臺灣博碩士論文加值系統

　　肺癌每年造成全世界上百萬人因而死亡，如此的高死亡率較其他癌症更具威脅性。在組織學分類上，肺癌又可因型態上的差異區分為小細胞癌(Small cell lung carcinoma)及非小細胞癌(Non-small cell lung carcinoma)，而非小細胞癌又可再區分為腺癌(Adenocarcinoma)、鱗狀細胞癌(Squamous cell carcinoma) 、大細胞癌(Large cell carcinoma) 、腺-鱗狀細胞混和癌(Adeno-Squamous carcinoma)。雖然在西方國家中，肺癌最主要的型態為鱗狀細胞癌，但在台灣地區，腺癌卻是發生率最高的肺癌型態，尤其好發於非吸煙女性。在肺腺癌女性中，常合併伴隨有惡性肋膜積水的產生，且預後較差。為了了解這樣的現象與病理機制上的相關性，同時在治療上能有更進一步的發展，我們收集肺腺癌病人的惡性肋膜積水作為研究的對象。利用二維電泳(2D-PAGE)與蛋白質譜分析-MALDI-MS (Matrix-assisted Laser Desorption/Ionization- Mass Spectrometry)-將惡性肋膜積水中的蛋白質做定性的分析。同時，分離出惡性肋膜積水中的癌細胞，利用微陣列生物晶片(Affymetrix oligonucleotide microarray)來對於癌細胞的基因做分析。在蛋白質體的研究中，藉由比對非吸煙女性族群與吸煙男性族群，找出三個表現量，在非吸煙女性表現量明顯高於吸煙男性，差異性較大的蛋白，分別是α-1 inhibitor III precursor, apolipoprotein E, coatomer protein complex, subunit β (COPB)，同時這些蛋白在肺炎所導致的惡性肋膜積水中表現是較少或甚至不表現的。此外，收集14個肺腺癌病人合併肋膜積水中癌細胞，正常肺組織，及正常肺細胞株(Bes2B)來進行微陣列生物晶片之基因分析，利用階層式叢集(Hierarchical clustering)，根據各個檢體在基因上表現的不同來進行分群的動作，發現正常肺組織、非吸煙女性、及吸煙男性會形成三個不同族群，並可藉由此方法找出在不同族群中表現差異大的基因群。另外利用TNoM (Threshold number of misclassification)方法，將病人區分成兩個族群-非吸煙女性及吸煙男性，找出在同一族群中表現較一致，但在不同族群表現差異性最大的29個基因，再用此29-gene-panel回推進行階層式叢集，發現此群基因的確可區分出不同的病人族群。而在蛋白質體研究中發現的COPB蛋白，在mRNA層次也同樣發現有非吸煙女性高於吸煙男性的趨勢。此外，APOE在免疫組織染色上發現多分布於癌細胞周圍，也可發現在某些癌細胞的細胞質中。在文獻中發現，APOE與癌症的侵略性、淋巴轉移相關，而COPB則與高基氏體和內質網間物質的運送及調控有關，因此，這兩個蛋白質間是否有互相影響的關聯性，或對於肺腺癌的病理機制及診斷有貢獻，則仍需更進一步確認。
　　因此，肺腺癌根據病理診斷上非吸煙導致肺腺癌，且常合併伴隨有惡性肋膜積水產生的女性，在基因表現及蛋白質表現上與男性均有差異性，在此研究中，我們找出差異性最大的29基因組，可作為診斷或檢驗之依據，此外，APOE及COPB對於此類肺腺癌可能也扮演相當重要的角色。

