血漿中肝球蛋白質表型和幾丁質分解酵素的分析及心血管疾病臨床運用研究

Plasma haptoglobin phenotypes and chitinolytic enzymes study and its clinical application in cardiovascular disease

廖純沂(Chun Yi Liau)

指導教授 ： 林中生

中山醫學大學/醫學院/醫學研究所/博士(2009年)

https://doi.org/10.6834/CSMU.2009.00105

摘要

背景
肝球蛋白質分子 (haptoglobin, Hp) 的表型 (phenotype) 和幾丁質分解酵素 (chitinolytic enzyme) 比活性與人類疾病有無關聯性的流行病學截至目前已知的證據很少。除了與血紅素有強的結合能力外，近年來的研究證實肝球蛋白質分子和促使發炎作用的激素、細胞和蛋白質的相關性，以及它在調節T淋巴球細胞群發展的角色，因而開始了一連串突破性的發現及流行病學相關研究的啟蒙；相對地，剛起步的人類幾丁質分解酵素在研究資料上就顯得單薄許多。但無論分子研究的進展如何，如何有效、快速及省時又省錢的的鑑定及分析技術應該是加速臨床流行病學研究的主要動力。
本研究除了著重於快速鑑定分析人類血漿中的肝球蛋白質表型和快速分析幾丁質分解酵素比活性等技術平台的建立外，更進一步利用已經建立的技術平台分析一般群體 (general population) 的血漿樣本和臨床群體 (clinical population) 的血漿樣本中肝球蛋白質表型和幾丁質分解酵素比活性的表現，透過流行病學尋找此兩種蛋白質和疾病間是否有相關性。
方法
利用新開發的mCBB-R250染色法和西方墨點法進行肝球蛋白質表型鑑定。Hp 1-1、Hp 2-1和Hp 2-2等三種肝球蛋白質表型在收集的血漿樣本中出現頻率百分比 (percentage of frequence) 和比例 (proportion) 則用敘述性統計法、單一樣本t檢定和Chi-square檢定進行分析。
利用乙二醇幾丁聚醣 (glycol chitosan) 做為研究人血漿幾丁質分解酵素反應的基質，建立使用鐵氰化鉀試劑 (K3Fe(CN)6) 溶液做為酵素反應產物呈色反應試劑的活性分析方法，用以標定人血漿中可以分解乙二醇幾丁聚醣的幾丁質分解酵素；利用已經開發的mCBB-G250活性染色方式直接鑑定電泳分離膠上幾丁質分解酵素的活性及分子量。利用常態檢定和獨立樣本母數或無母數檢定等方法進行幾丁質分解酵素比活性值的檢定分析。
結果
利用新開發的mCBB-R250染色法成功地分析了1148個血漿樣本中肝球蛋白質表型，提供臨床上有效且快速分析肝球蛋白質表型的一新方向。1148個血漿樣本中，肝球蛋白質表型為Hp 1-1者佔11.67%；肝球蛋白質表型為Hp 2-1者佔42.60%；肝球蛋白質表型為Hp 2-2者佔45.73%。對照151個罹患心血管疾病的血漿樣本，其肝球蛋白質表型為Hp 1-1者佔11.26%；肝球蛋白質表型為Hp 2-1者佔41.06%；肝球蛋白質表型為Hp 2-2者佔47.68%。經統計檢定分析，罹患心血管疾病患者肝球蛋白質表型的頻率百分比與總樣本的頻率百分比相似。然，151個罹患心血管疾病的血漿樣本中，有101位罹患穩定性心絞痛 (stable angina, CAD) 患者和50位罹患急性冠狀動脈綜合症 (acute coronary syndrome, ACS) 患者，兩者間Hp 2-2表型的頻率百分比的比例經統計檢定發現沒有差異，但是Hp 1-1和Hp 2-1表型的頻率百分比的比例是有明顯差異的，其中CAD患者Hp 2-1表型的頻率百分比較高，ACS患者Hp 1-1表型的頻率百分比較高。
利用乙二醇幾丁聚醣做為基質和使用鐵氰化鉀試劑溶液做為酵素反應呈色試劑的方法，成功地分析899個血漿樣本中幾丁質分解酵素比活性值。經統計檢定分析，899個血漿樣本中幾丁質分解酵素比活性值分佈為一常態分佈。其中，幾丁質分解酵素比活性平均值 (95%信賴) 為92.00 U/mg (90.584，93.416)，對照151個心血管疾病患者的幾丁質分解酵素比活性值，發現CAD患者血漿中幾丁質分解酵素比活性值 (102.724 U/mg) 高於ACS患者 (90.513 U/mg)。另外，利用mCBB-G250活性染色法直接鑑定電泳分離膠片中幾丁質分解酵素活性，在部分分離純化血漿幾丁質分解酵素的過程中，已經先行鑑定一新的幾丁質分解酵素，其分子量約為30 kDa。
結論
本研究已經完全確立有效、快速及省時又省錢的肝球蛋白質表型鑑定法、幾丁質分解酵素比活性分析法和幾丁質分解酵素活性鑑定法的技術平台。發展的技術平台應用於1178個收集的血漿樣本後，已獲得初步臨床數據。後續研究目標將進行相關流行病學的研究設計和分析臨床疾病的血漿樣本，增加檢定的可信度，釐清肝球蛋白質表型和幾丁質分解酵素比活性與疾病間的相關程度。

