朱善德臺灣大學:生化科學研究所洪蓴仁Hung, Chuan-JenChuan-JenHung2010-05-042018-07-062010-05-042018-07-062008U0001-2907200821231600http://ntur.lib.ntu.edu.tw//handle/246246/17885424p3 蛋白是小鼠子宮液中之分泌性蛋白，研究顯示24p3 蛋白的表現受到動情素黃體素之調節，主要表現於動情前期與動情期的子宮組織，於動情後期與動情期表現量很少，暗示24p3 蛋白於雌性生殖系統扮演重要的角色。利用人類子宮膜細胞株RL95-2 探討24p3 蛋白於子宮的生理功能，24p3 蛋白會造成子宮內膜胞caspase-3 活性增加並伴隨細胞凋亡，長時間處理下24p3 蛋白會誘發IL-8 的生並抑制caspase-3 活性而促使細胞存活，此現象可能與動情週期子宮組織重整關。因此本論文利用小鼠動情週期探討24p3 蛋白與IL-8 於子宮組織的相關表現了解24p3 蛋白與子宮組織重整的關係。西方點墨法、免疫組織化學染色法以及原位末端標記法確認動情期與動情後子宮組織有細胞凋亡的現象。於動情週期中24p3 mRNA 與三個IL-8 功能性同源白 (MIP-2、KC 以及GCP-2) 的mRNA 皆於動情期有顯著的表現，24p3 蛋白可藉由誘發小鼠IL-8 功能性同源蛋白而抑制24p3 蛋白引發的子宮內膜細胞凋亡，amp;#63851;與子宮組織重整的現象。由於巨噬細胞大量存在於動情期子宮組織並為細胞激重要來源，實驗中發現24p3 蛋白可活化小鼠巨噬細胞株RAW 264.7 促進一氧化 (NO) 的釋放，此一活化現象可能透過iNOS 以及TNF-α 基因表現量上升進而使巨噬細胞株凋亡。24p3 蛋白活化巨噬細胞亦會誘導MIP-2 與KC 的表現，對CP-2 表現則無影響，此結果與LPS 活化巨噬細胞的結果相同。根據實驗數據推24p3 蛋白可能藉由ERK 與JNK 來影響巨噬細胞相關基因之表現。同時本論文初步建立小鼠子宮內膜上皮細胞初級培養系統，以利研究24p3 蛋白與小鼠子宮膜細胞凋亡以及相關細胞激素分泌之關係，期望提供較多資訊以瞭解24p3 蛋白生殖系統中可能的生理功能。24p3 protein has been implicated in the cell death. Supplementation of 24p3 proteinn human endometrial cell line (RL95-2) causes the elevation of intracellular ROS andctivation of caspases, resulting in cell apoptosis. In addition, 24p3 protein induced-cellpoptosis could be suppressed by persistent incubation with 24p3 protein via cytokineecretion. It implied 24p3 protein involved in the uterine remodeling during thehysiological cycle. In order to demonstrate such hypothesize we attempt to investigatehe expression of 24p3 protein, IL-8 and apoptotic index in the uterus throughout theouse estrous cycle.4p3 protein secreted in proestrus and estrus abundantly, and then declined frometestrus to diestrus. Using Western analyses, IHC and TUNEL to measure apoptoticndex of endometrial cells during the mouse estrous cycle, we found that 24p3 proteinxpression coincided with the time scale of endometrial cell apoptosis during estrousycle. It suggests 24p3 protein play a role on the viability of endometrial cells directly.urthermore, the expression of 24p3 mRNA ,and three murine functional IL-8omologues (MIP-2, KC,and GCP-2) mRNA in uterus were much higher in estroustage during the estrous cycle. 24p3 protein may upregulate the expression of theseurine cytokines and inhibit 24p3 protein-induced apoptosis. Because of macrophagesre the major source of the cytokines in biological system and are abundant in thestrous stage, we have paid more attention on evaluating the influence of 24p3 proteinn murine macrophage. Using macrophage cell line (RAW 264.7) , we found that 24p3rotein can trigger the nitric oxide releasing in a time- and dose-dependent manner onhe cells via iNOS and TNF-α gene expression. Besides, 24p3 protein can induceacrophage activation by increasing iNOS and TNF-α gene to undergoictivation-induced cell death. After activation, the gene expressions of MIP-2 and KCut not of GCP-2 are induced by 24p3 protein in macrophages and the results areimilar to LPS-activated macrophages. In the meantime, we have established the mouseterine epithelial primary cell culture in order to extend our goal for elucidating theffect of 24p3 protein on endometrial epithelial cells. This study may provide thenformation regarding the pathogenesis of uterine disease.目錄 .................................................................................................................................. i文摘要 ......................................................................................................................... ivBSTRACT ........................................................................................................................ v寫表 ............................................................................................................................ vii一章 緒論 .................................................................................................................... 1、疏水性分子結合蛋白家族 .................................................................................. 1、24p3 蛋白 ............................................................................................................. 2、小鼠動情週期 ...................................................................................................... 7、子宮細胞激素 ...................................................................................................... 8、巨噬細胞在子宮扮演的角色 ............................................................................ 10amp;#63953;、巨噬細胞的活化 ................................................................................................ 12、本論文的研究目的 ............................................................................................ 13二章 材料與方法 ...................................................................................................... 16ART 1 小鼠動物實驗 ............................................................................................... 16、小鼠子宮液中24p3 蛋白純化 ...................................................................... 16.1 子宮液的誘發與存取 .................................................................................. 16.2 分子篩膠體管柱層析 .................................................................................. 16.3 蛋白質溶液的濃縮 ...................................................................................... 17.4 逆向高效能液相層析 .................................................................................. 17、小鼠動情週期各時期的判定 ........................................................................ 