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Enhancing efficacy of therapeutic EGFR DNA vaccine by depletion of CD25+ regulatory T cell in a mouse model of metastatic EGFR positive lung cancer

By 陳杏燕 and Hsing-Yen Chen

Abstract

肺癌指的是肺部組織內細胞生長失去控制的疾病。這種細胞生長可能造成轉移,就是侵入相鄰的組織和滲透到肺部意外。絕大多數肺癌是肺部惡性上皮細胞腫瘤,由上皮細胞病變而造成。肺癌是造成男性和女性癌症相關死亡的最主要原因。全球每年有130萬人死於肺癌。先前,我們報告在細胞外域經由操作基因槍送入naked DNA Plasmid coding的異種表皮生長因子受體,可誘發強烈的CTL活性和控制內源性表皮生長因子受體表現在小鼠皮下腫瘤LL2肺腫瘤的增長。然而,我們目前的研究顯示,操作基因槍送入EGFR DNA缺乏有效的治療患有轉移性LL2肺腫瘤的小鼠。而調節性T細胞 ( Tregs ) 對腫瘤免疫反應發揮重要的作用。因此,我們進一步確定是否利用CD25消耗抗體PC61剔除CD25 T細胞,增強DNA疫苗對抗轉移性肺腫瘤LL2的生長。我們發現,結合PC61的EGFR DNA疫苗顯著降低肺腫瘤的生長。在研究淋巴結細胞毒活性的結果顯示,結合PC61的EGFR DNA疫苗對於治療抗腫瘤作用和增加功能性CD8+ T細胞與CTL活性,有顯著的關連。 此外,在體內淋巴細胞耗竭實驗進一步證實,EGFR DNA疫苗與PC61抗體結合的療效是依賴於CD8+ T細胞。因此由這些觀察的結果顯示,可以經由操作基因槍送入EGFR DNA疫苗結合剔除CD25 T細胞的抗體,增加對大量表現EGFR之轉移性肺腫瘤的療效Previously, we reported that gene gun administration with naked DNA plasmids coding for the extracellular domains of the xenogenic EGFR gene can induce strong CTL activity and control endogenous EGFR -expressing LL2 lung tumors growth in subcutaneous tumor models in mice. However, our present study observed that gene gun administration EGFR DNA vaccines alone lack of effective against lung metastatic LL2 tumor in mouse model was established experimentally by an intravenous injection of tumors. Since regulatory T cells (Tregs) play an important role in immunosuppressive responses against tumors by immunotherapy. Thus, we further determine whether break suppressing immune responses by depletion of CD25+ Treg cells using CD25 depleting Ab PC61 would enhance the efficacy of DNA vaccine against metastatic LL2 lung tumor growth. We found that combination of EGFR DNA vaccine with PC61 vaccination significantly reduce the tumor growth in lung and prolong mice over either modality alone. In estimation of cytotoxicity activity studies of the lymph nodes revealed that therapeutic antitumor effect of combination with EGFR DNA vaccine and PC61 is associated with a significant increased the number of functional CD8+ T cell and level of CTL activity. Furthermore, in vivo lymphocyte depletion experiments further confirmed that the efficacy of combining with EGFR DNA vaccine and PC61 Ab, is dependent on CD8+ T cells. Thus, these observations suggest that increased therapeutic efficacy against metastatic EGFR positive lung tumor could be obtained by combining gene gun administration of naked DNA vaccine and depletion of CD25 Treg cells by using depleting antibody.目次 第一章、 緒論 ………………………………………………………………… 1 一、 轉移性肺癌 …………………………………………………………… 1 二、 表皮生長因子受器 …………………………………………………… 3 三、 調節性CD25 T細胞、CD8+ T cell及其相關之細胞激素 ………… 4 四、 Cytolytic T lymphocyte ( CTL ) 毒殺型T細胞 ……………………… 6 第二章、 研究目的 …………………………………………………………… 7 第三章、 材料與方法 …………………………………………………………… 9 一、 小鼠和細胞株 ………………………………………………………… 9 二、 EGFR DNA疫苗的製備……………………………………………… 9 三、 對於轉移性肺腫瘤LL2療效的建立 ……………………………… 9 四、 肺重定量 …………………………………………………………… 9 五、 消耗體內的T細胞 ………………………………………………… 10 六、 檢測抗原特異性CD8/INF-γ淋巴結中T淋巴細胞的數量 ……… 10 七、 細胞毒性分析 ……………………………………………………… 10 八、 統計分析 …………………………………………………………… 11 第四章、 結果 ………………………………………………………………… 12 一、 抗CD25抗體PC61有效的剔除經由靜脈注射帶有LL2肺腫瘤小鼠的CD4+CD25+T細胞………………………………………………… 12 二、 結合抗CD25抗體PC61的EGFR DNA疫苗提高抗腫瘤的療效 … 12 三、 剔除調節性CD25 T細胞提高EGFR DNA疫苗引起細胞組成的免疫反應 ……………………………………………………………………… 13 四、 結合抗CD25抗體的EGFR DNA疫苗的療效需要CD8+ T細胞 … 14 五、 EGFR DNA疫苗結合PC61抗體對腫瘤細胞的毒性測試 ………… 14 第五章、 討論 ………………………………………………………………… 15 參考文獻 …………………………………………………………………………… 21 圖目錄 圖一、抗CD25抗體PC61有效的剔除CD4+ CD25+ T細胞 …………………… 26 圖二、結合PC61抗體的EGFR DNA疫苗對於LL2 tumor的療效 ……………… 27 圖三、結合PC61抗體的EGFR DNA疫苗引起的細胞內免疫反應 ……………… 29 圖四、送入PC61和EGFR DNA疫苗再經由尾部靜脈注射LL2腫瘤C57B/6小鼠 剔除CD4+ T細胞或是CD8+ T細胞作用的療效 ………………………… 30 圖五、EGFR DNA疫苗結合PC61對於細胞毒性的影響 ………………………… 3

Topics: 轉移性肺癌, CD25調節性T細胞, CD8+, CTL, metastatic lung cancer, CD25+ regulatory T cell, CD8+, CTL
Publisher: 生命科學院碩士在職專班
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81162
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