發(fā)布時(shí)間：2018-04-23 17:53

  本文選題：免疫性肝損傷 + 脂多糖��； 參考：《華中科技大學(xué)》2013年碩士論文

【摘要】：免疫性肝損傷與自身機(jī)體免疫應(yīng)答有關(guān)。由卡介苗(BCG)+脂多糖(LPS)誘導(dǎo)的急性免疫性肝損傷模型更能模擬由病毒或內(nèi)毒素引發(fā)的急性肝炎模型。在肝臟疾病狀態(tài)下，藥物代謝酶的活性可能會(huì)發(fā)生改變，，從而會(huì)對(duì)藥物的藥物代謝動(dòng)力學(xué)及藥物療效產(chǎn)生影響。 因此，本研究首先采用BCG+LPS法建立免疫性肝損傷大鼠模型，將SD大鼠隨機(jī)分為3組，低劑量LPS組，高劑量LPS組和正常對(duì)照組，先腹腔注射BCG (8mg)，連續(xù)7d后，分別腹腔注射給予三組大鼠5mg·kg~(-1)LPS,10mg·kg~(-1)LPS和等體積的生理鹽水。12h后，檢測(cè)血清中谷丙轉(zhuǎn)氨酶（ALT）和谷草轉(zhuǎn)氨酶（AST）含量變化，將肝臟經(jīng)HE染色，在光鏡下觀察病理學(xué)變化。結(jié)果發(fā)現(xiàn)，與正常對(duì)照組相比，低劑量LPS組和高劑量LPS組大鼠血清ALT和AST活性顯著升高（P 0.05），且隨劑量的增加而升高；普通光鏡下觀察肝臟病理學(xué)變化，LPS組的大鼠肝臟出現(xiàn)炎癥細(xì)胞浸潤(rùn)和肝細(xì)胞壞死，且損傷程度隨LPS劑量的升高而加重。采用BCG+LPS（5mg·kg~(-1)）可以成功的建立免疫性肝損傷大鼠模型。 在模型建立成功的基礎(chǔ)上，我們考察了CES在免疫性肝損傷大鼠體內(nèi)的代謝活性，選取咪達(dá)普利和伊立替康(CPT-11)分別作為CES兩種同工酶CES1和CES2的特異性底物。我們首先建立了LC-MS/MS法和HPLC法測(cè)定血漿中咪達(dá)普利的活性代謝產(chǎn)物咪達(dá)普利拉和CPT-11的活性代謝產(chǎn)物SN-38的濃度，兩種方法均簡(jiǎn)便，快捷，專(zhuān)屬性好，在線性范圍內(nèi)靈敏，準(zhǔn)確，精密度高，為研究藥物體內(nèi)藥動(dòng)學(xué)打下了基礎(chǔ)。接著我們考察了咪達(dá)普利和CPT-11在免疫性肝損傷大鼠體內(nèi)的藥動(dòng)學(xué)特征。給模型組大鼠分別灌胃給予咪達(dá)普利(10mg·kg~(-1))和尾靜脈注射CPT-11(20mg·kg~(-1))，正常對(duì)照組大鼠給予等體積生理鹽水，于給藥后不同時(shí)間點(diǎn)采血，測(cè)定血漿中咪達(dá)普利拉和SN-38的濃度。結(jié)果顯示與正常對(duì)照組大鼠相比，模型組大鼠體內(nèi)咪達(dá)普利拉的AUC0-12h從1759.93±386.57mg·h-1·L-1降至620.93±197.16mg·h-1·L-1， Cmax從234.66±68.85mg·L-1下降至113.1±19.69mg·L-1(P0.05)；SN-38的AUC0-24h從646.1±110.9ng·h·mL-1降至275.9±52.5ng·h·mL-1，Cmax從145.6±15.9ng·mL-1下降至57.8±9.5ng·mL-1(P0.05)。以上結(jié)果表明咪達(dá)普利和CPT-11在免疫性肝損傷大鼠體內(nèi)的代謝均受到了抑制，藥動(dòng)學(xué)特征發(fā)生了改變。 最后，為進(jìn)一步考察CES1和CES2在免疫性肝損傷大鼠體內(nèi)表達(dá)特征的變化，我們采用熒光定量PCR (RT-PCR)和Western blot技術(shù)對(duì)肝臟中CES1和CES2的mRNA和蛋白表達(dá)水平進(jìn)行分析。RT-PCR結(jié)果顯示，CES1mRNA及CES2的兩種主要同工酶AB010635和AY034877的mRNA含量均比正常對(duì)照組含量低(P0.05)。Western blot結(jié)果顯示，CES1和CES2的蛋白表達(dá)水平均下降(P0.05)。因此，本研究發(fā)現(xiàn)免疫性肝損傷狀態(tài)下，CES1和CES2的表達(dá)和代謝活性顯著降低。本研究成果對(duì)臨床免疫性肝損傷患者服用經(jīng)CES代謝的藥物劑量調(diào)整具有一定的參考意義。
[Abstract]:Immune liver injury is associated with autoimmune response. The model of acute immune liver injury induced by BCG lipopolysaccharide (LPS) can simulate the acute hepatitis induced by virus or endotoxin. Under the condition of liver disease, the activity of drug metabolizing enzymes may change, which will affect the pharmacokinetics and efficacy of drugs. In this study, the immunological liver injury model was established by BCG LPS method. SD rats were randomly divided into three groups: low dose LPS group, high dose LPS group and normal control group. The rats were injected intraperitoneally with BCG 8 mg / g for 7 days. Three groups of rats were injected intraperitoneally with 10 mg kg~(-1)LPS and 10 mg kg~(-1)LPS of 5mg, and normal saline for 12 h. The changes of serum alanine aminotransferase (alt) and aspartate aminotransferase (AST) levels were detected. The liver was stained with HE and the pathological changes were observed under light microscope. The results showed that the activities of serum ALT and AST in the low dose LPS group and the high dose LPS