小白鼠子宮24p3蛋白與細胞激素之相關性研究

Abstract

24p3 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 與黃體素之調節，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 間期表現量很少，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 內膜細胞株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 細胞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 產生並抑制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 有關。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 以了解24p3 蛋白與子宮組織重整的關係。 以西方點墨法、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 期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 蛋白 (MIP-2、KC 以及GCP-2) 的mRNA 皆於動情期有顯著的表現，24p3 蛋白可 能藉由誘發小鼠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， 參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 素重要來源，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 氮 (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 促使巨噬細胞株凋亡。24p3 蛋白活化巨噬細胞亦會誘導MIP-2 與KC 的表現，對 GCP-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 測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 亦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 內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 於生殖系統中可能的生理功能。

24p3 protein has been implicated in the cell death. Supplementation of 24p3 protein in human endometrial cell line (RL95-2) causes the elevation of intracellular ROS and activation of caspases, resulting in cell apoptosis. In addition, 24p3 protein induced-cell apoptosis could be suppressed by persistent incubation with 24p3 protein via cytokine secretion. It implied 24p3 protein involved in the uterine remodeling during the physiological cycle. In order to demonstrate such hypothesize we attempt to investigate the expression of 24p3 protein, IL-8 and apoptotic index in the uterus throughout the mouse estrous cycle. 24p3 protein secreted in proestrus and estrus abundantly, and then declined from metestrus to diestrus. Using Western analyses, IHC and TUNEL to measure apoptotic index of endometrial cells during the mouse estrous cycle, we found that 24p3 protein expression coincided with the time scale of endometrial cell apoptosis during estrous cycle. It suggests 24p3 protein play a role on the viability of endometrial cells directly. Furthermore, the expression of 24p3 mRNA ,and three murine functional IL-8 homologues (MIP-2, KC,and GCP-2) mRNA in uterus were much higher in estrous stage during the estrous cycle. 24p3 protein may upregulate the expression of these murine cytokines and inhibit 24p3 protein-induced apoptosis. Because of macrophages are the major source of the cytokines in biological system and are abundant in the estrous stage, we have paid more attention on evaluating the influence of 24p3 protein on murine macrophage. Using macrophage cell line (RAW 264.7) , we found that 24p3 protein can trigger the nitric oxide releasing in a time- and dose-dependent manner on the cells via iNOS and TNF-α gene expression. Besides, 24p3 protein can induce macrophage activation by increasing iNOS and TNF-α gene to undergo vi activation-induced cell death. After activation, the gene expressions of MIP-2 and KC but not of GCP-2 are induced by 24p3 protein in macrophages and the results are similar to LPS-activated macrophages. In the meantime, we have established the mouse uterine epithelial primary cell culture in order to extend our goal for elucidating the effect of 24p3 protein on endometrial epithelial cells. This study may provide the information regarding the pathogenesis of uterine disease.