【摘要】：雙峰駝具有耐饑渴、耐酷暑嚴寒、耐粗飼料等生物學特點,而且還喜歡采食戈壁、荒漠草原上的鹽堿含量較高的植物和某些有毒植物,且不出現(xiàn)任何不良反應。然而,有關雙峰駝能夠在極端惡劣的環(huán)境下生存和對外源性化學物質(zhì)的生物轉(zhuǎn)化能力方面的研究卻處于空白狀態(tài)。CYP 1A酶作為參與機體內(nèi)源性和外源性物質(zhì)代謝的關鍵酶之一,在維持機體內(nèi)環(huán)境的相對平衡以及與外環(huán)境之間的相互作用上均起著至關重要的作用。為探明雙峰駝CYPlA酶的體外活性及其對外源性藥物代謝的影響,開展了本項研究。首先,采用差速離心法制備雙峰駝肝微粒體,并運用BCA法測定其蛋白濃度；CO還原差示光譜法分別測定CYP總酶含量和細胞色素b5含量；并通過CYP1A酶對7-乙氧基香豆素的脫烴基作用初步評價其體外活性。結(jié)果表明,所制備的雙峰駝肝微粒體懸液蛋白濃度為1.652±0.341mg/g、CYP總酶含量為0.08±0.014nmol/mg、細胞色素b5(Cybts)含量為0.113±0.036nmol/mg,且其中的CYP1A酶具有降解其特異性底物-7-乙氧基香豆素的活性。在此基礎上,為研究CYP1A2酶的體外活性,首先建立了適宜的CYP1A2酶體外孵育體系,并以該酶的特異性底物非那西丁與肝微粒體反應體系共同孵育一定時間,而后用混有內(nèi)標物的冰甲醇終止反應,使用高效液相色譜紫外檢測法(HPLC-UV)測定反應產(chǎn)物-對乙酰氨基酚的含量。流動相為甲醇：水=25：75,流速為0.5mL/min,檢測波長為235nm,柱溫為25℃,進樣量為20μL,等濃度洗脫30min。產(chǎn)物對乙酰氨基酚、內(nèi)標物間乙酰氨基酚、底物非那西丁的保留時間分別為5.046、6.304、23.452min,各峰分離良好。對肝微粒體孵育體系進行優(yōu)化后,得到最適肝微粒體濃度為4.95mg/mL,最適孵育時間為30min,最適底物濃度為200μg/mL。通過Lineweaver-Burk雙倒數(shù)作圖法,計算出最大反應速度Vmax為0.2268±0.0418nmol/min/mg,米氏常數(shù)Km為2.8205 ±0.5198μmol/mL,內(nèi)在代謝清除率CLint為0.0804 ± 0.0231μL·min/mg。最后進行了α-萘黃酮對CYP1A2酶活性的影響實驗。結(jié)果顯示,它對雙峰駝CYP1A2具有較強的、不可逆的競爭性抑制作用,其半抑制濃度IC50為0.5411±0.0405μmol/mL。因此,通過本實驗成功制備出能夠滿足雙峰駝CYP1A酶體外活性研究的肝微粒體懸浮液,建立了測定CYP1A2酶特異性探針藥非那西丁及其代謝產(chǎn)物的HPLC-UV檢測方法,初步闡明了CYP1A酶體外活性及其特異性抑制劑的影響。研究結(jié)果可為全面揭示雙峰駝CYP酶系的體內(nèi)活性奠定基礎,提供科學依據(jù)。
[Abstract]:Bactrian camel has the biological characteristics of hunger and thirst tolerance to heat and cold and forage and so on. It also likes to feed on Gobi desert steppe plants and some poisonous plants without any adverse reactions. However, studies on the ability of Bactrian camels to survive in extremely harsh environments and biotransformation of exogenous chemicals remain blank. CYP1A is one of the key enzymes involved in the metabolism of endogenous and exogenous substances in the body. It plays an important role in maintaining the relative balance of the internal environment and the interaction with the external environment. In order to investigate the in vitro activity of CYPlA enzyme and its effect on exogenous drug metabolism in Bactrian Camel, this study was carried out. Firstly, the liver microsomes of bactrian camels were prepared by differential centrifugation, and the total enzyme content of CYP and the content of cytochrome b _ 5 were measured by BCA method. The dealkylation of 7-ethoxy coumarin by CYP1A enzyme was evaluated in vitro. The results showed that the concentration of hepatic microsomal protein was 1.652 鹵0.341mg / g, the total enzyme content was 0.08 鹵0.014nmol / mg, the content of cytochrome B5 (Cybts) was 0.113 鹵0.036nmol / mg, and the CYP1A enzyme had the activity of degrading its specific substrate -7-ethoxycoumarin. On this basis, in order to study the activity of CYP1A2 enzyme in vitro, a suitable incubation system of CYP1A2 enzyme in vitro was established, and the specific substrate of the enzyme, phenacetin, was incubated with liver microsomal reaction system for a certain time. Then the ice methanol with internal standard was used to terminate the reaction and the content of paracetamol was determined by high performance liquid chromatography ultraviolet detection (HPLC-UV). The mobile phase consisted of methanol: water 25: 75, the flow rate was 0.5 mL / min, the detection wavelength was 235 nm, the column temperature was 25 鈩，

本文編號：2166218