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研究生:溫雅萍
論文名稱:血漿裂解肌肉蛋白-TroponinI影響因子之研究
論文名稱(外文):Study on my ofilament protein:Troponin I degradation in the plasma
指導教授:劉英明
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學院碩士在職專班
學門:生命科學學門
學類:生物學類
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
論文頁數:77
中文關鍵詞:旋轉肌球素-I凝血
外文關鍵詞:Troponin-IThrombin
相關次數:
  • 被引用被引用:1
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中文摘要
肌肉蛋白-Troponin I是調控蛋白(Troponin complex)之次結構蛋白,在肌肉收縮機制上扮演抑制ATP水解的活性,已被廣泛討論其生物性意義,希望能藉此協助診斷肌肉的病理性傷害及運動後的變化。本研究欲探討在肌肉損傷後TnI滲漏至血液中可能發生的連續變化。首先,先從雞隻的大胸肌粹取出TnI,再分別與雞血漿(雌雄各十隻)混合,置於41℃水浴中,經過不同時間點(3、6、24小時),此檢體經SDS-PAGE來分開各蛋白質,隨後再轉漬到PVDF膜上,膜上之sTnI及其裂片則使用一級抗體(小鼠的特異性單株抗體)及二級抗體(標識有鹼性磷酸之抗小鼠的抗體)來辨識。隨後用BCIP-NBT受質液來呈色。在20個檢體中,所有的雄性血漿皆具有將sTnI裂解成10kD裂片之能力;而10隻雌雞的血漿中,則只有4隻具有此裂解的反應。再分別測試不同血漿濃度及水解(Thrombin)處理下,裂解能力之差異,也發現在相同時間隨著血漿濃度提高,sTnI被裂解的程度有增加;而sTnI在Thrombin處理下,會被水解成2個裂片(10及15kD),同時也證實在Troponin complex中,sTnI受到TnT及TnC之保護,可延緩被血漿及酵素裂解。此外,sTnI在血漿中的濃度(量),也會影響被水解的能力。因此,當疾病發生或運動傷害,破壞肌肉細胞,瓦解結構蛋白,造成sTnI滲漏至血中後,我們推測thrombin可用來解釋sTnI在血漿中所發生的裂解反應,且血漿中此裂解反應與性別之間的關係,可能與thrombin及性別影響到血液凝固的活性有關。
英文摘要
Myofilament protein - Troponin I is one of the regulatory subunits (Troponin complex) of striated muscle. It inhibits the activity of ATPase during muscle contraction. The present study has discussed the biological significance of skeletal Troponin I (sTnI). We wish to assist diagnosing pathological damages to muscle or increasing in exercise.
In this study, we were interested in studying the successive events of TnI release into blood after muscle damages. Purified sTnI, prepared from chicken pectoral muscle, was incubated with the serum withdrawn from different lines of chickens. After different time intervals (3, 6, 24hours) of incubation(41℃, water bath), the samples were separated on 1D SDS-PAGE and then transferred to the PVDF membrane. The sTnI and its degrading fragments on the transblotted membrane were recognized by using a mouse specific monoclonal primary antibody against TnI, followed by a second anti-mouse antibody conjugated with alkaline phosphatase, and colorimetric detection by BCIP-NBT substrate reaction. All male chicken blood (ten male birds) degraded sTnI into the fragments at molecular weight of 10~15 kD, while only four serum samples of ten female chickens showed a sTnI degradation. Therefore, we suggest that sTnI degradation in the plasma, perhaps concern with the gender. And the difference from the species doesn’t decide either itself or genetics result from the fewer samples. To explore the possible candidate protease in the serum, a thrombin was used to cleave the sTnI. And then the sTnI was proteolyzed to 2 fragments (10 and 15 kDa) with the thrombin treatment. At the same time, we demonstrate that sTnI is protected from the TnT and TnC in the Troponin complex; therefore, it delayed enzyme digest. In addition, the concentration of sTnI in the plasma will influence the proteolysis.
With the result that sTnI release into the bloodstream by disruption of the muscle cell membrane, disintegrated cytoskeletal structure and contractile protein. It seems that thrombin in serum would answer for the sTnI degradation, and that the gender-dependent sTnI degradation in serum may be related to the sex-effect of the blood clotting activity by thrombin.
目 錄
口試委員會審定………………………………..………………………Ⅰ
博碩士論文授權書…………………………………………..…………Ⅱ
誌謝……………………………………………………………………..Ⅲ
目錄……………………………………………………………………..Ⅳ
圖目錄…………………………………………………..………………Ⅶ
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………3
第一章 緒論……………………………………………………………..5
第二章 文獻查證………………………………………………………..6
第一節 骨骼肌的構造與功能……………………………………...6
第二節 肌肉發生疾病時可選用之標記(markers)……………….10
第三節 Troponin I 分子之探討…………………………………..13
第四節 裂解反應之相關研究…………………………………….16
第五節 血液成分之探討………………………………………….18
第三章 問題與假設……………………………………………………20
第四章 研究材料及方法………………………………………………21
第一節 Troponin I及Troponin complex之純化…………………21
第二節 血樣製備………………………………………………….21
第三節 電泳前處理……………………………………………….25
第四節 鑄膠……………………………………………………….26
第五節 電泳(SDS-PAGE)…………………………………………27
第六節 Transferring the protein…………………………………...29
第七節 西轉漬法(Western-blotting)………………………………30
第五章 研究結果………………………………………………………31
第一節 SDS-PAGE and Western blot of specimen………………..31
第二節 TnI在雞血漿中隨時間變化之裂解情形………………...31
第三節 血漿裂解Troponin complex之探討……………………...34
第四節 sTnI在血漿中可能的裂解機制………………………….35
第六章 討論……………………………………………………………37
第一節 TnI協助診斷之可行性…………………………………...37
第二節 sTnI在不同個體裂解之差異…………………………….39
第三節 sTnI在血漿中裂解的分析……………………………….41
第四節 TnI結構之影響…………………………………………...43
第五節 sTnI濃度影響裂解之討論……………………………….44
第六節 性別與Thrombin activity之關係…………….…………..45
第七節 性別與sTnI及Thrombin 之探討……………………….48
第七章 結論……………………………………………………………49
參考資料………………………………………………………………..66
【附錄】
TnI sequencing…………………………………….……..75
Purification of chicken skeletal Troponin I………………76
圖目錄
<Fig.1> 肌原纖維之細微構造………………………………………48
<Fig.2> Troponin 分子之次結構蛋白……………………………...49
<Fig.3> 心肌之相關研究……………………………………………50
<Fig.4> 純化之Troponin I及Troponin complex……………………51
<Fig.5> 檢體於SDS-PAGE膠上的情形……………………………52
<Fig.6> Western blot membrane (Immunoblotting)…………………54
<Fig.7> Troponin complex study…………………………………….57
<Fig.8> sTnI在血漿中可能的裂解機制之探討……………………59
<Fig.9> 血漿中sTnI濃度不同影響裂解之探討…...………………62
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