臺灣博碩士論文加值系統

H1299細胞中的p53基因有homozygous partial deletion，因此不會表現p53蛋白。論文中是利用H1299細胞的特性，以DNA轉殖技術建立三種背景相同，但是p53 status不同的細胞株。分別是含有穩定表現野生型p53基因的H1299細胞株、含有穩定表現突變型p53基因的H1299細胞株以及只含有空載體不含任何p53基因的H1299細胞株。為了建立wtp53-H1299細胞株，首先，先建構pcDNA3-p53質體。將wtp53從已經鑑定為含有正確p53基因的pc53-SN質體中以BamHI切下轉接到pcDNA3載體上。利用轉殖技術分別將pcDNA3-p53及EGFP-N1質體轉殖進入H1299細胞建立wtp53/EGFP-H1299細胞株。本研究也轉殖了pcDNA3-mp53及EGFP-N1質體到細胞建立mp53/EGFP-H1299細胞株，以及轉殖了pcDNA3及EGFP-N1質體到細胞建立pcDNA3/EGFP-H1299細胞株。三種有轉殖質體的細胞分別在含G418的培養液中培養三週以上，形成穩定有質體的混合細胞族群（stable mix colonies）之後，以西方點墨法檢查三株細胞中p53及p21蛋白表現的情形，結果是：wtp53和mp53細胞中都可以檢查到p53蛋白的表現，無任何轉殖質體的H1299細胞株則完全無p53蛋白的表現。檢查各細胞株之p53下游基因p21蛋白的表現，則wtp53細胞株與無任何轉殖質體的細胞株相較，有較多的p21蛋白的表現。但是mp53細胞株與無任何轉殖質體細胞株，相較其p21蛋白表現，則沒有顯著性差異。由p53蛋白和p21蛋白的表現結果，可以確定這三種細胞株的建立是成功的。
完成以上細胞株建立後，本研究亦進行細胞株對於亞砷酸鈉的藥物敏感度實驗。三株細胞在不同濃度的亞砷酸納處理24小時之後，再培養五日後用SRB assay所得到的結果為：含有wtp53基因的細胞株，對於SA的IC50為3.9uM。含有mtp53基因的細胞株，對於 SA的IC50為6.6uM。negative control的細胞株，對於SA的IC50為15.4uM。因此，wtp53-H1299細胞株對於SA的敏感程度為原來的H1299細胞的4 倍，而mp53-H1299細胞株對於SA的敏感程度為原來的H1299細胞的2.3倍。換句話說，在H1299細胞中回復了p53蛋白的表現，可以增加細胞對藥物的敏感度。在H1299細胞中表現功能不正常的突變型p53蛋白，也使轉殖細胞對藥物的敏感性受到影響。這三種表現不同p53背景的H1299細胞繼續作為臨床上“ p53對應不同抗癌藥物的敏感度測試 ”，可以為本研究之繼續目標之一。

H1299 cells have a homozygous partial deletion of the p53 gene, and thus lack expression of p53 protein. In this study, three kinds of p53-transfected H1299 cells having different p53 gene status have been established from original p53-null H1299 cells. For establishment of wtp53-H1299 cells, normal p53 gene was be constructed from p53-SN into pCDNA3 plasmid , and named it pcDNA3-p53 plasmid. pCDNA3-p53 and EGFP-N1 plasmids were transfected into H1299 cells to establish wtp53/EGFP-H1299 cells by lipofectamine. Two other cell lines, mp53/EGFP-H1299 and pcDNA/EGFP-H1299 cells were established by transfecting pcDNA3-mp53 and pcDNA3, respectively, along with EGFP-N1 plasmids into H1299 cells.
Stable transfected cells were selected in G418-containing medium. They were characterized by Western blotting to examine the background expression of p53 and p21. P53 protein was detected in both wtp53/EGFP-H1299, and mp53/EGFP-H1299 cells. However, p21 protein was only markedly observed in wtp53-H1299 cells in comparison with those in mp53/EGFP-H1299 and pCDNA3/EGFP-H1299 cells. These indicated that all transfectants were have expected results.
The sensitivity to different concentrations of SA in above transfectants was further examined by SRB assay. The IC50 of SA in wtp53/EGFP-H1299, mtp53/EGFP-H1299, and pCDNA3/EGFP-H1299 cells was 3.9 mM, 6.6 mM, and 15.4 mM, respectively. Therefore, the sensitivity to SA was 4-fold and 2.3-fold in wtp53/EGFP-H1299 and mtp53/EGFP-H1299 cells, respectively, in comparison with that in pCDNA3/EGFP-H1299 cells. In other words, expression of wild type p53 could markedly enhance the sensitivity to SA in p53-transfected H1299 cells. However, expression of mutant type p53 also slightly affected the sensitivity to SA in mp3-transfected H1299 cells. We expected above thee transfected cells are suitable in using to investigate the relationship between p53 status and chemosensitivity to various chemotherapeutic agents.

一、中文摘要 -------------------------------------- 2
二、英文摘要 -------------------------------------- 4
三、緒 論 -------------------------------------- 6
四、研究動機 -------------------------------------- 11
五、材料與方法-------------------------------------- 13
六、結 果 -------------------------------------- 22
七、討 論 -------------------------------------- 31
八、參考文獻 -------------------------------------- 35
九、圖表及附錄-------------------------------------- 42

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