臺灣博碩士論文加值系統

血纖維蛋白溶酶系統（plasminogen activator, PA/plasmin system）與基質金屬蛋白酶（matrix metalloproteinases; MMPs）為細胞外基質（extracellular matrix; ECM）與基底膜（basement membrane）裂解之重要蛋白質水解酶，而乳腺退化時組織重建（remodeling）亦需要大量蛋白質水解酶存在。本試驗之目的在探討泌乳期別與乳中PA/plasmin system之關係，並同時比較血漿中及乳中嗜中性白血球PA/plasmin system在不同乳期之變化，以確認乳中PA/plasmin system變化的機制。
山羊乳腺於逐漸退化期（gradual involution; GI期）和穩定退化期（steady involution; SI期），乳中plasmin之活性較泌乳期高，而血液中之plasmin濃度在泌乳期與退化期沒有變化，因此退化期乳中　plasmin活性之變化並非來自全身性因素。由於plasmin是參與乳腺退化時分解細胞外基質最直接之酵素，其在退化期活性的增加顯示對於乳腺之退化及重建的重要。Plasminogen的活性在SI期顯著高於極活躍泌乳期（very active lactation; VL期）、低活性泌乳期（less active lactation; LL期）和GI期，其濃度在乳中變化趨勢與plasmin結果相同，而在血液中乳腺退化期間則無顯著差異存在。因此乳中plasminogen可以作為乳中plasmin的主要前驅物。乳中uPA（urokinase-type PA）活性在退化期逹到最高，但同樣在血液中其活性均無明顯差異。與本試驗同時測得之plasmin 和plasminogen之變化趨勢相符，因此，uPA活化plasminogen形成plasmin可能是造成乳中plasmin增加的重要原因。
SI和GI期之乳清MMP-2和MMP-9的活性顯著比VL和LL期高，血液方面無顯著差異。推論退化期間乳中plasmin及uPA活性增加，相對也會提高MMP-9之活性與細胞表面pro-MMP-2活化。退化期之乳中嗜中性白血球釋出MMP-9的能力顯著比泌乳期強。由於退化期uPA活性上升，活化乳中嗜中性白血球，分泌更多MMP-9。羊乳汁中plasmin分子量約為75 kDa，血中plasmin分子量約為85 kDa。羊乳uPA分子量約為60 kDa。山羊乳中plasmin分子量與牛乳88 kDa型之plasmin有些微差異，但是羊乳中之uPA之型式則與牛乳之型式相符。
利用Western blot分析淘汰不搾乳之羊隻乳腺組織uPA及uPAR（uPA-receptor）之表現，主要表現在細胞膜。顯示uPA的活化可能因結合膜上之uPAR而強化，而有較強之plasminogen活化效率，及促進MMPs之分泌。退化期uPA與uPAR在嗜中性白血球表現均增加。由於uPA可以經由uPAR之路徑增加自己之表現，所以當乳中uPA增多時，乳中嗜中性白血球之uPA與uPAR之表現也因此增加。
綜合本研究之結果證明，由於乳腺退化時期組織uPA與uPAR表現量增加，強化plasminogen轉變為plasmin之能力，同時也提升乳中MMP-9與MMP-2之活性；也因為退化期乳汁中uPA量增加，刺激乳中嗜中性白血球uPA與uPAR量表現增加，更活化嗜中性白血球而釋出更多MMP-9活性，因此PA/plasmin system與MMP二者呈現層層（cascade）互相調控之關係，所以乳腺退化PA/plasmin system與MMP共同參與乳腺之退化過程，其活性之變化成為乳腺退化程度之明顯指標。

