型 鈣 粘 蛋 白 其 生 物 功 能 主 要 是 促 進 細 胞 接 合 (homophilic cell adhesion)，在胚胎發育(embryonic development) 中主導 組織器官的形成，並和細胞的移動(cell migration)、組織的分化(tissue remodeling) 有關 [74,75,85]。在胚胎發育時，不同的鈣粘蛋白在適當時間 及地點的表現(spatiotemporal expression) 受到高度調控，引導不同組織的 成型。例如在老鼠胚胎的一個細胞期間(one-cell stage)有 E-cad 之表現 [86]，而 N-cad 則表現在腸胚(gastrula)的中胚層細胞[87]，P-cad 則表現在 blastocyst 的滋養外胚層(trophectoderm)[88]。Type 1 典型鈣粘蛋白在表 皮細胞的極性(polarity)之維持，佔有重要的地位[89,90]。而且 E-cad 的表 現可維持表皮細胞的分化(differentiated)及非侵襲性 (non-invasive)的 型 態，例如在成熟的表皮細胞若喪失 E-cad 的功能則細胞會發育成像纖維母 細 胞 (fibroblast-like)的型態，且具有侵襲性 [91]，甚至導致贅瘤的形成 (neoplastic transformation)[92,93]，現今研究指出缺少 E-cad 可能與直腸 癌[94,95]、胃癌[96,97]、胰臟癌[98]、食道癌[99,100]、肝癌[101]、肺癌 [102,103]、膀胱癌[104,105]、前列腺癌[106-108]、乳癌[109-115]、子宮癌 [116]、卵巢癌[117]、甲狀腺癌[118]、皮膚癌及口腔癌等有關[119-123]。而 N-cad 則 與 中 胚 層 及 神 經 外 胚 層 的 腫 瘤 有 關 ， 如 pleural mesothelioma[124]. 、. astrocytoma. 、. oligoblastoma[125]. 、. rhadomyosacroma[126]。而 P-cad 則與膀胱癌[127]、乳癌[128]等有關。. 18.
(19) 1-3-2： Type 2 典型鈣粘蛋白： Type 2 典型鈣粘蛋白：包括人類 cadherin-5、-6、-8、-9、-11、 -12 、 -14[129] 及 其 他 動 物 鈣 粘 蛋 白 如 rodent[130] 、 chicken[131] 及 Xenopus[132]。Type 2 典型鈣粘蛋白 與 type 1 典型鈣粘蛋白的結構類似 只是在靠近 N 端的細胞外部分不含有 HAV 的 CAR sequence。 Type 2 典型鈣粘蛋白其生物功能與 type-1 典型鈣粘蛋白類似， 和細胞接合及組織形成(morphogenesis)及腫瘤的形成(tumorigenesis)有 關。例如以往的報告顯示，cadherin-6 與人類及齧鼠類(rodent)腎臟的發育 有關[133,134]，且 cadherin-6 與腎臟癌的形成有關。Cadherin-11 則與齧 鼠 類 (rodent)骨 頭 的 形 成 [135]及 中 樞 神 經 的 發 育 有 關[136,137]， 而 且 cadherin-11 可能與 trophoblast cell 及胎盤的形成有關[138,139]。另外 cadherin-11 也與子宮內膜基質細胞的分化有關[138-140]。. 1-3-3： Desmosomal 鈣粘蛋白： Desmosomal 鈣粘蛋白：包括 desmocollins (Dscs 1,2,3) 及 desmogleins (Dsgs 1,2,3)，其結構與典型鈣粘蛋白類似，但細胞質部分較 長 且 胺 基 酸 較 少 雷 同 。 Desmosomal 鈣 粘 蛋 白 主 要 功 能 則 是 形 成 desmosomes，以接受外力的機械性壓力(mechanical stress)保持細胞的完 整性 ，如位於皮膚、牙齦及子宮頸 ，若受到損傷會導致皮膚的病變如. 19.
(20) pemphigus vulgaris、severe skin blistering[141]。. 1-3-4： Protocadherins： Protocadherins：包括 Pcdh-1、 –2、-3、-8、-9，其結構與典型 鈣粘蛋白類似，但細胞外部分其 cadherin-repeats sequence 超過五組，且 protocadherins 不與 catenins 相接合，此可能反映出 protocadherins 與其 他的鈣粘蛋白有不同的生物特性[78]。Protocadherins 可存在於非脊椎動物 (invertebrates)中，與胚胎腦部及中樞神經的發育有關[142]。. 1-3-5： Cadherin-related proteins： Truncated 鈣 粘 蛋 白 ： 包括 T-cad (cadherin-13)、 H-cad 及 L-cad，此群鈣粘蛋白缺少細胞外部分，此外 T-cad 尚缺少穿細胞膜部分而 以 phosphoglycolipid 與細胞膜接合。至於 truncated 鈣粘蛋白的功能，至 目前為止並不十分確定。. 1-4：鈣粘蛋白與子宮內膜異位症的關係： 過去只有少數文獻提及 E-cad、N-cad、P-cad 和子宮內膜異位症 [58-70]。 Gaetje 提及 E-cad-negative 的 子 宮 內 膜 與 轉 移 的 癌 症 細 胞 (metastatic carcinoma cell)具有侵襲的特性(invasive phenotype)[68]，而. 20.
