臺灣博碩士論文加值系統

鼻咽癌為東南亞特別盛行的腫瘤，宿有「中國癌之稱」；雖然多種與鼻咽癌有關的危險因子已被發現，如：種族、遺傳、環境、EB病毒…等，但鼻咽癌形成的分子機制仍不是很清楚。在真核細胞，細胞週期的進行受到cyclins、CDKs、CDKIs這些蛋白質的交互作用被調控著，這些細胞週期調節蛋白對正常細胞與腫瘤細胞的生長扮演相當重要的角色。
p27Kip1為Cip/Kip家族的一名成員，p27Kip1蛋白會抑制cyclin D-CDK4與cyclin E-CDK2，使細胞停留在G1期。雖然在人類腫瘤中，p27Kip1基因很少發生突變，但在幾種癌症中，像：直腸癌、乳癌、食道癌發現p27Kip1蛋白的表現量較低。
先前的實驗研究顯示，在兩株鼻咽細胞其p27Kip1的表現量都明顯低於正常鼻咽上皮細胞(NNE)與293(HEK)；因此我們將p27Kip1基因與誘導載體結合後，形成一Doxycycline誘導載體pBIG2r/p27Kip1，且轉染到兩株鼻咽癌細胞，觀察一些細胞週期調節蛋白的表現情形。
實驗發現過量表現p27Kip1在兩株鼻咽癌細胞會使cyclin E與CDK2表現量增加，並使較多鼻咽癌細胞停滯在G1期，細胞倍增時間增加，甚至有部分鼻咽癌細胞發生自然凋亡，因此，過量表現p27Kip1或許可以使鼻咽癌細胞休止於G1期且導致凋亡。

Nasopharyngeal carcinoma is a commonly occuring tumor in Southern China. However, the genetic basis underlying its tumorigenicity is still unclear. In eukaryotic cells, progression of the cell cycle is regulated by interactions of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors (CDKIs). These cell cycle-regulator proteins play important roles in growth of both normal and tumor cells. Many human tumors exhibit deregulation of one or more genes which involved in regulation of cell cycle progression.
p27Kip1, a member of the Cip/Kip family, inhibits both cyclin D-CDK4, and cyclin E-CDK2 complexes and regulates progression of the cell cycle from G1 to S phase. Although p27Kip1 gene mutations are rare in human tumors, low expression of p27Kip1 are observed in several cancers, such as colon, breast and esophagus.
In our previous study, p27Kip1 shown lower expression in two NPC cell lines compared with NNE and 293 (HEK). A doxycycline inducible construct, pBIG2r/p27Kip1, included a full length of human p27Kip1 cDNA was transfected into two NPC cell lines. Expression of several cell cycle-related genes were analyzed.
By increasing p27Kip1 in NPC cell lines, we found that the G1 phase and the doubling time were lengthened. Protein expression of cyclin E and CDK2 were up-regulated. These data suggest that the overexpression of p27Kip1 might be cause NPC cells to arrest at G1 phase and might lead to apoptosis.

壹、緖論
一、前言---------------------------------------------------------------------1
二、細胞週期---------------------------------------------------------------2
三、細胞週期調節基因p27Kip1------------------------------------------5
四、鼻咽癌------------------------------------------------------------------6
貳、研究目的---------------------------------------------------------------------11
參、材料與方法
一、鼻咽癌細胞與正常鼻咽上皮細胞之培養(Cell Lines)--------12
二、生長曲線(Growth Curve)-------------------------------------------13
三、反轉錄聚合酵素連鎖反應(RT-PCR)----------------------------13
四、勝任細胞(Competent Cell)的製備---------------------------------15
五、pUCmT與可誘導式載體pBIG2r的製備----------------------16
六、具有p27Kip1基因重組質體的製備-------------------------------17
(1)pUCmT/p27Kip1質體的製備
(2) pBIG2r /p27Kip1質體的製備
七、轉染細胞(Transfection)---------------------------------------------21
八、西方墨點法(Western Blot Analysis)------------------------------22
九、多重探針核糖核酸分解酵素保護分析(RPA)------------------26
十、流式細胞儀分析細胞週期之變化(Flow Cytometry)----------30
肆、結果與討論------------------------------------------------------------------32
伍、結論---------------------------------------------------------------------------37
陸、參考文獻---------------------------------------------------------------------68

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