臺灣博碩士論文加值系統

粒線體在細胞中之功能包括細胞生長、分裂、能量代謝、呼吸作用(respiration)及細胞凋亡的調控(apoptosis)。粒線體功能的失調，會造成人類疾病及進一步導致癌症的發生。所以粒線體形態對於細胞維持正常生理代謝和功能有重要的作用，一旦調控出現障礙會導致嚴重的疾病。粒線體融合和分裂調控粒線體的形態與分怖。粒線體融合蛋白-2是粒線體外膜的蛋白，為多功能膜蛋白，其參與細胞增殖與代謝，也調控了內質網及粒線體的形態。現今並不清楚粒線體融合蛋白-2在癌症的角色。本研究，是探討粒線體融合蛋白-2在肺癌的角色，我們利用免疫組織化學染色分析粒線體融合蛋白-2在非小細胞肺癌檢體的表現：腫瘤組織 80％和非腫瘤組織 20％（卡方分佈；p＜0.05）。利用西方墨點分析腫瘤組織和非腫瘤組織粒線體融合蛋白-2蛋白的表現。粒線體融合蛋白-2 在惡性腫瘤中有高度表現，在非小細胞肺癌的惡化也扮演一個重要之角色。粒線體融合蛋白-2是在於診斷和治療非小細胞肺癌新的指標。

Mitochondria are key players in several cellular functions
including growth, division,energy metabolism, and apoptosis.
Mitochondrial dysfunction contributes to a number of human disorders and may aid cancer progression. Although normal
mitochondrial morphology is important for the function and physiology of cells, defects in mitochondrial dynamics may cause severe diseases. Fusion and fission of mitochondria regulate their morphology and distribution. Mitofusin-2（Mfn-2）is an outer mitochondrial membrane protein. Recent observations indicate that Mfn2 is a multifunctional membrane protein that participates in cell proliferation and metabolism and that it is required for normal endoplasmic reticulum morphology. The roles of Mfn-2 in cancers are largely unknown. In this study, which aims at defining the role of Mfn-2 in lung cancer, we analyzed the expression of Mfn-2 in non-small cell lung cancer（NSCLS） using immunohistochemistry . Mfn-2 expression that was detected in the cytoplasm in average
immunostaining of Mfn2 was 80％ for tumor and 20％ for non-tumor（Pearson Chi-Square；p＜0.05）.We analyzed the expression of Mfn-2 in tumor and non-tumor pairs by using western blot. Increased expression of Mfn-2 in malignant cells compared to the non-tumor cells suggests that Mfn-2 expression may play a role in NSCLS aggravation. It may be a novel marker for diagnosis and treatment of NSCLS.

目錄
中文摘要……………………………………………………………I
英文摘要……………………………………………………………II
第一章 前言……………………………………………………………1
1、 粒線體…………………………………………………………1
1.1 粒線體的外觀與構造………………………………………1
1.2 粒線體的功能………………………………………………2
1.3 粒線體的DNA……………………………………………… 3
1.4 粒線體的分裂與融合………………………………………3
1.5 粒線體與疾病………………………………………………5
1.6 粒線體與癌症………………………………………………6
2、 粒線體融合蛋白………………………………………………8
2.1 fzo 基因……………………………………………………8
2.2 人類的粒線體融合蛋白……………………………………9
3、 肺癌……………………………………………………………12
4、 研究動機與目標………………………………………………15
第二章 材料與方法…………………………………………………16
1、細胞培養（Cell culture）………………………………………16
1.1 解凍細胞……………………………………………………16
1.2 細胞繼代培養……………………………………………16
2、全蛋白質萃取（Protein extraction）…………………………17
2.1 細胞全蛋白質萃取…………………………………………17
3、蛋白質定量（Protein concentration determination）……………17
4、西方墨點法（Western immunoblotting）………………………18
4.1 電泳膠的配置………………………………………………18
4.2 膠體電泳及轉漬……………………………………………18
4.3 免疫偵測……………………………………………………19
5、免疫組織化學染色（Immunohistochemistry）…………………20
5.1 脫蠟…………………………………………………………20
5.2 抗原修復……………………………………………………20
5.3 染色…………………………………………………………20
6、免疫細胞化學染色（Immunocytochemistry）………………21
7、統計分析(statistical analysis)…………………………………22
第三章 結果……………………………………………………………23
1、Mfn2在非小細胞肺癌組織有高度表現………………………23
2、西方墨點法下腫瘤組織與非腫瘤組織，Mfn2蛋白的表現……24
3、西方墨點法下腫瘤細胞株Mfn2蛋白的表現…………………25
4、Mfn2蛋白與細胞凋亡…………………………………………25
第四章 討論…………………………………………………………28
第五章 結論…………………………………………………………30
參考文獻………………………………………………………………31
圖表目錄
表一 、組織蠟塊檢體，性別及病理診斷一覽表…………………39
表二、以免疫組織化學染色的方式，觀察在肺癌組織中Mfn2在的腫瘤組織及非腫瘤組織的表現……………………………40
圖一、以免疫組織化學染色的方式，分別以抗體Ck7、Ck20及TTF-1
確認檢體為原發性而非轉移性之肺癌組織………………41
圖二、以免疫組織化學染色的方式，觀察在腫瘤組織中 Mfn2的
表現…………………………………………………………42
圖三、以STS藥物作用於腫瘤細胞株（A431）Mfn2的表現………43
圖四、以西方墨點法，探討在腫瘤組織與非腫瘤組織，Mfn2的表
現……………………………………………………………44
圖五、以西方墨點法，探討腫瘤細胞株Mfn2的表現……………45

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