(21) Starzinski 也有相似的報告[63]。Gaetje 同時指出在子宮內膜異位症的 E-cad-negative 細胞較正常子宮內膜增加[68]，這與 Scotti 的發現相似 [61]。但 Beliard 則發現，E-cad 在子宮內膜細胞與子宮內膜異位細胞中， 未有明顯的改變[69]。Ota 則指出 E-cad 在而分泌期較增殖期增加，且子宮 內膜異位細胞皆較子宮內膜細胞稍多[66]。Peralta 則指出在子宮內膜異位 症其 N-cad 的表現(expression)與 cystoadenoma、borderline ovarian tumor 相類似，因此認為子宮內膜異位症具有贅瘤特性(neoplastic potentially)， 且卵巢的子宮內膜異位瘤可能與其他腫瘤一般，是由 mesodermal cells 化 生(metaplasia)來[67]。Van der Linden 認為 P-cad 存在於增殖期子宮內膜 及子宮內膜異位並推論它與子宮內膜異位症可能有關[73]。. 1-5： 假說與基本原理(Hypothesis and Rationale)： 子宮內膜異位症雖然是一種良性的疾病，但其特性確是十分具有侵襲 性，即使經過外科手術治療，仍然有很高的復發的機率。由於其發生的病 理機轉至今尚未十分清楚，而異位的子宮內膜細胞，其表現的特性有些類 似癌細胞的增生和侵犯性。為了瞭解異位的子宮內膜細胞如何附著宿主細 胞及增生，研究其沾粘的機轉是有必要的。本研究選擇的鈣粘蛋白為研究 方向，希望找出異位子宮內膜細胞中典型鈣粘蛋白的種類，及子宮內膜異 位組織與子宮腔內之子宮內膜細胞間的典型鈣粘蛋白的表現差異，以便瞭. 21.
(22) 解 子 宮 內 膜 異 位 症 的 形 成 。 本 研 究 分 為 兩 個 階 段 ： 第一階段 ： 利 用 degenerate reverse transcription-polymerase chain reaction (RT-PCR) 的方法找出子宮內膜異位組織的典型鈣粘蛋白的種類。第二階段： 以 semi-quantitative RT-PCR 的方法分析子宮內膜異位組織與子宮腔內之子 宮內膜中各種典型鈣粘蛋白的表現差異。. 22.
(23) 第二章 鑑定人類子宮內膜異位症的典型鈣粘蛋白 (Identification of Classical Cadherin subtypes expressed in endometriosis). 2-1： 材料與方法 2-1-1： 檢體與取樣(tissues) 本實驗檢體取自在中國醫藥學院附設醫院婦產科接受子宮內膜異 位囊腫切除手術所之檢體，選擇三位患者，年齡分別為 23、28、32 歲，開 刀前三個月未曾使用過賀爾蒙藥物。取出之檢體剝取內膜囊腫的裏層子宮 內膜異位組織，而子宮腔內之子宮內膜的組織則以刮匙刮取少許子宮腔內 之子宮內膜，部份以液態氮急速冷凍隨即保存於-80°C 冰箱中，另一部份以 4% formaldehyde/2% glutaraldehyde 固定，以 hematoxylin & eosin 染色， 確定子宮內膜的組織是處於增殖期或分泌期，其中增殖期為 2 位，分泌期 1 位。. 23.
(24) 2-1-2. RNA 之萃取(Trizol RNA extraction)： 2-1-2-A： 儀器 1.. Source capture system (Germfree Laboraties). 2.. Centrifuge 5810R (Eppendorf, Germany). 3.. Microcentrifuge (Uover Laboratories, USA). 4.. Spectrafuge 16M (National Labnet, USA). 5.. TM firestek B206 (Firstek Scientific). 6.. Minigel Migration Trough Mupid-2 (Cosmo Bio Co LTD, USA). 7.. Rocker platform (Bellco Biotechnology, USA). 8.. UV box TFX 20 M (vukber Lourmat, France). 9.. DS34 Polaroid Electrophoresis hood (UK). 2-1-2-B： 試劑 1.. Trizol Reagent (Life Technologies, Inc., Gaithersburg, MD). 2.. 1X TBE Buffer. 3.. 2 % Agarose. 4.. Chloroform. 5.. Isopropanal. 6.. Ethyl glycol. 7.. Diethyl pyrocarbonate. 24.