19.1 動情週期同步化 .......................................................................................... 19.2 動情週期的判定 .......................................................................................... 19、子宮組織 RNA 的分析 ................................................................................. 20.1 組織總核醣核酸的萃取 .............................................................................. 20.2 反轉錄反應 .................................................................................................. 21.3 聚合酶連鎖反應 .......................................................................................... 22.4 洋菜膠體電泳 .............................................................................................. 24、子宮組織蛋白的萃取 .................................................................................... 24、BCA 蛋白質定量 ........................................................................................... 25amp;#63953;、十二酯硫酸鈉-聚丙烯醯胺膠體電泳 ........................................................... 26、西方點墨法 .................................................................................................... 27、免疫組織化學染色 ........................................................................................ 29.1 組織包埋與切片製作 ................................................................................. 29i.2 免疫組織化學染色 ...................................................................................... 31、原位末端雜交法 ............................................................................................ 33、三明治免疫法染細胞激素抗體陣列膜 ........................................................ 34一、小鼠子宮內膜上皮細胞初級培養 ............................................................ 35二、免疫螢光染色 ............................................................................................ 36ART 2 小鼠巨噬細胞株實驗 ................................................................................... 37三、小鼠巨噬細胞株RAW 264.7 (ATCC TIB-71) 細胞培養 ........................ 373.1 細胞培養 .................................................................................................... 373.2 細胞冷凍 .................................................................................................... 383.3 細胞解凍 .................................................................................................... 39四、細胞RNA 的萃取 ...................................................................................... 39五、即時定量聚合酶連鎖反應 ........................................................................ 39六、一氧化氮的測量 ........................................................................................ 40七、 細胞蛋白質的萃取 .................................................................................. 42八、Caspase-3 活性偵測 (PhiPhiLux/ CaspaLux kit) ..................................... 43三章 結果 .................................................................................................................. 44、小鼠動情週期的判定 ........................................................................................ 44、小鼠動情週期子宮內膜細胞的凋亡 ................................................................ 44、子宮組織中相關基因於動情週期表現之變化 ................................................ 46、表現於小鼠動情期子宮液的細胞激素 ............................................................ 47、小鼠巨噬細胞株RAW 264.7 具有24p3 蛋白受體 ......................................... 48amp;#63953;、24p3 蛋白活化小鼠巨噬細胞株RAW 264.7 ................................................... 49amp;#63953;、24p3 蛋白引發小鼠巨噬細胞株RAW 264.7 細胞凋亡現象 .......................... 51、24p3 蛋白誘導小鼠巨噬細胞株RAW 264.7 細胞激素的分泌 ...................... 51、24p3 蛋白影響小鼠巨噬細胞MAPK 磷酸化 .................................................. 52、小鼠子宮內膜上皮細胞初級培養系統的建立 ................................................ 53次 ............................................................................................................................... 54一、小鼠24p3 蛋白的純化 .................................................................................. 55二、小鼠動情週期不同時期的陰道抹片 ............................................................ 57三、活化態caspase-3 在小鼠動情週期各時期子宮組織蛋白的表現 ............... 59四、免疫組織化學染色法偵測活化態caspase-3 在子宮組織的表現位置 ....... 61五、TUNEL 法偵測子宮組織凋亡細胞 .............................................................. 63六、分析小鼠子宮組織24p3、24p3 蛋白受體、MIP-2、KC、GCP-2 、IL-6 以MCP-1 mRNA 於動情週期不同時期的變化 .......................................... 65七、以細胞激素抗體陣列膜偵測動情期子宮液的細胞激素 ............................ 67九、以RT-PCR 確認小鼠巨噬細胞株RAW 264.7 具有24p3 蛋白受體基因 . 71ii十、24p3 蛋白以及LPS 對小鼠巨噬細胞株產生一氧化氮的影響 .................. 73十一、24p3 蛋白以及LPS 處理小鼠巨噬細胞株對iNOS mRNA 表現量的影響.................................................................................................................................. 75十二、24p3 蛋白與LPS 處理小鼠巨噬細胞株對TNF-α mRNA 表現量的影響.................................................................................................................................. 77十三、24p3 蛋白處理誘發小鼠巨噬細胞株細胞凋亡的現象 ........................... 79十四、24p3 蛋白與LPS 處理小鼠巨噬細胞株對MIP-2、KC 與GCP-2 mRNA現量的影響 ............................................................................................ 81十五、24p3 蛋白對小鼠巨噬細胞株MAPK 活性於十二小時內之影響 .......... 83十六、小鼠子宮內膜上皮細胞初級培養之純度鑑定 ........................................ 85四章 討論 .................................................................................................................. 86五章 參考文獻 .......................................................................................................... 93application/pdf3843627 bytesapplication/pdfen-US24p3蛋白細胞激素巨噬細胞24p3 proteincytokinesmacrophage[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178854/1/ntu-97-R95b46023-1.pdf