本文選題：膽固醇7α-羥化酶 + HPLC　； 參考：《山東大學》2010年碩士論文

【摘要】： 背景：膽汁酸是在肝細胞內由游離膽固醇生成的,第一步及限速反應是膽固醇在膽固醇7a-羥化酶的作用下進行的7a-羥化作用。膽固醇7a-羥化酶(CYP7A1)位于微粒體,屬細胞色素P450酶系。據(jù)報道,它的活性受膽汁酸池體積、組成和疏水性,膽汁酸的腸肝循環(huán),激素、腸道因子以及各種調節(jié)CYP7A1轉錄的核受體的表達的負反饋作用所影響。 測定膽固醇7a-羥化酶活性的方法主要有三種：(1)用薄層色譜技術,采用同位素結合法將放射性標記的膽固醇與7a-羥膽固醇分離；(2)GLC-MS法；(3)用HLPC-分光光度法。 用高效液相色譜法(HPLC)測定CYP7A1酶活性的基本原理是在肝微粒體的催化下,內源性膽固醇作為膽固醇7a-羥化酶的底物,適合的反應溫度、時間等因素協(xié)同作用下,生成7a-羥膽固醇；我們通過加入膽固醇氧化酶后將7α-羥膽固醇轉化為7α-羥-4-膽甾烯-3-酮(HCO),然后用HPLC對7a-羥膽固醇進行定量。以HCO產(chǎn)物量的多少來判斷該CYP7A1活性的大小。本研究對前有的研究方法進行了改進,其一是采用了7β-羥膽固醇作為內標,其二是采用反相HPLC法對酶反應產(chǎn)物進行分離與定量。 目的：對大鼠肝微粒體中膽固醇代謝酶(CYP7A1)活性測定方法的建立。在本研究中,我們采用反相液相色譜法(RP-HPLC)來測定膽固醇7a-羥化酶的活性。 方法：采用XDB-C18 (4.6×250mm,5μm)色譜柱；柱溫為室溫；以乙腈-甲醇(95：5)為流動相,流速：0.8mL/min;檢測器為可變波長掃描紫外檢測器(VWD),VWD=240nm。以7β-羥膽固醇為內標與大鼠的肝微粒體進行體外孵育,以反相液相色譜法測定膽固醇的反應產(chǎn)物7α-羥-4-膽甾烯-3-酮(HCO)。 結果： 1.以待測物濃度為橫坐標,待測物與內標物的峰面積比值為縱坐標,求得線性回歸方程,結果為：Y=1.31362786X+0.0268326, R2=0.99603。根據(jù)標準曲線,7a-羥膽固醇的線性范圍為10-500ng/ml。當信噪比(S／N)為2.3時,方法最低檢測限lOng/ml,回收率及精密度均符合檢測要求。 2.在所建立的RP-HPLC條件下,膽固醇的產(chǎn)物(HCO)保留時間約為13.6min。雜質峰不干擾測定。 3.大鼠肝微粒體膽固醇7a-羥化酶的活性：1.214±1.153 nmol/h/mg protein。 結論：該HPLC能夠準確的檢測出在大鼠肝微粒體中的膽固醇7a-羥化酶的活性。
[Abstract]:Background: bile acids are produced by free cholesterol in hepatocytes. The first step and rate-limiting reaction is the 7a-hydroxylation of cholesterol by cholesterol 7a-hydroxylase. Cholesterol 7a- hydroxylase CYP7A1) is located in microsomes and belongs to cytochrome P450 enzyme system. Its activity is reported to be affected by the volume, composition and hydrophobicity of bile acid pools, the intestinal and hepatic circulation of bile acids, hormones, intestinal factors and the negative feedback effects of various nuclear receptors regulating CYP7A1 transcription. Determination of cholesterol 7a-hydroxylase activity by TLC, radioisotope binding method was used to separate radioactive cholesterol from 7a-hydroxyl cholesterol by GLC-MS) HLPC-spectrophotometry was used to determine the activity of cholesterol 7a-hydroxylase. The basic principle of determining the activity of CYP7A1 by HPLC was that under the catalysis of liver microsomes, endogenous cholesterol was used as substrate of cholesterol 7a- hydroxylase, suitable reaction temperature, time and other factors to produce 7a-hydroxycholesterol. After the addition of cholesterol oxidase, 7 偽 -hydroxycholesterol was transformed into 7 偽 -hydroxy-4-cholestene -3-one HCO, and then 7a-hydroxycholesterol was quantified by HPLC. The activity of HCO was judged by the amount of the product. In this study, the former research methods were improved, one was to use 7 尾 -hydroxyl cholesterol as internal standard, the other was to separate and quantify the enzyme reaction products by reverse phase HPLC. Aim: to establish a method for the determination of cholesterol metabolism enzyme CYP7A1 in rat liver microsomes. In this study, RP-HPLC was used to determine the activity of cholesterol 7a-hydroxylase. Methods: a XDB-C18 column with a column temperature of 4.6 脳 250 mm ~ (5 渭 m) was used at room temperature, acetonitrile-methanol 95: 5) as mobile phase with a flow rate of 1: 0.8 mL / min and a variable wavelength scanning ultraviolet detector (VWDX) at 240 nm. Rat liver microsomes were incubated with 7 尾 -hydroxycholesterol as internal standard in vitro. The reaction product 7 偽 -hydroxy-4-cholesten-3-one was determined by reversed-phase liquid chromatography (RP-HPLC). Results: 1. The linear regression equation was obtained by using the concentration of the object under test as the horizontal coordinate and the ratio of the peak area of the object to the internal standard as the vertical coordinate. The results were as follows: 1. 31362786X 0.0268326, R2 + 0.99603. According to the standard curve, the linear range of 7 a-hydroxycholesterol is 10-500 ng / ml. When S / N = 2.3, the minimum detection limit of the method is lOng / ml, and the recovery rate and precision are in line with the detection requirements. 2. Under the conditions of RP-HPLC, the retention time of cholesterol product was about 13.6 mins. Impurity peaks do not interfere with the determination. 3. The activity of rat liver microsomal cholesterol 7a- hydroxylase: 1. 214 鹵1.153 nmol/h/mg protein. Conclusion: the HPLC can accurately detect the activity of cholesterol 7a- hydroxylase in rat liver microsomes.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R341

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