(25) 8.. Ethidium bromide (10mg/ml) u. Ethidium bromide. u. Dissolved in 100 ml distilled H2O ， store at room. 1 g. temperature，避光。 2-1-2-C： 步驟 â. 100 mg 的組織剪碎在加入 1 ml Trizol reagent，再進一步磨碎。. â. 於 4°C 、10000 rpm 離心 1 分鐘後，取上清萃取液。. â. 加入 0.2 ml chloroform 混合均勻，室溫靜置 5 分鐘後。. â. 於 4°C 、12000 rpm 離心 15 分鐘後，取最上面透明之 RNA 萃取液。. â. 再加入 0.5 ml cold isopropanol 混合均勻，室溫靜置 10 分鐘後。. â. 於 4°C 、13000 rpm 離心 8 分鐘後，移除上清液，留下沉澱物。. â. 加入 1 ml 100% alcohol，混合均勻。. â. 於 4°C 、10000 rpm 離心 5 分鐘，去除上清液，再靜置 5 分鐘(需 避免沉澱物過度乾燥)。. â. 加入 40 µl RNAse and DNAse free 水。. â. 於 58°C 加熱 10 分鐘使沉澱物溶解。. â. 將所得的 RNA 產物以電泳分析法來評估含量。. â. 置於-80°C 冰箱中保存。. 2-1-2： 反轉錄(Reverse transcription, Promega)：. 25.
(26) 2-1-2-A：儀器： 1.. TM firestek HB-100 (Firstek Scientific). 2.. TM firestek B206 (Firstek Scientific). 2-1-2-B： 試劑： 1.. Random primer (0.5µg/µl of Promega). 2.. Oligo-dT primer (0.5µg/µl of Promega). 3.. MMLV RT buffer (Promega). 4.. 0.1 M DDT(dithiothreitol). 5.. dNTP(100 mM, 25mM each, Protech). 6.. RNAsin (Invitrogen, 10 units/µl). 7.. MMLV RTase (Promega, 200 units/µl). 2-1-2-C： 步驟： â. 將 1-5 µg 之 RNA (2-23µl) 加 RNAse and DNAse free 水至 23µl。. â. 加入 2µl random primer 及 0.5µl Oligo-dT primer 總共 25.5µl。. â. 加熱 70°C 5 分鐘之後，置於冰上 2 分鐘，然後短暫離心。. â. 再加入 8µ MMLV RT buffer、2µl 0.1 M DDT、2µl dNTP、0.5µl RNAsin、2µl MMLV RTase，總共 40µl。. â. 於 37°C 置放 3 小時。. â. 於 94°C 加熱 5 分鐘。. 26.
(27) â. 置於-80°C 冰箱中保存。. 2-1-3：Polymerase chain reaction using degenerate primers 2-1-3-A：儀器： 1.. PCR system 2400 (Perkin Elmer, USA). 2.. Minigel Migration Trough Mupid-2 (Cosmo Bio Co LTD, USA). 3.. Rocker platform (Bellco Biotechnology, USA). 4.. UV box TFX 20 M (vukber Lourmat, France). 5.. DS34 Polaroid Electrophoresis hood (UK). 2-1-3-B：試劑 1.. 10 X PCR buffer. 2.. 2.5 mM dNTP. 3.. MgCl2. 4.. 20 µM cadherin degenerate forward primer (sequence 見 Table 1). 5.. 20 µM cadherin degenerate forward primer (sequence 見 Table 1). 6.. Taq polymerase. 27.
(28) 7.. DNA- Gel-loading buffer (6X) u. Bromophenol blue. 0.25 ml. u. Xylene cyanol FF. 0.25 ml. u. Ficoll. 15 ml. u. Add H2O to 100 ml. 2-1-3-C：步驟： â. 將 2-4 µl cDNA 加入 ddH2O 至 35 µl. â. 加入 5µl of 10X PCR buffer、5µl of 2.5 mM dNTP、2µl of MgCl2、 1 µl of 20 µM forward primer、1µl of 20 µM reverse primper、1µl of Taq polymerase，總共 50µl。. â. 以聚合連鎖反應(PCR)之儀器進行反應。(反應條件見 Table 2). â. 將所得的 DNA 產物將用來植入 PCR-II vector。(見 Figure 3). 28.
