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研究生:朱家正
研究生(外文):Chia-Cheng Chu
論文名稱:牛隻血清中Haptoglobin之純化、分析與基礎值的建立
論文名稱(外文):Purification, Analysis, and BasalLevel of Serum Haptoglobin in Cattle
指導教授:馮翰鵬
指導教授(外文):Hang-Poung Fung
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:Haptoglobin急性期蛋白分析純化基礎值血清
外文關鍵詞:HaptoglobinAcute phase proteincattleassaypurificationbasal levelserum
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為了對牛隻疾病作早期的預警,以利於在疾病爆發之前早日加以預防控制,急性期反應的監控是首要之務。在動物發生感染或炎症反應初期,由單核球或巨噬細胞所產生的一些細胞素IL-1、TNF a和IL-6會刺激肝臟製造數種蛋白質,稱之為急性期蛋白。急性期蛋白的種類繁多,可分為主要蛋白、金屬結合蛋白、凝血因子和補體成分等,在動物體內的防禦系統上扮演多種角色,其中對牛隻疾病反應性最高者為haptoglobin (Hp)。肝臟受到IL-6的刺激後合成並分泌Hp,血液中的Hp會和游離的hemoglobin (Hb)結合,形成Hp-Hb複合體,加速肝臟將游離Hb從循環中移除。除了協助Hb的代謝外,Hp亦具有炎症反應與免疫的調節作用。在文獻中,正常健康牛隻血清中的Hp濃度相當低,甚至測不到。因此,本實驗中欲以自行純化的Hp標準品,配合本實驗所建立之Hp檢測方法,對健康女牛進行採樣調查,建立健康牛隻的基礎值,在往後的研究中則可以此基礎值作為判定的標準。本實驗以炎症反應牛隻的血清為材料,進行50-65%的硫酸銨飽和度劃分,經PBS回溶並透析,以PBS為移動相進行液相層析分子篩管柱HiPrepTM Sephacryl® S-200 HR column (16 mm ×60 cm)的分離,Hp主要在首先出現的高峰中。以此方法純化得Hp純度72.53%,濃縮後可為檢測時之標準品。此外,自行開發Hp的檢測方法,敏感度為1.89 mg/L,分析內與分析間變異係數皆小於5%,顯示精確度穩定,並以市售檢測套組PhaseTM Range Haptoglobin Assay與西方免疫墨點法確認此方法之可行性。然後,以此自行建立的方法檢測10頭6月齡女牛1年的樣品共454件,將所測定的Hp濃度(mg/L)取常用對數值進行資料轉換。樣品轉換後之值的分布以2.1為界限呈現兩個集中的區域,小於2.1的樣品佔所有樣品的93.8% (426/454),較符合常態分布,表示在大部分時間裡血清中Hp基礎而穩定的分泌情形,將基礎值上限定為其mean + 2 ×SD所對應的濃度值73.6 mg/L;而所有健康女牛樣品轉換值之mean + 2 ×SD所對應的濃度值213.4 mg/L則定為臨床表現正常,但可能受到輕微或潛伏性的感染或緊迫而造成Hp濃度升高的上限。女牛血清中Hp之基礎值並不受季節的影響,個體間亦無差異。對不同的臨床狀況進行初步分析發現,分娩後正常的牛隻(n = 11) 血清中的Hp值與健康女牛之間並無顯著差異(P > 0.05),而產後疾病牛隻(n = 15)則顯著高於健康女牛(P < 0.05),顯示血清中的Hp確實有助於臨床上的診斷。未來期望能將急性期蛋白的例行檢測實際運用於獸醫的臨床使用。

目錄
表次 II
圖次 III
中文摘要 IV
英文摘要 VI
第一章、前言 1
第二章、文獻探討 3
第一節、急性期反應與急性期蛋白 3
第二節、Haptoglobin的特性與功能 8
第三節、Haptoglobin的分析方法 11
第四節、Haptoglobin在牛隻臨床上的研究與運用 14
第三章、材料與方法 17
第一節、Haptoglobin的純化 17
第二節、Haptoglobin檢測方法的建立 20
第三節、乳牛血清haptoglobin基礎值的建立 25
第四章、結果 27
第一節、Haptoglobin的純化 27
第二節、Haptoglobin檢測方法的建立 29
第三節、乳牛血清haptoglobin基礎值的建立 38
第五章、討論 48
附錄 54
參考文獻 57

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