臺灣博碩士論文加值系統

p53是人類腫瘤抑癌基因，在腫瘤組織中經常發現p53有異常的情形發生。透過對肺癌患者的組織分析中，篩選後決定選用7種不同的突變的p53，分別是H179Y、S240R、R249S、A276D、E286Q、P67C/L194R和G361R，利用基因選殖的方式表現於p53 null的H1299肺癌細胞株中，並且另外選殖了一個MDM2 p53-dependent promoter，並接於luciferase的reporter 質體DNA上，共同送入H1299細胞中，觀察這些突變的p53蛋白轉錄活化的情形。結果顯示在S240R和E286Q仍分別約佔有wild type p53的43%和28%轉錄活化功能的表現，至於其他突變的p53轉錄活化能力都表現低落。而進一步分析這些突變的p53蛋白與MDM2 p53-RE結合的能力，發現除了在S240R有明顯的結合，在E286Q、V143A和P67C/L194R也有微弱的結合反應，其餘突變的p53皆無任何的結合反應，顯示p53蛋白會因突變的位置不同，而影響與DNA結合的能力。此外還利用細胞群落形成效率觀察這些突變的p53蛋白對於抑制癌細胞生長的能力，結果證實S240R具有抑制細胞生長的能力，仍保有部分的抑癌的功能，並與肺癌患者的組織分析結果核對，符合患者的MDM2 mRNA有表現，種種結果顯示突變的p53 S240雖然發生點突變，但是仍然保有部份p53 wild type抑癌的能力。此外有文獻指出利用p53的codon 361~382的peptides可將突變p53原本失去功能恢復，於是利用基因選殖的方式將p53的codon 340~382選殖出來，與p53一起送入細胞，觀察其轉錄活化的情形，結果發現p53 codon 340~382不但不能恢復突變p53蛋白的功能，反而會抑制wild type p53轉錄活化的能力。

p53 is a critical tumor suppressor gene, which can respond to multiple signals of cellular gatekeeper for growth and division. MDM2 gene is one of the downstream target genes for transcriptional activation by the product of p53 tumor suppressor gene. Transactivation of MDM2 gene expression is represented by the presence of a functional p53 protein. To understand the biological function of the mutant p53 that may play the role in tumorigenesis, we construct a number of p53 mutants by site directed mutagenesis (H179Y, P67C/L194R, S240R, R249S, A276D, E286Q, G361R), and followed by characterization with its ability to transactivate MDM2. The MDM2 promoter was ligated into pGL-3 luciferase reporter MDM2-p53RE-pGL3 gene for this assay. We analyzed the expression of these mutants p53 proteins by Western blotting, and the p53 mutants protein-DNA binding activity by gel retardation assay. The transactivation properties of p53 mutants were compared by co-transfecting on MDM2-p53RE-pGL3 into the p53 null cell line H1299 that derived from non small cell lung carcinoma. In order to study the p53 mutant grow suppression ability in tumor cells, performed colony formation assay and the colony number were compared. Mutant p53 S240R and E286Q exhobited the luciferase activity of MDM2-p53RE-pGL3 at about 40% and 30% of the wt p53 vector, respectively. The binding of mutant p53 to the p53 respontive element in MDM2 was analyed by EMSA. Our findings indicated the presence of mutant p53 in the protein-DNA comples and revealed that p53 mutants S240R maintain partial MDM2-p53RE binding activity when compared to wild type p53. It showed that S240R induces apoptosis with just over 21.5 % the efficiency of empty vector (pcDNA 3). The attemp to rescue the mutant p53 protein transactivation by a trans-elememt of trunscated p53 polypeptide correspondary to the carboxy-terminal residues 340-382. Susprisely, this trunscated p53 polypeptide could not restore the transactivation activity of mutant p53. Moreover, it inhibit both the mutant and wt p53 transactivation activity significatly.

一、中文摘要 -------------------------------------------- 2
二、英文摘要 -------------------------------------------- 4
三、緒 言 -------------------------------------------- 6
四、研究動機 ------------------------------------------- 18
五、實驗材料與儀器 ------------------------------------- 20
六、實驗方法 ------------------------------------------- 23
七、實驗結果 ------------------------------------------- 46
八、討 論 ------------------------------------------- 56
九、圖表及圖表說明 ------------------------------------- 66
十、參考文獻 ------------------------------------------- 79

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