(29) Table 1. Primers used in degenerate and semiquantitative RT-PCR assays. Gene. primer. sequence. size. Degenerate cadherin. Forward. 5’-GAATTCACNGCNCCNCCNTAYAYGA. 170 bp. Reverse. 5’-GAATTCTCNGC NARYTTYTTRAAR *. Forward. 5’-TCCATTTCTTGGTCTACGCC. Reverse. 5’-CACCTTCAGCCAACCTGTTT. Forward. 5’-GACCAACGAGGCCCCTTTTGTGCTG 357 bp. Reverse. 5’-GTGGTGGGAGGGCTTCCATTGTCCA. Forward. 5’-GTGCCATTAGCCAAGGGAATTCAGC. Reverse. 5’-GCGTTCCTGTTCCACTCATAGGAGG. Forward. 5’-TTCTTGCTGCTCTTTTGGGT. Reverse. 5’-CCTGCTCCATCTCCTGAAAG. Forward. 5’-CAAAACCTGGGCAGTTGATT. Reverse. 5’-CCTCTTCAATGCAGCAAACA. Forward. 5’-ACCAGATGTCTGTGTCAGA. Reverse. 5’-GTCATCCTTGTCATCTGCA. Forward. 5’-CCAGCCGAGCCACATCGCTC. Reverse. 5’-ATGAGCCCCAGCCTTCTCCAT. E-cadherin. P-cadherin. N-cadherin. Cadherin-6. Cadherin-9. Cadherin-11. GAPDH. *R = either A or G, Y = either C or T, N = either A, C, G, or T. 29. 361. bp. 373 bp. 275 bp. 416 bp. 742 bp. 359 bp.
(30) Table 2, PCR conditions Cad-11. Cad-6、Cad-9、E-、P-、N-cad. 94°C. hold 5 min. 94°C. hold 5 min. 94°C. 30 sec. 94°C. 30 sec. 55°C. 30 sec. 60°C. 30 sec. 72°C. 2 min. 72°C. 1 min. 30-40 cycles 72°C. 30-40 cycles 8 min. 72°C. 8 min. Cad Degenerate PCR. GAPDH. 95°C. hold 5 min. 94°C. hold 5 min. 95°C. 1.5 min. 94°C. 30 sec. 45°C. 2 min. 60°C. 30 sec. 72°C. 3 min. 72°C. 1 min. 35 cycles 72°C. 20-30 cycles 8 min. 72°C. 30. 10 min.
(31) 2-1-5：Ligation、transformation and bacterial growth 2-1-5-A：儀器 1.. TA cloning Kit Dual Promoter. 2.. Microcentrifuge. 3.. Thermocycler. 4.. 14°C water bath. 5.. 42°C water bath. 6.. 37°C water bath. 7.. Bucket with ice. 2-1-5-B：藥劑 1.. Taq DNA polymerase. 2.. TE buffer. 3.. Mineral oil. 4.. Luria-Bertani (LB) medium and agar. 5.. Dimethylformamide (DMF). 6.. 40 mg/ml 5-bromo-4-chloro-3indolyl-β-galactoside (X-Gal) in DMF. 7.. 50 mg/ml ampicillin stock. 8.. 50 mg/ml kanamycin. 9.. 100 mM isopropyl-β-D-thiogalactoside (IPTG). 31.
(32) 10. One Shot cells ( INVáF’ One Shot Competent Cells & TOP10F’ One Shot Competent Cells ) 2-1-5-C： 步驟 [. Ligation (Clone into PCR II vector ) â. 離心一瓶(vial)PCR II (見 Figure 3. )，然後收集上清液。. â. 計算 PCR product 所需的量(公式 X ng PCR product = Y bp PCR product 乘以 x 50 ng PCR vector 除以 size in bp of the PCR II vector：~3900). â. 轉換成為體積 X µl. â. PCR product X µl 加入 10 X ligation buffer 1µl 再加入 PCR II vector(25ng/µl) 2µl. â. 加入無菌的純水(sterile water)至 9µl. â. 加入 T4 DNA ligase (4.0 Weiss units) 1µ總共 10µl. â. 放置於 14°C 水槽(water bath)至少 4 小時，最好 overnight. â. store -20°C 冰箱中。. 32.
(33) Figure 3. Schematic representation of PCR-II vector. 33.
