| 摘要: | 本論文之目的在於利用雙色基因微陣技術探討H1299人類肺癌細胞中受野生型p53蛋白所調控的下游基因之表現。本實驗利用H1299-p53V173L細胞株中一段突變型p53基因進行研究，此基因產物具有溫度敏感的特性，培養於37℃及32℃可分別表現為突變型及野生型p53蛋白結構。將細胞分別培養於37℃及32℃ 0、6、8、10、12、14、16、18、20、22及24小時後萃取mRNA以37℃為對照組，32℃為實驗組，在反轉錄反應時分別標記biotin及digoxigenin，並針對9600點基因微陣作雙色雜交及呈色。經影像數據化及統計分析後，篩選出495個基因行SOM (self-organizing map)群集分析，以分成24種表現型態，結果共選取八組呈現趨勢性表現增加或降低的基因(144個)進行定序分析及探討，其中大部分基因與訊息傳遞、細胞生長代謝調節及DNA修補有關。此結果說明在細胞未受到低氧壓或X-ray照射等刺激的情況下，野生型p53蛋白也可能具有調控許多基因表現的功能。在此亦發現了具有調節免疫功能的MHC class I基因群一致且明顯地被野生型p53所誘導，顯示這類基因的表現與p53有密不可分的關係，且似乎意味著野生型p53蛋白在此細胞中可能正扮演著調節腫瘤監控系統之角色。過去研究中，p53與免疫調節機制間的關係是被忽略的。因此這個有趣的發現，有待進一步深入研究。 p53 is a well-studied tumor suppressor gene. In the past, many conventional methods were used to identify p53 function-associated genes. In order to identified unknown genes whose function were related with p53, colormetric cDNA microarray were used to study the genome-wide transcriptional expression pattern of genes, which are regulated by tumor suppressor gene p53 in human non small cell lung cancer cell line H1299. To chase the downstream genes of p53, the cell line H1299-p53V173L was used for experiments since it expresses wild-type p53 once the growth temperature was shifted from 37℃ to 32℃. Post temperature shift from 37℃ to 32℃, cells were harvested at the following time intervals: 0, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours. The derived cDNA were labeled and hybridized to microarray membranes containing 9600 cDNA dots. The signal density of these cDNA dots were acquired with image processing for statistic analysis. Through such process, totally 144 genes were identified as p53-upregulated or p53-downregulated, and were further sequence verified. The majority of these genes are related with signal transduction, cell cycle, metabolic regulation and DNA repair. Some genes found associated with p53 in the literature were successfully identified, for instance, PCNA, ku80, APEX etc. According to the data in this thesis, p53 might control many genes expression, even though when cells were not stimulated by X-ray or hypoxia. Among those genes, the most interesting one is the MHC (major compatibility complex) class I, which plays a major role in immune response. Different alleles of MHC class I was observed significantly and consistently induced by p53. This indicates that p53 may be involved in some immune pathway to target stressed or tumor cells for elimination. The association between p53 and immune system was all the time totally ignored. With cDNA microarray technology, this association is confirmed and is worth with further investigation. |
