- 學位論文
放射線感受性與放射線抵抗性大腸直腸癌細胞株基因表現圖譜差異之研究
Differentially Expression Profile of Radio-sensitive and Radio-resistant Colorectal Cancer cell lines
摘要
放射線治療對於惡性腫瘤局部控制扮演著重要的角色，然而放射線治療的效果卻因癌症異質性，導致相同的放射線治療在不同個體間療效反應存在極大差異。本實驗目的為分析放射線感受性與放射線抵抗性大腸直腸癌細胞株基因表現圖譜差異來探討此基因群在大腸直腸癌放射線治療療效中所扮演的角色與影響。首先以 X 光射線照射刺激大腸直腸癌細胞株 SW620 進行寡核苷酸晶片分析，實驗結果顯示共1601個基因受到上升調控 (基因表現放射線處理後 / 放射線處理前≧1.5) ，而共57個基因受到下降調控(基因表現放射線處理後 / 放射線處理前≦0.5)，進一步將上升調控基因經由生物資訊線上資料庫進行分析；歸納此基因群所參與之生物反應路徑，主要與 DNA 受損相關反應、免疫反應以及補體與血液凝集連鎖反應 (Complement and Coagulation Cascade) 之反應路徑相關。在上述生物反應路徑中，吾人共篩選 30 個目標基因並以基因陣列尼龍膜片進行上升調控基因表現驗證。同時，以購自 ATCC 六株大腸直腸癌細胞株 COLO205、 T84、 HCT116、 SW480、 SW620 和 SW403 利用 ATPlite assay 進行細胞放射線敏感度評估。結果顯示六株細胞株之細胞存活分數值 (survival fraction at 2 gray, SF2)具顯著性的差異 (p=0.0074) ，其中 SW403 、 SW480、 T84 和 Colo205 為放射線抵抗性細胞株，而 SW620 和 HCT116 為放射線感受性細胞株。另一方面，利用基因陣列尼龍膜片比較放射線感受性與放射線抵抗性細胞株經放射線照射後基因表現圖譜異同，結果顯示 30 個基因中 breast cancer 1 (BRCA1)、prostaglandin-endoperoxide synthase 1 (PTGS1)、 complement factor B (CFB)、 zeta-chain (TCR) associated protein kinase 70kDa (Zap70)、 interleukin 8 (IL8)、 chemokine (C-X-C motif) receptor 4 (CXCR4)、 vav 1 guanine nucleotide exchange factor (VAV1)、 caspase 1, apoptosis-related cysteine peptidase (CASP1)、 DNA-binding protein B (dbpB)、 RAD51 homolog (RAD51)、 alanyl (membrane) aminopeptidase (ANPEP)、cell division cycle 25 homolog A(CDC25A)和 tumor protein p73 (TP73) 共13個基因在放射線感受性細胞株與放射線抵抗性細胞株間具有表現圖譜的差異。期待此一基因表現圖譜差異將具有相當潛力成為大腸直腸癌放射線治療反應相關研究之依據。
並列摘要
Radiotherapy plays a critical role in the local and regional control of malignant tumors. However, the principal limitation is large variability in response to radiotherapy among different individuals due to tumor heterogeneity. The purpose of this study is to compare gene expression profile between radio-sensitive and radio-resistant colorectal cancer cell lines to identify radiation related molecules which can evaluate the effect of radiation for colorectal cancer cell lines. Colorectal cancer cell line, SW620 was irradiated with high energy photo beam. Following radiation treatment, RNA was extracted from non-irradiated and irradiated cells, respectively, and gene expression analysis was performed by oliogonucleotide microarray. The result of oliogonucleotide microarray shows that 1601 genes were up-regulated (gene expression ratio of post- to pre-radiation treatment≧1.5), and 57 genes were down-regulated (gene expression ratio of post- to pre-radiation treatment≦0.5). After bioinformatics database analysis, majority of genes are involved in DNA damage response pathway, immune response pathway and complement and coagulation cascades pathway .We selected thirty up-regulated genes related to these biological reactions of colorectal cancer cells to radiation for further validation using membrane array. To further confirm the result above, six colorectal cell lines, COLO205, T84, HCT116, SW480, SW620 and SW403 were purchased from ATCC. The radiosensitivities of them were determined by survival fraction at 2 gray (SF2 ) of the surviving cells using the ATPlite assay. Result showed that the SF2 of six colorectal cancer cells are significantly different (p<0.0074). SW403, SW480 and T84 are the radio-resistant cell lines; SW620 and HCT116 are the radio-sensitive cell lines. Furthermore, we compared the gene expression after irradiation among the six cell lines by membrane array analysis. Of the 30 genes, breast cancer 1 (BRCA1), prostaglandin-endoperoxide synthase 1 (PTGS1), complement factor B (CFB),zeta-chain (TCR) associated protein kinase 70kDa (Zap70), interleukin 8 (IL8), chemokine (C-X-C motif) receptor 4 (CXCR4), vav 1 guanine nucleotide exchange factor (VAV1), caspase 1, apoptosis-related cysteine peptidase (CASP1), DNA-binding protein B (dbpB), RAD51 homolog (RAD51), alanyl (membrane) aminopeptidase (ANPEP), cell division cycle 25 homolog A(CDC25A) and tumor protein p73 (TP73) were differentially expressed between radio-sensitive and radio-resistant colorectal cancer cell lines. The data may help in the better understanding of radiation response and these genes may also serve as potential candidates of predictive markers for colorectal cancer patients’ radiotherapy responses.
參考文獻
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