(34) [. Transformation â. 解凍先前已被 PCR product clone 的 PCR II vector(ligation reaction). â. 離心，置於冰上. â. 解凍 50µl One Shot competent cells ( TOP110F’ or INVαF’) 及 0.5M β-mercaptoethanol (β-ME). â. INVαF’ cells only：加入 2µlβ-mercaptoethanol (β-ME)，輕輕混 合。. â. 加入 2µl ligation reaction 至 competent cells，輕輕混合。. â. 置於冰上 30 分鐘。. â. 置於 42°C 水槽 30 秒，之後置於冰上。. â. 加入 250µl SOC medium(室溫). â. 置於 37°C shaking incubator 以 225rpm 平行搖晃 1 小時。. â. 取 50 及 200µl 分別塗於 LB agar plates 上(含有 X-Gal 及 50µg/ml kanamycin or ampicillin). â. 每種至少放入兩種不同量，至少有一種 well-spaced colonies. â. 確定凝固後，將 plates 顛倒置於 37°C 的 incubator 至少 18 小 時. â. 置於 4°C 冰箱 2~3 小時以利顏色之形成。(見 Figure 4). 34.
(35) Figure 4.. Agar plate for transformed bacterial selection. 當 被 含 有. cadherin product 所 clone 的 PCR II vector 轉植成功的 TOP10F’細胞則形 成白色菌落，若不含有 cadherin product 所 clone 的 PCR II vector 轉植成 功的 TOP10F’細胞則形成藍色菌落，若轉植不成功的 TOP10F’細胞則無法 成長形成菌落。. 35.
(36) [. Bacterial growth â. 取出至少 10 個白色菌落(white colonies)(本篇為 30 個). â. 置於 2~5 ml LB broth ( 加入 50µg/ml ampicillin or 50µg/ml kanamycin) overnight 日後做 plasmid 分析。. 2-1-6：Plasmid DNA extraction and DNA sequence 2-1-6-A：儀器 1.. Microcentrifuge. 2.. Gene-spin Miniprep Purification Kit( Beckman CEQ2000, ABI377, LICOR IR2, Pharmacia A.L.F.). 2-1-6-B：藥劑 1.. Gene-Spin Kit reagents u. Solution I. u. Solution II. u. Solution III. u. Wash Solution. 36.
(37) 2.. 3.. Restriction Enzyme 10 X buffer H u. 900mM Tris-HCl (pH 7.5). u. 500mM NaCl. u. 100mM MgCl2. Enzymes storage buffer u. 10mM Tris-HCl (pH 7.4). u. 400mM NaCl. u. 0.1mM EDTA. u. 1mM DTT. u. 0.15 % Triton X-100. u. 0.5mg/ml BSA. u. 50 % glycerol. 2-1-6-C：步驟 [. Plasmid DNA extraction â. 取 1~2 ml 放置 overnight 的 culture. â. 置於 12000~14000rpm 離心 30~60 秒. â. 加入 200 µl Solution I，Vortex 直至細胞完全溶解. â. 加入 200 µl Solution II，輕輕上下搖晃 5~6 次. â. 加入 200 µl Solution III，輕輕上下搖晃 5~6 次. 37.
(38) â. 置於 12000~14000rpm 離心之 5 分鐘. â. 將 spin column 放入收集管(collection tube)，小心將上清液移 入 spin column. â. 離心 30 秒. â. 將 spin column 移出收集管. â. 拋棄過濾液，再加入 700µl washing solution，離心 60 秒. â. 拋棄過濾液，離心 3 分鐘，已去除殘餘的 ethanol. â. 將 spin column 移出，. â. 再將新的 microcentrifuge tube 放入 column. â. 加 50~100 µl H2O or TE 至 column ( 若 plasmid > 7kb 則使用 60~70°C 的 H2O or TE. [. â. 離心 1 分鐘，以移出 DNA，置於-20°C 冰箱保存。. â. 重覆上述兩個步驟可多得 10~15% DNA. DNA sequencing â. 將所得到的 plasmid DNA 取出 1µg. â. 加入 EcoR I restriction Enzyme 1µl. â. 加入 assay buffer 50 µl. â. 加入 Acetylated BSA 直至 0.1 mg/ml. â. 置於 37°C 1 小時. 38.
(39) â. 將所得的產物以電泳分析是否含有約 170 bp 的典型鈣粘蛋白 (見 Fig 5). â. 使用 automated DNA sequncer 分析 DNA sequence. â. 得到的 sequence 利用網路上 BLAST computer program 與 Genbank/EMBL 的資料比對以確認找到的鈣粘蛋白亞型. 2-2： 結果 第一階段實驗我們發現在子宮內膜異位組織共有六種典型鈣粘蛋 白亞型：分別為 E-cad、N-cad、P-cad、Cad-6、Cad-9、Cad-11 其出現 比例如 Table 3。. 39.
