莊曜宇Chuang, Yao-Yu臺灣大學:電機工程學研究所康 瑜Kang, YuYuKang2010-07-012018-07-062010-07-012018-07-062008U0001-2907200812044200http://ntur.lib.ntu.edu.tw//handle/246246/187944肺癌是全世界最普及、致死率最高的癌症。非小細胞肺腺癌佔肺癌病人的百分之八十，不論是否為轉移的腫瘤，經由放射治療後的五年存活率僅達百分之五。然而，關於肺癌細胞對輻射線的反應機制仍不清楚。本研究以具有不同遷移能力之人類肺腺癌細胞CL1-0和CL1-5作為離體的研究對象。本研究目的在於利用微陣列晶片探討造成兩株肺腺癌細胞對輻射線敏感度差異之成因以及Jagged 1 基因在此兩細胞株中，細胞敏感度差異之中扮演的角色。首先使用群落形成測試法來比較兩株細胞對於輻射線敏感度的差異，接著以流式細胞分析儀分析在輻射線照射過後，細胞在不同時間點的細胞週期分佈。同時，以微陣列晶片實驗來探討在輻射線照射過後，兩細胞株相關基因變化以及分子機制。並且，在微陣列晶片資料中發現，Jagged 1的表現量在兩細胞的差異極大，而此表現形式也以即時聚合連鎖反應以及西方墨點法得到驗證。為了更近一步探討Jagged 1 在輻射線敏感度差異中扮演的角色，在CL1-0中大量表現Jagged 1，並且進行功能測試。結果顯示，CL1-5 相較於CL1-0來說，對於輻射線具有較高的敏感性。經由10 Gy 加瑪射線照射之後，CL1-0和CL1-5 均在24小時的時間點，具有最高比率的細胞停滯在G2/M期，並且均缺乏G1/S期的滯留。將微陣列的資料經由密集叢聚分析結果可知，有六個集群在兩細胞株之中表現形式差異極大，這些集群中的基因和細胞死亡、細胞週期、細胞生長、增殖與細胞功能維持有關。在其中一個不同表現的集群中，具有和G2/M細胞檢查點相關的基因，與實驗中G2/M 細胞週期停滯之引發吻合。 在CL1-0中大量表現Jagged 1之功能測試結果中發現，相較於CL1-0，其經由10 Gy輻射線照射之後，存活率上升，而經由即時聚合連鎖反應可知，其細胞中NFκBIA 的含量改變，由流式細胞分析儀的結果可知，其在24 小時，sub G1 以及 G2/M 的細胞分佈比例介於CL1-0 與CL1-5之間。 综合上述，CL1-5較CL1-0來得具有輻射線敏感性，而CL1-5主要的死亡機制是透過增殖死亡，除此之外，Jagged 1 與CL1-0以及 CL1-5輻射敏感性差異有相關連，可能與NFκBIA具有交互作用，進而影響存活率。Lung cancer is the most common and lethal cancer in the world. NSCLC accounts for 80% lung cancer patients, and the 5-year survival rate after radiotherapy is about 5% whether the patients are with less or more extensive tumors. However, the mechanism behind radiation response of lung cancer is still not clear. Here, lung adenocarcinoma cell lines CL1-0 and CL1-5 with different metastasis abilities were used as the objects of our in vitro study. The aim of this thesis was to investigate the radiosensitivity in CL1-0 and CL1-5 via global gene expression profiles and the role of a notch ligand, Jagged 1 played in radiosensitivity. Clonogenic assay was used to obtain radio sensitivity in CL1-0 and CL1-5. To better understand the cell cycle distributions followed by radiation, flow cytometry experiment was performed at different time points. At the same time, the temporal global gene expression profile was obtained to understand the radiosenstivity via global transcriptional activities. In addition, the expression difference of Jagged 1 between CL1-0 and CL1-5 were significant in microarray, and were further confirmed by real time PCR and western blotting. To investigate the role of Jagged 1 in radiosensitivity, Jagged 1 overexpressed CL1-0 was obtained to perform functional assay. The data indicated that CL1-5 was more radiosensitive than CL1-0 after 10 Gy radiation. The irradiated CL1-0 and CL1-5 exhibited G2/M blockade and with the lack of G1/S checkpoint. Moreover, the subG1 area was significantly higher in the irradiated CL1-0 than the CL1-5 cells. From microarray data and through tight clustering analysis, there were six clusters expressed significantly different between CL1-0 and CL1-5. Genes among them were related to cell death, cell cycle, cell growth and proliferation, cellular function and maintenance. A different expression gene group between CL1-0 and CL1-5 showed genes related to G2/M checkpoint, which was consistent with the G2/M arrest activities. The Jagged 1 functional assay results showed an increasing survival rate in Jagged 1 overexpressed CL1-0 compared to CL1-0.Also, the transcription level of NFκBIA were changed in Jagged 1 overexpressed CL1-0. Compared to CL1-0 and CL1-5, Jagged 1 overexpressed CL1-0 cell line showed a mid percentage in sub G1 and G2/M phase. In conclusion, CL1-5 was more radio