2010-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657755摘要：子宮内膜癌是女性生殖系統最常見的侵襲癌，自1950年以來，雖然子宮内膜癌的死亡率降低超過60%，其發生率卻明顯增加，而且年齡層有逐漸降低的趨勢。我們知道腫瘤細胞在癌化過程中，一方面需要大量繁殖增生，另一方面則需對抗來自宿主體內免疫系統的清除作用，才能達到其侵入深部組織與蔓延的目的。腫瘤內浸潤淋巴球(tumor infiltrating lymphocyte, TILs)一直被探討是否影響癌症病患本身的免疫反應及疾病的預後，而T細胞在TIL中對腫瘤免疫監督與免疫逃脫上扮演一重要的角色。在T細胞中具有調節免疫作用的被稱為調節T細胞(regulatory T cell, Treg)。CD25，為interleukin-2 receptor的α-chain，具有CD4+CD25+的T細胞是主要自然產生的Treg，此類細胞僅佔CD4+T細胞的一小部分(約5-6%)，主要由胸腺衍生，不靠特定的抗原刺激，在細胞表面高度表現出如Foxp3等分子，而主要的免疫抑制是靠細胞與細胞接觸的機制。Treg細胞目前已在多種腫瘤上被發現與疾病的預後有相關聯。主要發現在TIL中，CD4+CD25+T細胞的比例會增加，CD4+CD25+T細胞會分泌TGFβ，並抑制CD8+T細胞分泌INFγ。TIL中，CD8+T細胞常被認為受到各種因素影響而無法使腫瘤消退。輔助T細胞(help T cell, Th)，在誘導與維持抗原專屬性CD8+記憶T細胞(memory T lymphocyte)的作用上，具有關鍵的作用，而Treg則對於CD8+記憶T細胞具有負向的調節。吾人之前的研究(NSC91-2314-B002-302, NSC91-2314-B002-397, NSC92-2314-B002-307)已證實人類癌症細胞會影響癌組織附近免疫細胞的組成，也發現腫瘤內浸潤淋巴球(TILs)有Th2/Tc2 模式。吾人證實了HLA基因的突變造成癌細胞上HLA抗原的表現的減少，也發現免疫細胞抑制受體CD94/NKG2A明顯在TILs毒殺性T細胞的表現增加。吾人進一步欲了解子宮内膜癌病患TILs中Treg與CD8+記憶T細胞及effector cell的消長與疾病的關係。吾人欲利用機械式研磨萃取法(mechanical dispersal technique) 來獲得子宮内膜癌之腫瘤內浸潤淋巴球及癌細胞，探討TILs中CD4+CD25+ Tregs及其上Foxp3、GITR、CD103和CTLA-4的表現，進一步探討Tregs分泌的cytokine表現，最後探討Tregs的表現對毒殺性T細胞其毒殺力之影響。第一年之研究主要利用吾人所發展出的mechanical dispersal technique分離腫瘤內浸潤淋巴球，以流體細胞儀分析CD4+CD25+ Tregs及其上FoxP3、GITR、CD103和CTLA-4的表現，另外也會分析Tregs與CD8+記憶T細胞及effector cell表現之相關。第二年之研究我們將以ELISA檢測CD4+CD25+Tregs分泌Th1 cytokines (IFN-γ、IL-12和TNF-α)和Th2 cytokines (IL-4和IL-10)的狀態，進一步以流體細胞儀分析Tregs與毒殺性T細胞內毒殺顆粒（包括granzyme B及perforin）的表現。第三年之研究主要建立腫瘤細胞與自體免疫細胞之毒殺模式(Cytotoxicity Assay)及免疫抑制模式（immunosuppression assay），來測定Tregs的表現與毒殺性T細胞之相互作用對其毒殺力之影響，以期能找出癌細胞調控免疫細胞之途徑，可用於了解腫瘤癌細胞與免疫細胞間的互動關係。以進行腫瘤免疫調控(immuno -regulation)之研究。吾人預期可找出子宮内膜癌之腫瘤細胞與宿主免疫系統間之互動關係，進而了解癌細胞於微環境中所產生免疫抑制現象之機轉。我們的研究目的在於找出子宮内膜癌細胞逃避宿主的免疫監控與調節T細胞之相關性，進一步希望對於未來發展免疫調節治療有所幫助。<br> Abstract: Endometrial carcinoma (EC) is the most common malignancy of the female genital tract, accounting for almost half of all gynecologic cancer, and is ranked fourth in age-adjusted cancer incidence among women in the United States. In 2002, about 39,300 new cases are diagnosed annually, resulting in more than 6,600 deaths. Uterine corpus cancer is the seventh leading cause of death from malignancy in women. Overall, about 2% to 3% of women develop EC during their lifetime. Because human endometrial carcinoma has a characteristic stepwise progression, the anticancer immune reactions are especially important for localizing the spread of this malignancy. In our previous studies (NSC91-2314-B002-302, NSC91-2314-B002-397, NSC92-2314-B002-307), we have developed a new mechanical dispersal technique for the isolation of TILs and cancer cells from cancer tissue. Our studies utilize such technique to investigate the population of Tregs in TILs of patients with EC.Since little is known about the immunoregulatory effects of CD4+CD25+ Tregs in human EC, our further studies try to establish a novel immuno-suppressive role of Tregs with expression of FoxP3, GITR and CTLA-4, thus dysregulating the cytokines milieu and down-regulating the immunity in EC. This study will analyze the immune status of EC patients in detail to obtain a clear idea of their innate, humoral and cellular immune functions.In the first year project, we will investigate the the expression of FoxP3, GITR, CD103 and CTLA-4 on CD4+CD25+ Treg cells from PBMC and TILs in human EC. Moreover, the phenotye of CD8+ T cell will be also studied. In the second year project, we will assess the cytokine secretory status of CD4+CD25+ Tregs from PBMCs and TILs of Th1 cytokines(IFN-γ, IL-12 and TNF-α) and Th2 cytokines (IL-4 and IL-10). In addition, the intracellular cytotoxic molecules, perforin and granzymeB, will be assessed. In the third year project, we will determine whether the CD4+CD25+ T cells from EC patients are functional Tregs. The correlation between prevalence of Tregs and clinical outcome will be clarified for the patients.調節T細胞子宮内膜癌CD4+CD25+T細胞腫瘤內浸潤淋巴球Foxp3T regulatory cellsCD4+CD25+tumor-infiltrating lymphocytesendometrial cancerFoxp3