(40) Figure. 5.. A. representative. photomicrographs. of. ethidium. bromide-stained gels containing plasmid DNA (3.9 kb of PCR II vector) and PCR products (~170 bp) generated by using degenerate primers, which extracted from transformed cell and digested by using the restriction enzyme EcoRI.. 40. . ~3.9 kb. . ~170 bp.
(41) Table 3. Analysis of cadherin cDNA clones generated from endometriotic lesions ________________________________________________________________________ Cadherin subtype. No of clones. Clones analyzed/patient (%). ________________________________________________________________________ Patient 1. E-cad P-cad N-cad Cad-6 Cad-9 Cad-11. 4 5 6 5 3 7. 13 (4/30) 17 (5/30) 20 (6/30) 17 (5/30) 10 (3/30) 23 (7/30). Patient 2. E-cad P-cad N-cad Cad-6 Cad-9 Cad-11. 6 4 4 6 2 8. 20 (6/30) 13 (4/30) 13 (4/30) 20 (6/30) 7 (2/30) 27 (8/30). Patient 3. E-cad 5 17 (5/30) P-cad 3 10 (3/30) N-cad 5 17 (5/30) Cad-6 7 23 (7/30) Cad-9 3 10 (3/30) Cad-11 7 23 (7/30) ________________________________________________________________________. 41.
(42) 第三章：六種鈣粘蛋白在子宮內膜異位組織與正常子 宮內膜的不同表現 (Comparison of Six Cadherin Subtypes in Endometriosis and Eutopic Endometrium). 3-1： 材料與方法 2-1-1： 檢體與取樣(tissues) 本實驗檢體取自在中國醫藥學院附設醫院婦產科接受子宮內膜異 位囊腫切除手術所之檢體，患者年齡為 20~42 歲的生殖年齡(reproductive age)，開刀前三個月未曾使用過賀爾蒙藥物。取出之檢體剝取內膜囊腫的 裡層子宮內膜異位組織，而正常子宮內膜的組織則以刮匙刮取少許子宮腔 內之子宮內膜，部份以液態氮急速冷凍隨即保存於-80°C 冰箱中，另一部份 以 4% formaldehyde/2% glutaraldehyde 固定，以 hematoxylin & eosin 染 色，確定子宮內膜的組織是處於增殖期或分泌期，其中增殖期 5 位，分泌 期 4 位。. 42.
(43) 3-1-2：RNA 之萃取(Trizol RNA extraction)： 其所需儀器、藥劑及步驟如前所述(見第 22 頁) RNA 產物之定量 â. 2µl RNA +3µl ddH2O + 5µl RNA denature buffer. â. 加熱 58°C15 分鐘. â. 置於冰上 2 分鐘. â. 短暫離心. â. 加入 2µl RNA loading buffer. â. 跑膠. â. 置於 EBTBE 內染色 30 分鐘. â. 置於 UV box 照相. â. 評估 RNA 相對含量。. 3-1-3： RT(Reverse transcription, Promega) 其所需儀器、藥劑及步驟如前所述(見第 24 頁). 43.
(44) 3-1-4：PCR 其所需儀器、藥劑及步驟如前所述(見第 25 頁) â. 首先以 PCR 的方式得到的 GAPDH(a house keeping gene)，再以 不同的 PCR cycle 求得最適當的 cycles 為 25 cycles. â. 再以相同的 PCR 的方式求得 E-cad、P-cad、N-cad、cad-6、cad-9、 cad-11，最適當的 cycles。. â. 結果以不同的 PCR cycle 求得最適當的 cycles 分別為 GAPDH 25 cycles，E-cad、P-cad、N-cad、cad-6、cad-9、cad-11 為 35 cycles (見 Figure 6-9). â. 算出子宮內膜異位細胞與子宮腔內之子宮內膜細胞之間所需的 mRNA 量. â. 以六種不同鈣粘蛋白亞型的specific cadherin primers 製造鈣粘蛋白 產物(cadherin products). â. 將所得的鈣粘蛋白產物以電泳分析法來評估 DNA 之含量。 u. DNA 產物之定量 â. 加入 2µl DNA loading buffer. â. 跑膠，然後置於 EBTBE 內染色 30 分鐘. â. 置於 UV box 照相. â. 評估 PCR product (DNA)相對含量(見 3-1-5). 44.
(45) Figure 6. Validation of semiquantitative RT-PCR for GAPDH or E-cad. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for GAPDH or E-cad using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown). 45.
(46) Figure 7. Validation of semiquantitative RT-PCR for P-cad or N-cad. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for P-cad or N-cad using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown). 46.
(47) Figure 8. Validation of semiquantitative RT-PCR for cad-6 or cad-9. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for cad-11 using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown). 47.
