膽結石成份研究及基因突變分析

膽結石在國人的盛行率約20%，是常見的消化道疾病，膽結石依組成成份可分膽固醇結石與血色素結石。本論文主要分成三個部分，首先我們使用微分干涉顯微鏡與傅立葉轉換紅外線光譜儀進行膽結石成份的分析測試，並對臨床檢體加以分類。其次，由於Gpbar1基因剔除的小鼠不易因攝取過量膽固醇造成膽固醇結石的產生，顯示Gpbar1基因功能可能與膽結石發生有關；因此，我們分析了膽固醇結石患者及非膽固醇結石患者共179例檢體的Gpbar1基因密碼區的序列，發現在非膽固醇結石患者中有4例出現一個相同的可能單一核苷酸多型性(SNP)，有2例出現造成胺基酸改變的核酸位點變異，其中一例為肝癌患者；而在膽固醇結石患者中也發現一例造成胺基酸改變的核酸位點變異。有趣的是，在肝癌患者和膽固醇結石患者所發現的變異胺基酸在GPBAR1蛋白質中位於演化高度保留區。另外，膽結石除了膽固醇與血色素之外還包含一些脂肪酸，因為膽汁由肝臟製造，我們想了解肝臟脂肪酸含量與肝內結石的相關性；為此，我利用棕櫚酸和硬脂酸作為標準品建立一套脂肪酸衍生化及高效能液相層析分析程序，用以檢測檢體中脂肪酸的含量。

關鍵字

膽結石； GPBAR1基因；高效能液相層析；脂肪酸衍生化

並列摘要

Gallstone is a common digestive disease. Its prevalence in Taiwan is approximately 20%. Gallstones can be divided into cholesterol stones and hemoglobin stones according to the composition. The thesis consists of three parts. First, we used the differential interference microscope and Fourier transform infrared spectroscopy to analyze the compositions of gallstones, and to classify the clinical gallstone specimens. Second, Gpbar1 gene knockout mice were resistant to cholesterol gallstone formation under excessive intake of cholesterol, indicating Gpbar1 gene function may be associated with the occurrence of gallstones. Therefore, we analyzed the coding sequence of the Gpbar1 gene from a total of 179 cases of cholesterol gallstone patients and non-cholesterol gallstone individuals. We found 4 cases with a same potential single nucleotide polymorphism (SNP), and two cases (one with hepatoma) with nucleotide variants that cause corresponding amino acid change of the Gpbar1 gene in the non-cholesterol gallstone group; on the other hand, a case with an amino acid-changing nucleotide variant was also found in the cholesterol gallstone patients. Interestingly, the altered amino acids found in the cases with hepatoma and cholesterol gallstone are in the highly conserved positions of the GPBAR1 protein. Moreover, the gallstone contains some fatty acids in addition to cholesterol and hemoglobin, as bile is made from the liver, we would like to know the relevance of liver fatty acid level and intrahepatic stone formation. Thus, I utilized the palmitic acid and stearic acid as standards to establish an analysis protocol for fatty acid derivation and high-performance liquid chromatography, for future detection of the fatty acid levels in liver specimens.

並列關鍵字

Gallstones ； GPBAR1 gene ； High performance liquid chromatography ； Fatty acid derivative

參考文獻

Anderson, K. E., E. Kok, et al. (1972). "Bile acid synthesis in man: metabolism of 7 -hydroxycholesterol- 14 C and 26-hydroxycholesterol- 3 H." J Clin Invest 51(1): 112-7.

Bligh, E. G. and W. J. Dyer (1959). "A rapid method of total lipid extraction and purification." Can J Biochem Physiol 37(8): 911-7.

Carey, M. C. (1978). "Critical tables for calculating the cholesterol saturation of native bile." J Lipid Res 19(8): 945-55.

Cirillo, D. J., R. B. Wallace, et al. (2005). "Effect of estrogen therapy on gallbladder disease." JAMA 293(3): 330-9.

Diehl, A. K. (1991). "Epidemiology and natural history of gallstone disease." Gastroenterol Clin North Am 20(1): 1-19.

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膽結石成份研究及基因突變分析

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