sensitive than CL1-0 and the death cause was mainly from mitotic death. In addition, Jagged 1 was associated partially with the radiosenstivity in CL1-0 and CL1-5, and may have a cross talk with NFκBIA.Table of Contents文摘要 ibstract ivable of Contents viist of Figures viiiist of Tables ixhapter 1 Introduction 1.1 Lung cancer 1.2 Cellular responses to ionizing radiation DNA damage 2.3 Global gene expression analysis by microarray 3.4 Notch Signaling 4.5 Role of Jagged 1 in cancer 5.6 NF-κB pathway 6.7 Motivation and specific aims 7hapter 2 Materials and Methods 9.1 Cell culture 9.2 Radiation Treatment 9.3 Clonogenic survival assay 10.4 RNA Preparation 10.5 Illumina microarray hybridization 11.6 Synthesis of cDNA and cRNA for Affymetrix microarray analysis 11.7 Oligonucleotide microarray hybridization 12.8 Validation of Microarray Results by Real-Time PCR 13.9 Flow cytometry analysis and apoptosis determination 14.10 Western Blot Analysis 15hapter 3 Results 16.1 Clonogenic survival assay of CL1-0 and CL1-5 16.2 Flow cytometry analysis and apoptosis determination of CL1-0 and CL1-5 16.3 Identification of differentially expressed genes and the gene expression profile 18.4 Microarray data validation by real time PCR 21.5 Western blot of Jagged 1 after 10Gy radiation treatment 22.6 Jagged 1 over expressed in CL1-0 23.7 Clonogenic survival assay of Jagged 1 overexpressed CL1-0 23.8 Flow cytometry analysis and apoptosis determination of Jagged 1 over expressed CL1-0 24.9 Real time PCR results of Jagged 1, NFκBIA and NFκBIZ in Jagged 1 over expressed CL1-0 24hapter 4 Discussion 26.1 Elucidation of different cell survival mechanisms in CL1-0 and CL1-5- from microarray and flow cytometry results 26.2 Elucidation of different cell survival mechanisms in CL1-0 and CL1-5- from Jagged 1 related functional assay 29hapter 5 Conclusion 32igures 33ables 49eference 56ist of Figuresig 1. Survival rate of CL1-0 and CL1-5 after 5Gy and 10Gy radiation treatments 33ig 2. Cell cycle distribution in CL1-0 and CL1-5 34ig 3.Cell Cycle distributions in CL1-0 and CL1-5 after 10Gy radiation treatment 36ig 4. The top 5 gene functions and canonical pathways of 1595 genes analyzed by IPA 37a) Top 5 gene functions of 1595 genes 37b) Top 5 canonical pathways of 1595 genes 37ig 5. Tight cluster analysis results of six differentially expressed clusters in CL1-0 and CL1-5 after 10 Gy radiation treatments 39ig 6. Validation of Jagged 1 expressions in microarray by real-time PCR 41ig 7. Validation of NFκBIA expressions in microarray by real-time PCR 42ig 8. Validation of NFκBIZ expressions in microarray by real-time PCR 43ig 9. Western Blot Analysis of Jagged 1 protein induction by 10 Gy radiation 44ig 10. Western blot analysis of Jagged 1 in Jagged 1 over- expressed CL1-0 45ig 11. Survival rate of CL1-0, CL1-5, and Jagged 1 overexpressed CL1-0 after 10Gy radiation treatments 46ig 12. Sub G1 and G2M phase distributions in CL1-0, CL1-5 and Jagged 1 over expressed in CL1-0 after 10Gy radiation treatment 47ig 13. TNF-α is induced after 10Gy radiation in CL1-5. 48ist of Tablesable 1. The top functions of 5 major networks in 1595 genes 49able 2 The related networks of six differentially expressed clusters 50luster a 50luster b 50luster c 51luster d 52luster e 53luster f 541134610 bytesapplication/pdfen-US肺癌輻射輻射敏感度微陣列晶片Jagged 1lung cancerradiationradiosensitivitymicroarray[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/187944/1/ntu-97-R95921057-1.pdf