| Summary: | 碩士 === 國立中興大學 === 畜產學系所 === 94 === 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.
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