Plasminogen activator（PA）/plasmin system and matrix metalloproteins（MMPs）are vital proteases which breakdown extracellular matrix（ECM）and basement membrane. Large amount of proteolysis is required for tissue remodeling during involution of mammary gland. The present study explored the activity of PA/plasmin system at various lactation states. Furthermore their activities in plasma and milk neutrophils were also compared to establish the sources of variation in PA/plamsin activity in milk of goat.
Plasmin activity in milk of goat was significantly higher（P < 0.05）in involution stage（including gradual involution; GI and steady involution; SI）than lactation stage but not for plasma. Therefore, the change in plasmin activity of milk was not systemic. Plasmin seemed directly participate in ECM breakdown of mammary gland during involution, and served a very important role in tissue remodeling. Plasminogen activity in milk of SI stage was higher（P < 0.05） compare to those of very active latation（VL）, less active lactation and GI stage but not in plasma. Plasminogen might be supplied as abundant precursor of plasmin during involution of mammary gland. Urokinase-PA（uPA）activity in milk of goat displayed the similar tendency as plasmin and plasminogen. Since uPA activated plasminogen to plasmin that contributed the increased plasmin activity in mammary gland during involution.
Activities of MMP-2 and MMP-9 in milk of SI and GI were higher（P < 0.05）compare to those of VL and LL, but not in plasma. During involution, the increased activity of plasmin and uPA enhanced the activation of pro-MMP-9 and pro-MMP-2 in milk. Also, the release of MMP-9 from milk neutrophils was higher（P < 0.05） at involution stage compare to lactation stage. It is suggested that the increased activity of uPA during involution further stimulated the released of gelatinase from milk neutrophils. The results of zymography showed that plasmin in milk and plasma were approximately 75 kDa and 85 kDa in molecular weight, respectively. uPA in milk was approximately 60 kDa. The type of plasmin in milk of goats were slightly different from the 88 kDa plasmin of cow milk but the type of uPA in milk of these two species were similar.
Study of the expression of uPA and uPAR（uPA-receptor） in mammary tissue of dried goats using Western blot analysis indicated that both expressed mostly in plasma membrane. uPA bound to uPAR on cell membrane was enhanced in activity towards plasminogen activation and gelatinase release. Expressions of uPA and uPAR on neutrophils from milk were also increased during involution stage. uPA up-regulates its own expression via the uPAR pathway. Therefore, increase uPA in milk elevated, the expression of both uPA and uPAR on milk neutrophils.
The results of the present study suggest that the expression of uPA and uPAR in mammary tissue of involution satge increased to enhance the conversion of plasminogen to plasmin. Simultaneously, MMP-2 and MMP-9 activity in milk were also elevated by uPA. In turn, increase uPA activity in milk during involution stage stimulated uPA and uPAR expression on neutrophils which further released more MMP-9. Therefore, the cascades interplayed by PA/plasmin system and MMPs within mammary gland during involution work together to promote the process of tissue regression. Changes in activity of these proteases are reliable indicators of the extent of mammary involution.

壹、中文摘要 I
貳、前言 3
參、文獻檢討 4
一、乳腺之結構 4
二、泌乳功能維持 4
三、乳腺退化（involution）與細胞凋亡（apoptosis） 6
四、乳汁中之蛋白質酶 10
五、Plasminogen 和plasmin構造及功能性 11
(一)乳汁中plasmin之特色 11
(二) Plasmin inhibitor 14
六、PA和PAI結構及功能性 14
七、uPA-receptor（uPAR）結構及調控 17
八、基質金屬蛋白酶（Matrix Metalloproteinases; MMPs） 21
(一) MMPs之家族成員 22
(二) MMPs之基本結構 23
(三) MMPs活性之調節 24
(四) MMPs於乳腺退化及癌症發展所扮演之角色 27
肆、材料與方法 30
一、試驗動物與飼養管理 30
二、試驗藥品 30
三、嗜中性白血球之分離 33
(一)血液 33
(二)乳汁 33
四、分離乳腺實質細胞之細胞膜與細胞質液 34
五、製備嗜中性白血球萃取液（cell lysate） 34
六、羊乳中plasmin和plasminogen之萃取 35
七、Plasmin zymography 36
八、Plasminogen activator zymography 38
九、Gelatinase zymography 38
十、Plasmin chromogenic Assay 39
十一、Plasminogen chormogenic assay 40
十二、uPA chromogenic assay 40
十三、Western blotting for uPA and uPAR 40
十四、蛋白質定量分析 41
十五、統計分析 42
伍、結果 43
一、山羊泌乳期與退化期之定義及乳中可沈澱酪蛋白在期間之分布 43
二、山羊泌乳期與退化期乳汁和血液中plasmin活性之比較 46
(一)乳汁中plasmin活性 46
(二)血液中plasmin活性之比較 53
三、山羊泌乳期與退化期乳汁和血液中plasminogen活性之比較 58
(一)乳汁中plasminogen活性之比較 58
(二)血液中plasminogen活性之比較 61
四、山羊泌乳期與退化期乳汁和血液中uPA活性之比較 65
(一)乳汁中uPA活性之比較 65
(二)血液中uPA活性之比較 72
五、泌乳期與退化期乳汁和血液中gelatinase活性之比較 76
(一)乳汁中gelatinase活性之比較 76
(二)血液中gelatinase活性之比較 80
六、泌乳期與退化期乳汁中嗜中性白血球gelatinase活性之比較 83
七、利用Western blot 偵測乳腺組織、嗜中性白血球uPAR和uPA表現及
確立乳中之uPA表現 83
(一)退化期乳腺組織uPAR和uPA之表現及與其化組織之比較 86
(二)不同乳期乳中嗜中性白血球uPAR和uPA之表現 86
(三)乳中uPA之表型確認 87
陸、討論 92
柒、結論 101
捌、參考文獻 103
玖、英文摘要 121

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