(48) Figure 9. Validation of semiquantitative RT-PCR for cad-11. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for cad-11 using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown). 48.
(49) 3-1-5：以半定量 RT-PCR 分析內膜異位組織和子宮內膜組織中六種鈣黏蛋 白亞型的不同表現 â. 將 所 得 到 的 鈣 粘 蛋 白 產 物 (cadherin products) 相 片 以 Hewlett Packer Digital scanner 掃瞄至電腦分析之. â. 使用 UN-SCAN-IT Gel Version 5.1 system (Silk Scientific, Orm, Utah)分析求得 Cadherin/GAPDH mRNA 之比值 。 ( Figure 10 & 11). 3-1-6：統計方法 以 Wilcoxon signed ranks test 來作檢定，以 p<0.05 為有顯著意義。. 3-2： 結果 第二階段實驗我們發現這六種鈣粘蛋白同樣也存在子宮腔內之 子宮內膜組織中。其中 N-cad 和 Cad-9 文獻上尚未有人報告過存在於子宮 內膜，屬於新的發現。還有，E-cad、N-cad、Cad-6、Cad-9 及 Cad-11 的 表現，在子宮內膜異位組織與子宮腔內之子宮內膜之間並無統計學上的差 異。唯有 P-cad 在子宮內膜異位組織的表現較較子宮腔內之子宮內膜高， 呈統計學上有意義的差別，這樣的差異無論是在增生期或分泌期都是存在 的(見 Figure 10 & 11)。. 49.
(50) Figure 10. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the proliferative phase of the menstrual cycle.. Photomicrographs of ethidium bromide-stained. gels containing PCR products generated using primers for E-cad P-cad or N-cad at 361, 357 and 373 base pairs respectively (Panel A) or cad-6, cad-9, or cad-11 at 275, 416 and 742 base pairs respectively (panel B). For each cadherin subtype, PCR products were generated using template cDNAs generated from eutopic endometrium (lane a) or endometriotic lesions (lane b). A PCR reaction in which the first strand cDNA was omitted was performed as a negative control (lane c). Products amplified using GAPDH specific primers at 359 base pairs are also shown for each sample. DNA markers are presented on the left hand-side of each gel. The absorbance values obtained for each cadherin subtype were normalized to the values obtained from for the corresponding GAPDH. The results derived from this analysis as well as from two other independent. experiments. (photomicrographs. not. shown). were. standardized to the control. The values obtained from this study, as well as from 4 other studies are presented (mean ±SEM; n=5) in the bar graphs. (*p<0.05).. 50.
(51) Figure 10. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the proliferative phase of the menstrual cycle.. 51.
(52) Figure 11. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the secretary phase of the menstrual cycle.. Photomicrographs of ethidium bromide-stained. gels containing PCR products generated using primers for E-cad P-cad or N-cad at 361, 357 and 373 base pairs respectively (Panel A) or cad-6, cad-9, or cad-11 at 275, 416 and 742 base pairs respectively (panel B). For each cadherin subtype, PCR products were generated using template cDNAs generated from eutopic endometrium (lane a) or endometriotic lesions (lane b). A PCR reaction in which the first strand cDNA was omitted was performed as a negative control (lane c). Products amplified using GAPDH specific primers at 359 base pairs are also shown for each sample. DNA markers are presented on the left hand-side of each gel. The absorbence values obtained for each cadherin subtype were normalized to the values obtained from for the corresponding GAPDH. The results derived from this analysis as well as from two other independent. experiments. (photomicrographs. not. shown). were. standardized to the control. The values obtained from this study, as well as from 4 other studies are presented (mean ±SEM; n=4) in the bar graphs. (*p<0.05).. 52.
(53) Figure 11. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the secretary phase of the menstrual cycle.. 53.