關鍵字

肝球蛋白質表型 ； 幾丁質分解酵素 ； 心血管疾病

並列摘要

Backgrounds
Little is known about the association between human haptoglobin phenotypes and diseases. Also, not much is known about the association between blood chitinolytic enzyme levels and human diseases. Recent studies have suggested a link between haptoglobin and the molecules associated with inflammation. Subsequent discoveries on its roles in T-cell development and the governing signaling pathways have further enlightened both basic and epidermiological studies. The understanding of human chitinases, on the other hand, is relatively limited. To our knowledge, lacking of rapid and convenient analytical methods has limited the progress on studying both molecules in human.
Our study aimed to develop rapid and accurate biochemical methods in analyzing (1) the haptoglobin phenotypes in human plasma, and (2) the specific activity of chitinolytic enzymes. These analytical methods were then applied to plasma samples from clinical and general populations in investigating the possible associations between human diseases and these traits.
Methods
We have developed a new method, termed mCBB-R250 staining, for rapid and sensitive typing of haptoglobin phenotypes from human plasma. The percentage of frequencies and the proportion of the three haptoglobin phenotypes, Hp 1-1, Hp 2-1, and Hp 2-2, in our collection of human plasma samples were analyzed using descriptive statistic, one sample t test and Chi-square tests.
Using glycol chitosan as substrate, an analysis method using (K3Fe(CN)6) as indicator were developed to determine the chitioson-degrading enzymes in human plasma. An in-gel activity staining method was also developed for the detection and separation of chitinolytic enzymes. The specific activities of chitinolytic enzymes were analyzed using the normality test, independent-sample parametrical test, or non-parametrical test.
Results
We have successfully applied the mCBB-R250 method to determine the Hp phenotypes of 1148 samples. Among these, 11.67% are Hp 1-1, 42.60% are Hp 2-1, and 45.73% are Hp 2-2 type. As a comparison, the distribution of the three Hp phenotypes, as determined for 151 plasma samples from patients with cardiovascular diseases, are 11.26%, 41.06%, and 47.68% respectively for Hp 1-1, 2-1, and 2-2 type. These percentages are similar to that of the general population. Among the 151 samples from cardiovascular disease patients, 101 are stable angina (CAD), and the rest 50 are acute coronary syndrome (ACS) patients. While the percentages of Hp 2-2 type are statistically indifferent between these groups, the percentages of the Hp 1-1 and Hp 2-1 types are significantly different among CAD and ACS groups, in which we found that Hp 2-1 type is higher in CAD, but Hp 1-1 is higher in ACS.
Using glycol chitosan and (K3Fe(CN)6) solution, the specific activities of chitinoltic enzymes, after analyzing 899 human plasma samples, was determined to be a normal distribution, with an average specific activity of 92.00 U/mg (95% CI 90.584，93.416). We have found that, among 151 patients of cardiovascular diseases, the specific chitinolytic enzyme activities in CAD group (102.724 U/mg) are higher than those in the ACS group (90.513 U/mg). We have also developed an in-gel detection method for rapid separation and detection of chitinolytic enzymes. With this new method, we have identified a new chitinolytic enzyme with a molecular weight of about 30 kDa.
Conclusion
We have developed rapid, accurate, and inexpensive methods for haptoglobin phenotyping and chitinolytic enzyme activity staining. The methods were applied to the Hp phenotyping and chitinolytic enzyme activity detection of 1178 plasma samples. With their application in clinical studies, these methods may grant us further understandings on the possible association with human diseases.

並列關鍵字

haptoglobin phenotype ； chitinolytic enzyme ； cardiovascular disease

參考文獻

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