group were significantly higher than those in the normal control group (P 0.05), and increased with the increase of the dose. The pathological changes of lipopolysaccharide group were observed under light microscope. Inflammatory cell infiltration and hepatocyte necrosis occurred in lipopolysaccharide group, and the degree of injury increased with the increase of LPS dose. The immunological liver injury model could be established successfully by BCG LPS(5mg. On the basis of successful establishment of the model, we investigated the metabolic activity of CES in immunological liver injury rats, and selected midazapril and Ilitecan CPT-11 as specific substrates of CES isozyme CES1 and CES2, respectively. We first established a LC-MS/MS and HPLC method for the determination of the concentrations of the active metabolites of midazapril and CPT-11 in plasma. The two methods were simple, rapid and specific, and were sensitive and accurate in the linear range. The high precision laid the foundation for the study of pharmacokinetics in vivo. Then we investigated the pharmacokinetic characteristics of imidapril and CPT-11 in immune liver injury rats. Rats in the model group were given intragastric administration of imidapril (10 mg / kg) and caudal vein injection of CPT-11(20mg (KGG) and normal control group (control group). Blood samples were collected at different time points after administration, and the concentrations of midapriline and SN-38 in plasma were determined. The results showed that compared with the normal control group, the AUC0-12h of the model group was decreased from 1759.93 鹵386.57mg h -1 to 620.93 鹵197.16mg h -1 L -1, and the AUC0-24h of Cmax from 234.66 鹵68.85mg L -1 to 113.1 鹵19.69mg L -1 P0.05N 38 decreased from 646.1 鹵110.9ng h mL-1 to 275.9 鹵15.9ng h ml -1 C max from 145.6 鹵15.9ng mL-1 to 57.8 鹵9.5ng ml -1 P 0.05N. These results indicated that both imidapril and CPT-11 were inhibited in vivo and pharmacokinetic characteristics were changed in immune liver injury rats. Finally, in order to investigate the expression characteristics of CES1 and CES2 in immunological liver injury rats, The expression levels of mRNA and protein of CES1 and CES2 in liver were analyzed by fluorescence quantitative PCR RT-PCR and Western blot. The results of RT-PCR showed that the contents of AB010635 and AY034877, the two main isozymes of CES2, were lower than those of normal control group (P 0.05). Western blot The results showed that the protein expression levels of CES1 and CES2 were both decreased (P 0.05). Therefore, we found that the expression and metabolic activity of CES1 and CES2 decreased significantly in immune liver injury. The results of this study have some reference significance for the dosage adjustment of CES metabolism in patients with immune liver injury.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R563.8

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相關(guān)期刊論文 前4條

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本文編號(hào)：1792992