　　Lung cancer causes about a million deaths per year worldwide, which is more than any other malignant disease. The major histological types of lung cancers are adenocarcinoma, squamous cell carcinoma, and small cell lung carcinoma. Whereas squamous cell carcinoma is the most common type of lung cancer in Western countries, adenocarcinoma is the predominant lung cancer type in Taiwan, especially in female non-smokers. Among the female patients with lung adenocarcinoma, malignant pleural effusion (MPE) is frequently associated with and is a poor prognostic sign. To understand the mechanisms underlie the pathobiology and to identify biomarkers for better diagnosis of malignant pleural effusion associated lung adenocarcinoma. We collected MPEs from patients with lung adenocarcinomas for study. Proteins precipitated from MPE were separated by 2D-PAGE and interested spots were identified by MALDI-MS (Matrix-assisted Laser Desorption/Ionization- Mass Spectrometry). RNA specimens extracted from cancer cells in MPE were subjected to Affymetrix oligonucleotide microarray analysis. In proteomic studies, by comparing protein expression patterns of smoking male with non-smoking female lung cancer patients, we have identified three proteins overexpressed in female, non-smoking group. They were α-1 inhibitor III precursor, apolipoprotein E, coatomer protein complex, subunit β (COPB). The expression of these three proteins was low or undetected in pleural effusion pooled from 10 patients with pneumonia. Fourteen specimens of cancer cells in MPE, three of normal lung epithelial cell line (Bes2B), and ten normal part of lung tissue were available for RNA isolation and the extracted RNAs were sent for Affymetrix oligonucleotide microarray analyses. Using non-supervised, hierarchical clustering analysis, normal lung tissue, lung adenocarcinoma cells from non-smoking females, and lung adenocarcinoma from smoking males were separated into three groups according to the genetic expression pattern. Besides, we use the statistical method-TNoM (Threshold number of misclassification) to find out a 29-gene panel which differentially expressed in non-smoking female and smoking male. The 29-gene panel has a potential to be used for differential diagnosis of lung adenocarcinomas resulted from different etiology. The higher expression of COPB and APOE was found both in proteomic and microarray studies, suggesting a correlation exists between mRNA and protein expression. Since APOE has been implicated in the invasiveness of human cancers, the findings from the study are not only markers for diagnosis but also could be pathobiological factors. Therefore, studying cancer cells and proteins in pleural effusion by proteomic and microarray approaches is feasible. Further works are warranted to verify the data and disclose more important factors.

List of Tables………………………………………………………………………………………I
List of Figures……………………………………………………………………………………II
Abstract in Chinese…………………………………………………………………………… III
Abstract in English………………………………………………………………………………IV Acknowledgement……………………………………………………………………………………VI

1. Introduction
1.1 Lung cancer………………………………………………………………………………… 1
1.2 Lung cancer subtypes………………………………………………………………………2
1.3 Lung adenocarcinoma……………………………………………………………………… 3
1.4 Malignant pleural effusion accompanies adenocarcinoma………………………… 5
1.5 Proteomic study of malignant pleural effusion from lung adenocarcinoma………………………………………………………………………………………6
1.6 Genetic expression of cancer cells from malignant pleural
effusion………………………………………………………………………………………7
1.7 Microarray data analysis…………………………………………………………………9
2. Material and methods
2.1 Two-dimentional electrophoresis, microarray materials, and histocytological
specimens……………………………………………………………………………………11
2.2 Tumor cells purification……………………………………………………………… 11
2.3 Isolation of total RNA – TRIzol method……………………………………………12
2.4 Affymetrix array hybridization and image processing……………………………13
2.5 Reverse transcriptase-polymerase chain reaction
(RT-PCR)…………………………………………………………………………………… 13
2.6 Protein concentration assay……………………………………………………………13
2.7 Protein precipitation……………………………………………………………………14
2.8 Two-dimension gel electrophoresis……………………………………………………15
2.9 Western Blotting………………………………………………………………………… 15
2.10 Immunohistochemistry staining……………………………………………………… 17

3. Results
3.1 Tumor cells were purified from malignant pleural effusion……………………21
3.2 Gene expression patterns are different in normal and diseased, and
differential gene expression can classify female non-smokers and male
smokers by Hierarchical
clustering………………………………………………………………………………… 21
3.3 29-gene panel identified by TNoM classification can separate female
non-smokers and male smokers………………………………………………………… 23
3.4 α-1-inhibitor III precursor, ApoE, and COPB are found differentially
expressed in female non-smokers and male smokers in 2-D
electrophoresis……………………………………………………………………………25
3.5 Overexpression of COPB protein parallels upregulation of gene expression in
tumor cells…………………………………………………………………………………26
3.6 Immunohistochemistry staining showing COPB protein is not absolutely
expressed by tumor cells……………………………………………………………… 26
4. Discussion………………………………………………………………………………………28
5. Appendix…………………………………………………………………………………………33
6. References………………………………………………………………………………………34

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