(54) 第四章 討論、總結與未來研究方向. 4-1：討論 為了瞭解子宮內膜異位症的形成機轉，探討它們的細胞接合物質 有助於解開為什麼有 3~10%的婦女，她們的月經逆流會產生子宮內膜異位 症這個謎。本研究檢測出六種 classical cadherins 存在於子宮內膜異位症的 組織中，除了 E-cad、N-cad、P-cad 曾被報告存在子宮內膜異位細胞外， 我們還首先指出有 Cad-6、Cad-9、和 Cad-11。隨後在比較子宮腔內之子 宮內膜與異位子宮內膜時，這六種鈣黏蛋白也都存在子宮腔內之子宮內膜 中。過去的文獻未曾報告 N-cad 或 cad-9 在子宮腔內之子宮內膜當中。本 研究也首次分析出這兩種鈣黏蛋白存在於子宮內膜，雖然子宮內膜上皮細 胞或基質細胞並未曾被測出含有 N-cad，但是血管的內皮細胞則具有 N-cad [143]，因此我們認為本研究的 N-cad 可能來自組織中的血管上皮細胞。同 樣地，cad-9 在文獻中未曾有人報告存在子宮內膜上皮或基質細胞[139]， 因此，cad-9 在子宮內膜的分佈與功能有待往後繼續研究。 本研究的結果，我們認為這六種鈣粘蛋白負責子宮內膜異位組織 的成長和維持。雖然我們僅分析 mRNA，但是過去我們以及別人的研究報 告均顯示 mRNA 和蛋白質的表現是具有良好的對應性的[127, 144-146]。 過去的文獻中Gaetje 曾指出在子宮內膜異位細胞 E-cad-negative. 54.
(55) 較子宮內膜細胞增加[68]，而 Scotti [61] 報告 E-cad 在子宮內膜異位症的 表現略為減少。然而 van der Linden 和 Beliard 則發現，E-cad 在子宮腔內 之子宮內膜細胞與子宮內膜異位細胞中，未有明顯的改變[69, 72]。Ota 則 指出 E-cad 在而分泌期較增殖期多，且子宮內膜異位細胞皆較子宮腔內之 子宮內膜細胞稍多[66]。本研究發現 E-cad 不管在增殖期或是分泌期，於子 宮內膜異位組織與子宮腔內之子宮內膜之間的表現，並無明顯的差別。因 此我們認為 E-cad 在子宮內膜的形成可能不是關鍵性的角色。這些研究結 果的差異有可能是來自於使用不同的研究方法學或是取樣時的差異。 另外 cad-6、cad-9 和 cad-11，不管在增殖期或是分泌期，於子 宮內膜異位組織和子宮腔內之子宮內膜的表現並無明顯差異，因此它們在 子宮內膜異位症可能不是關鍵性的角色。 另 外 ， Peralta 指 出 在 子 宮 內 膜 異 位 症 其 N-cad 的 表 現 與 cystoadenoma、borderline ovarian tumor 相類似，因此認為子宮內膜異位 症具有贅瘤特性(neoplastic potentially)，且卵巢的子宮內膜異位瘤可能與 其他腫瘤一樣是由 mesodermal cells 化生來[67]。然而本研究並未發現 N-cad 在子宮內膜異位組織與子宮腔內之子宮內膜之間有任何差異，這樣 的結果顯示 N-cad 與子宮內膜異位的發生可能無關。 至 於 P-cad， 文獻顯示它主要表現在複層上皮細胞 (stratified epithelium)的基底層(basal layer)或是副基底層(parabasal layer)，這與細胞. 55.
(56) 的分化(differentiation)有關[147]。van der Linden 曾推測 P-cad 可能在子宮 內膜異位症的發生佔有一定的角色[73]。本研究發現不管在增殖期或是分泌 期，子宮內膜異位組織的 P-cad 都比子宮腔內之子宮內膜明顯高出很多， 呈統計學上有意義的差別。因此，我們認為 P-cad 在子宮內膜異位組織與 腹膜等細胞間的沾粘、侵入及增殖扮演著重要的角色。所以當婦女逆流的 子宮內膜細胞，若因為某種原因導致過量的 P-cad 表現，便能夠與宿主細 胞如腹膜，相互沾粘進而發展成子宮內膜病灶。. 4-2 總結 本研究為首次發現 N-cad 和 cad-9 存在於子宮腔內之子宮內膜， 其定位及功能仍有待進一步研究。另外我們也首先提出共有六種典型鈣粘 蛋白(E-cad、P-cad、N-cad、cad-6、cad-9、cad-11)存在於子宮內膜異位 組織中，與子宮腔內之子宮內膜作比較時更發現唯有 P-cad 呈現有意義的 差別。由於 P-cad 在子宮內膜異位組織的表現明顯高於子宮腔內之子宮內 膜，因此我們認為 P-cad 在子宮內膜異位症的形成，甚至於維持扮演著關 鍵性的角色。. 4-3 未來的研究方向 1.. 我們將利用免疫組織染色法定位 N-cad 和 cad-9 在子宮腔內之子宮. 56.
(57) 內膜的正確位置，以及六種典型鈣粘蛋白在子宮內膜異位組織的分 佈。 2.. 其次我們將分析腹膜的典型鈣粘蛋白是否含有 P-cad。. 3.. 最後我們將作子宮腔內之子宮內膜及子宮內膜異位細胞的體外培 養，以便尋找 P-cad 的調控因子。. 57.
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