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標題: 乳牛多形核嗜中性白血球上TNF-α自泌調控血纖維酶原活化系統對乳腺組織重組之探討
Bovine PMN-associated TNF-α autocrine regulation of plasminogen activation system in mammary tissue remodeling
作者: 周文科
Chou, Wen-Ko
關鍵字: involution;乳腺退化;dry period;PMN;casein;PA system;plasmin;uPA;TNF-α;autocrine;somatic cell;乾乳期;酪蛋白;PA系統;PMN;plasmin;uPA;TNF-α;自泌作用;體細胞
出版社: 畜產學系所
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本試驗的目的在測定乾乳時乳腺分泌物之蛋白質水解與體細胞上plasminogen activation system (PA system) 的活化程度。同時應用試管中polymorphonuclear neutrophil (PMN) 為細胞模型,探討乾乳分泌物調控PA系統的生物活性及tumor necrosis factor-α (TNF-α) 可能角色。本試驗每週一次收集停止泌乳後一至三週之乳腺分泌物,分別利用100,000 x g 沉澱酪蛋白 (casein, CN),或是1700 x g製備體細胞以及乳清。結果發現casein比例隨乾乳期間而逐漸下降,總蛋白量則隨乾乳時間而漸增。體細胞數以及neutrophil/macrophage ratio (N/M ratio) 會在第一週乾乳顯著的上升 (P < 0.05)。乳清中urokinase-type plasminogen activator (uPA) 以及plasmin在乾乳時的活性,發現在乾乳第一週會比第二以及第三週有最顯著的plasmin活性上升 (P < 0.05),uPA在乾乳期間則無顯著的增加。體細胞中uPA以及TNF-α的表現,發現uPA基因會隨乾乳進行顯著降低在體細胞的表現,TNF-α基因則會在第一週乾乳時表現最顯著 (P < 0.05),第二以及第三週下降。分離血液中PMN作為細胞模型,觀察添加第一週乾乳分泌物處理對PA系統的影響,並添加TNF-α抗體觀察TNF-α在PA系統活化上的角色。試驗發現,乾乳分泌物並不能很顯著的增加游離態uPA以及plasmin在的活性,但是添加TNF-α抗體後可以顯著的抑制PMN釋放plasmin以及uPA。乾乳分泌物對PMN TNF-α以及uPA基因表現的影響發現,乾乳分泌物顯著的增加了兩者的表現,加入TNF-α抗體後PMN TNF-α以及uPA基因的表現會部分的下降。從uPA以及plasmin在乾乳乳腺分泌物中活性的測量結果可以推測, plasmin會在乾乳期間上升活性,可能藉此參與乳線的退化。乾乳時uPA活性並不會產生改變,因此plasmin活性的上升可能是因為乾乳時tight junction滲透性改變,血液中PMN 以及plasminogen轉移到乳腔中被活化,而導致plasmin活性的上升,進而參與extracellular matrix (ECM)重組以及酪蛋白的水解。從in vitro試驗結果可以推測乳腺乾乳分泌物可以刺激PMN TNF-α以及uPA重新合成,而TNF-α的路徑也會部分的參與其中。添加TNF-α抗體後可以顯著的抑制PMN釋放plasmin以及uPA,顯示在PMN PA系統活化時內生性的TNF-α會以自泌的方式參與,進而影響PA系統的活性。綜合以上的結果可以得到以下結論,停止泌乳後乳腺分泌物內有大量的酪蛋白水解以及plasminogen活化產生。而早期乾乳分泌物中分離出來的體細胞可以發現TNF-α以及uPA基因顯著的表現,可以推測乾乳牛乳腺內體細胞所分泌的TNF-α會以自泌作用的方式來調節本身的PA系統的活性。進一步藉由血液中分離出的PMN為模型發現,PMN基礎釋出uPA以及plasmin的能力部分必須依靠自泌方式作用的TNF-α途徑才能維持。添加乾乳分泌物可以部分的經由TNF-α自泌作用的途徑稍微的使PMN釋出的uPA以及plasmin活性增加,同添加乾乳分泌物後發現PMN TNF-α會藉由重新合成,並以自泌的方式提升自身uPA的活性,因而增加釋出plasmin的能力,使PMN在乳腺組織重組的能力上升。

The research objective of this thesis was to investigate the extent of proteolysis of mammary secretion as well as the activation of plasminogen activation (PA) system on somatic cells in dried-off cows. An in vitro model using polymorphonuclear neutrophil (PMN) isolated from peripheral blood was used to explore the bioactivity of dry-cow mammary secretion in PA system activation and the involvement of TNF-α. Dry secretion was collected at weeks 0 to 3 (wk0-wk3) posterior to milk stasis. Precipitation with 100,000 x g was used for casein determination while 1700 x g was used for separation somatic cells and milk serum. Results showed that the ratio of casein to total protein decreased with the advancement of dry period while microscopc somatic cell count (MSCC) and neutrophil/macrophage ratio (M/N) increased significant (P<0.05) since wk1. On the other hand, plasmin activity in milk serum of dry mammary secretion was significant (P<0.05) higher at wk1 compared with wk0 while the activity of urokinase plasminogen activator (uPA) did not show difference thoughtout wk0-wk3. The expression of uPA on somatic cells decreased along with the process of drying-off while the expression of TNF-α was significant increased (P<0.05) at wk1 then drcreased at wk 2 and wk3. In vitro study using blood PMN and antiTNFα antibody indicated that although dry mammary secretion did not significantly stimulate PMN-associated activities of uPA and plasmin, antiTNFα significantly (P<0.05) reduced these PMN-associated activities. Results of in vitro study also indicated that dry mammary secretion significant (P<0.05) induced TNF-α and uPA expressions on PMN which were partially blocked by antiTNFα antibody. Since plasmin activity, but not uPA activity, in dry secretion elevated in parallel with the progress of dry period, suggesting that plasmin might participate in mammary gland involution and its elevation was likely due to plasminogen migration from circulation to mammary gland through tight junction. In vitro study demonstreated that dry mammary secretion of cow contains factors responsible to the induction of neosyntheses of TNF-α and uPA in PMN. The finding that antiTNFα significantly decreased PMN-associated uPA and plasmin activity suggests the involvement of autocrine loop of PMN endogenous TNF-α. Combining of the in vivo and in vitro results we conclude that extensive caseinolysis and activation of plasminogen were observed in mammary gland secretion after stasis. Expressions of TNF-α and uPA on somatic cells were greater at earlier stage post milk stasis suggesting a role of somatic cell-associated TNF-α in autocrine regulation of plasmin system in mammary gland of dry cows. The constitutive plasmin and uPA activities of PMN were partially dependent on an autocrine TNF-α pathway. Early dry secretion of cows slightly increased PMN-associated plasmin activity and significantly stimulated uPA releasing by PMN partially via an autocrine TNF-α pathway. Early dry secretion of cow significantly up-regulated expressions of uPA and TNF-α on PMN via an autocrine TNF-α pathway and contribute to mammary tissue remodeling.
其他識別: U0005-1208200623023900
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