朱善德臺灣大學：生化科學研究所李倍慈Li, Pei-TzuPei-TzuLi2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/52817小白鼠子宮能分泌24p3醣蛋白，在過去的研究中發現血球、纖維母細胞及肝細胞受IL-6、TNF-a、glucocorticoid、LPS及松節油等刺激後可誘導24p3蛋白質的生成，推測24p3蛋白質於發炎反應、壓力環境中及細胞凋亡過程扮演相當重要的角色，但真正之生理弁鄖疇憬24p3 protein is a secreted glycoprotein, which is estrogen-dependently expressed in mouse uterus, but is constitutively expressed in epididymis. The protein could associate with sperm head and enhance the sperm motility, and was also suggested as a stressor, apoptotic factor or inflammatory factor in blood cells, the function is not clear yet. Using mouse connective tissue cell line L929as a model, we are going to elucidate the reaction mechanism of 24p3 protein on the cells. First of all, dexamethasone could induce the 24p3 gene expression and protein secretion in this cell line, which suggested the existing of autocrine mechanism of 24p3 protein. According to the fluorescence microscopy observation, 24p3 protein could internalize into the cell in temperature-dependent status. Upon the internalization of the 24p3 protein into the cell, the intracellular level of ROS would be increased and the intracellualr level of GSH be decreased at beginning for protecting the cells and then be regenerated before 48 hours. Thereafter, the intracellular biochemical events would be triggered for responsive to the stress, including the changes of Bcl-2 and Bax ratio, DNA synthesis and p53 protein translocation. Incubation of the L929 cells with 10 mM 24p3 protein for 72 hours, the cell cycle would be arrested at S phase and the obvious DNA synthesis increase would be observed in 24p3 protein treated-cell via BrdU assay. At the same time, the mRNA expression ratio of Bcl-2/Bax decreased and cell viability could be reduced significantly. Besides, the translocation of p53 protein from cytosol to nuclei could be observed with fluorescence microscopy via FITC-labeled antibody, and it also suggested the increment of p53 protein synthesis. All of these may trigger by the 24p3 protein internalization to elevate the level of ROS in cytosol, which keeps the cell under stress then undergo the apoptosis. Further study is needed to elucidate the complicated mechanism.目錄 中文摘要………………………………………………………………………………I 英文摘要……………………………………………………………………………...II 縮寫表………………………………………………………………………………..III 第一章 緒論…………………………………………………….……………………1 前言……………………………………………………………….…………………..1 第二章 實驗材料與方法……………………………………….……………………7 材料……………………………………………………………….…………………..7 試劑配方……………………………………………………………………………..11 方法…………………………………………………………………………………..12 1. 小白鼠子宮液之製備…………………………………...………………….12 1.1 子宮液之取得………………………………………………………....12 1.2 子宮液之儲存…………………………………………………………12 2. 小白鼠子宮液之24p3蛋白純化…………………………………………..13 2.1 分子篩膠體層析………………………………………………………13 2.2 蛋白質溶液之濃縮……………………………………………………13 2.3 逆向高效液相層析法…………………………………………………13 2.4 蛋白質定量……………………………………………………………14 3. 小白鼠結締組織細胞株細胞培養…………………………………………15 4. MTT測試法…………………………………………………………………15 5. 細胞RNA的萃取…………………………………………………………..16 6. 逆轉錄-聚合酶連鎖反應…………...………………………………………17 7. 洋菜膠體電泳…………………………………………...………………….18 8. 磁力珠免疫沉澱法…………………………………………………………19 9. SDS-聚丙烯醯胺膠體電泳…………………………………………………19 10. 西方墨點法………………………………………………………………..20 11.FITC標定24p3蛋白質……………………………………………………..21 12. 24p3蛋白質免疫染色………………………………...…………………...21 13. 流式細胞儀（FASC）判別細胞死亡…………………………………….22 14. ROS測定………………………………………………… ……………..…23 15. Glutathione的測定…………………………………………………………23 16. 細胞週期之判定…………………………………………………………..24 17. 利用BrdU染色測定的DNA合成………………………………………..24 18. p53蛋白質螢光免疫染色……………………………………………...….25 19. 錐蟲藍染色法……………………………………………………………..25 20. 統計分析 …………………………………………………………………25 第三章 實驗結果…......…………………..……………………….………………...26 實驗結果……………………………………………………………………………..26 第四章 討論…..…………………………………………………...………………...30 討論…………………………………………………………………………………..30 圖……………………………………………………………………………………..35 參考文獻…………………………………………………………………………..…49 附錄…………………………………………………………………………………..541376473 bytesapplication/pdfen-US小白鼠分泌性蛋白24p324p3 proteinotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52817/1/ntu-93-R91242004-1.pdf