臺灣博碩士論文加值系統

本研究透過修飾型環糊精-毛細管微胞電動層析法(cyclodextrin- Micellar electrokinetic chromatography, CD-MEKC )探討人體內極低密度脂蛋白(very low-density lipoprotein, VLDL)中的載體脂蛋白。針對CD-MEKC的條件優化，在分離緩衝溶液中測試了幾種環糊精、pH值，以及膽鹽、磷酸鹽和Reagert B濃度的影響。在儀器分離條件中，也針對樣品進樣時間、分離電壓及毛細管溫度進行探討。本實驗的最佳分離條件為分離緩衝溶液: 20 mM 磷酸鹽(sodium phosphate, PB)、Y5 mM Bile Salts、X6 mM Reagert B、pH Z2、進樣時間為12 秒、分離電壓為 15 kV、毛細管溫度為 15 °C。最佳化的CD-MEKC分離條件顯示出VLDL載體脂蛋白良好的分離效果。
藉由人體VLDL和載體脂蛋白標準品做電泳遷移率比較和共注射實驗，本CD-MEKC方法可在人體VLDL的載體脂蛋白中鑑定出Apo CI、Apo CIII及Apo E。本實驗還使用載體脂蛋白標準品Apo CI、Apo CIII、Apo E進行定量分析，均呈現良好的線性關係，Apo CIII、Apo E相關係數(correlation coefficient, R2)大於0.99，而Apo CI則等於0.989。這三種載體脂蛋白之實驗偵測極限 ( limit of detection, LOD )皆小於0.02 mg/mL，而定量極限(limit of quantitation, LOQ)則皆小於0.06 mg/mL。
使用本實驗最佳化的CD-MEKC方法對尿毒症病人VLDL上的載體脂蛋白進行分析，結果發現尿毒症病人Apo CIII與Apo E含量顯著高於正常人的含量。此新分離方法在未來可能有助於尿毒症的診斷、預防及治療。

A cyclodextrin-micellar electrokinetic chromatography (CD-MEKC) method has been optimized to investigate the apolipoproteins of human very low-density lipoprotein (VLDL). In the separation buffer, the effects of various CDs and pH values were examined. Meanwhile, the concentrations of bile salts, sodium phosphate and Reagert B were also tested. For MEKC separation, the sample injection time, separation voltage and capillary temperature were selected. The optimal CD-MEKC conditions were: separation buffer (20 mM sodium phosphate, Y5 mM Bile Salts, X6 mM Reagert B, pH Z2), injection time (12 s), separation voltage (15 kV) and capillary temperature (15℃). The optimized CD-MEKC separation conditions showed good separation efficiency for VLDL apolipoproteins.
Apolipoproteins CI, CIII and E were identified in human VLDL by the effective mobilities and co-injections of human apolipoprotein standards. Quantitation has also been studied for apolipoprotein standards including CI, CIII and E. Good linear relationships with correlation coefficient (R2) greater than 0.99 were obtained for apos CIII and E, and apo CI had a correlation coefficient of 0.989. For these three apolipoproteins, LOD was lower than 0.02 mg/mL, and LOQ was lower than 0.06 mg/mL.
Furthermore, VLDL apolipoproteins from uremic patients have been studied by the optimal CD-MEKC method. It was found that the levels of apos CIII and E were significantly higher for uremic patients than for healthy subjects. This study might be helpful for diagnostic, prevention and treatment of uremia in the future.

摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 VIII
第一章、緒論 1
第一節、前言 1
第二節、心血管疾病 2
1-2-1、心血管疾病介紹 2
1-2-2、動脈血管的組成與功能 2
1-2-3、動脈粥狀硬化過程 3
1-2-4、脂蛋白介紹 5
1-2-5、脂蛋白的分類 5
1-2-6、脂蛋白代謝途徑 8
1-2-7、載體脂蛋白的介紹 10
1-2-7-1、Apolipoprotein A 11
1-2-7-2、Apolipoprotein C 12
1-2-7-3、Apolipoprotein E 13
1-2-7-4、Apolipoprotein B 14
第三節、尿毒症 14
1-3-1、慢性腎臟病 14
1-3-2、尿毒症介紹 15
1-3-3、尿毒症與心血管疾病 16
1-3-4、高三酸甘油酯血症 16
1-3-5、低密度脂蛋白變異 16
1-3-6、尿毒症患者的極低密度脂蛋白組成異常 17
第四節、環糊精與膽鹽 18
1-4-1、環糊精基本性質 18
1-4-2、環糊精衍生物 19
1-4-3、膽鹽 21
1-4-4、環糊精與膽鹽的包合複合物 22
第五節、毛細管電泳 23
1-5-1、毛細管電泳簡介 23
1-5-2、儀器架構 23
1-5-3、分離原理 24
1-5-3-1、電滲流（electroosmotic flow, EOF） 24
1-5-3-2、電泳流（electrophoretic flow） 26
1-5-4、毛細管電泳的進樣方法 28
1-5-5、毛細管電泳分離模式 28
1-5-5-1、毛細管帶狀電泳 29
1-5-5-2、毛細管凝膠電泳 29
1-5-5-3、毛細管電層析 30
1-5-5-4、毛細管等電聚焦電泳 30
1-5-5-5、微胞電動層析 31
1-5-6、毛細管電泳法之偵測器 31
1-5-7、線上濃縮機制 33
第六節、研究目的 35
第二章、實驗藥品與方法 36
第一節、藥品來源與儀器設備 36
2-1-1、藥品來源 36
2-1-2、儀器設備 38
2-1-3、材料 38
第二節、樣品前處理 39
2-2-1、超高速離心分離人體血漿脂蛋白 39
2-2-2、載體脂蛋白的沉澱 41
第三節、毛細管電泳儀參數設定 42
第三章、結果與討論 43
第一節、毛細管電泳法分離 VLDL 的載體脂蛋白之最佳化條件探討 43
3-1-1、環糊精種類 44
3-1-2、膽鹽濃度的探討 51
3-1-3、Reagent B 濃度的探討 57
3-1-4、pH值的探討 65
3-1-5、分離電壓的探討 69
3-1-6、毛細管溫度的探討 73
3-1-7、磷酸鹽濃度的探討 78
3-1-8、進樣時間探討 83
第二節、載體脂蛋白的定性與定量 88
3-2-1、載體脂蛋白的定性分析 88
3-2-1-1、利用標準品的遷移率進行定性分析 88
3-2-1-2、以標準品共注射進行定性分析 93
3-2-2、載體脂蛋白的定量分析 98
3-2-3、健康和尿毒症病人的 VLDL 101
第四章、結論 111
參考文獻 112

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