【作者】 王宁；

【导师】 郭仁宣；

【作者基本信息】 中国医科大学 ， 外科学， 2004， 博士

【摘要】 肝脏有很多重要的功能，包括解毒和很多生理功能。肝功不全将导致一些如氨、酚、硫醇等水溶性毒素，以及芳香氨基酸、游离脂肪酸、胆酸、非结合型胆红素、内生性苯二氮(艹卓)及一些假性神经递质等需与蛋白结合的非水溶性毒素的蓄积。上述毒素均被在肝功不全病人的血清中检测到过，并被认为是肝性脑病、脑水肿、肺水肿、肾功和心功不全的病因(Awad SS，etal．，2000)。在药物代谢方面，肝硬化病人的安定(Diazepam，DZP)半衰期比正常人长约5倍。另外，安定和其他苯二氮(艹卓)类药物常被用于有躁动的肝性脑病的病人(Andreasen PB，et al．，1976)。 肝脏可清除多种毒素。有人认为内生性苯二氮(艹卓)在肝性脑病的发病机制中发挥了很重要的作用(Itzhak Y，et al．，1995)。 近年来人们寄予人工生物肝脏很大希望，使之成为治疗肝功不全晚期一种有效手段。目前有很多种人工生物肝脏设备被应用于基础和亚临床研究。本实验采用了一种3维立体的容器来培养肝细胞作为人工生物肝脏设备。该设备有很多优点，如培养细胞密度高，长期应用可靠性良好，可在细胞和培养液之间更有效地交换营养、废物和气体(Matsumura，1993)。Enosawa等人曾以该设备培养肝细胞株，并降低了急性缺血性肝功不全猪的血浆氨浓度。他们应用的是转染的HepG2细胞(HepG2-GS)，该细胞可过表达谷氨酰胺合成酶(glutamine synthetase，GS)。谷氨酰胺合成酶是一种氨代谢旁路中的酶，它主要分布于肝小叶中央静脉的周围，它对氨的亲和力比氨基甲酰-磷酸合成酶要高(Enosawa S，et al．，2000)。HepG2-GS的功能主要限于代谢氨；而很多毒素和药物如睾丸酮、利多卡因、红霉素和苯二氮(艹卓)类则是由细胞色素P450 3A4(cytochrome P450 3A4，CYP3A4)代谢的。 目的 本实验拟建立一种转染GS和CYP3A4基因，过表达此两种酶的HepG2-GS-3A4细胞系，体外测定其氨和苯二氮(艹卓)类代表性药物——安定的代谢率；并将其应用于上述人工生物肝脏设备，体外测定其氨和苯二氮(艹卓)类代表性药物安定的代谢率;最后，建立急性肝功不全加安定中毒犬模型，并以上述人工生物肝脏系统治疗以辅助代谢血清安定和氨。 因此，本实验目的为: 1.建立HePGZ一Gs一3A4细胞系，体外测定其安定与氨的代谢率。 2.人工生物肝脏循环器中培养HePGZ一GS一3A4细胞，并体外测定其安定与氨的代谢率。 3.建立急性肝功不全加安定中毒犬模型，并以上述人工生物肝脏系统治疗以辅助代谢血清安定和氨。、方法、结果 首先，在建立HePGZ一GS一3A4细胞系之后，在培养皿中测定其体外安定和氨的代谢率。安定和它的代谢产物以HPLC法检测。结果发现0 .1一30林岁血DZP在4小时内被HePGZ一GS一3A4细胞呈时间一浓度依赖性代谢。DZP代谢率约为0.12土0.006一3.213土0.210林扩mF丫mg Pro-tein。HePGZ一GS一3A4对DZP的N一去甲基化和3一经基化Km为204.34，61 .55卜M;HepGZ一GS一3A4对DZp的N一去甲基化和3一经基化Vm分别为106.90，85.13PmoF而可mg Protein。 0.1、lmM氨被HePGZ一GS一3A4细胞代谢6小时后，代谢量分别为34.3%和43.1%。HePGZ一GS一3A4细胞对0.lmM和lmM氨的代谢率分别为1 8 .6，234.0林酬h/mg protein。 然后，将约4.0 x 108的HePGZ一GS一3A4细胞接种于Enosawas等应用的同样的人工生物肝脏循环器中(EnosawaS，etal·，2000;US patent5270207【1993」)。在循环器的反应器中，肝细胞附着于一种缠绕于中轴的玻璃纤维膜上。该反应器外腔内有温水循环，使其内腔保持37OC。培养液、02和C02可从反应器上面不断地输人。24小时后，新鲜的培养液以luday的速度输人反应器，在反应器内充分混合后，以同样速度排出。接种细胞后约7天，通过检测反应器内培养液内葡萄糖和乳酸含量，了解到反应器内细胞近汇合时，开始测定DZP和氨的代谢率。停止培养液循环。然后将含DzP和NH4CI的培养液充人反应器，使其终浓度达到DzPS林扩而;N玩Cl，2.smM。保持反应器内恒温于37，C左右。持续充人含5%CO:的02。分别于O，1，4，10，24小时取样。并以前述同样方法检测DZP及其代谢产物浓度和氨浓度。结果在未接种有肝细胞的人工生物肝脏循环器中，DZP浓度无明显降低(P>0.05);在接种有HePGZ细胞的人工生物肝脏循环器中，DZP浓度有降低，然而无统计学意义(P>0 .05)，且无NDZP，TZP和OZP被检测到(图14)。在接种有HePGZ一GS一3A4细胞的人工生物肝脏循环器中，DZP浓度明显降低(P<0.05)。且有NDZP、咒P产生，咒P产量约为NDZp的5倍。DZp被代谢约542林g，NDZp、化p分别86.2、440·4林g产生。DZP浓度约降低18 .1%，开始的4小时内平均速度为0.0180p岁扮eell，而24小时平均速度为0.01 14p扩h/eell。 在未接种有肝细胞的人工生物肝脏循环器中，氨浓度无明显降低(P>0.05);在接种有HePGZ细胞的人工生物肝脏循环器中，氨浓度有降低，然而无统计学意义(P>0.05);在接种有HePGZ一Gs一3A4细胞的人工生物肝脏循环器中，氨浓度明显降低(P<0.05)。氨浓度约降低31.7%，开始的4小时内更多还原

【Abstract】 Many kinds of drugs are metabolized by the liver. In case of hepatic failure , drugs and toxic substances will accumulate in the body. From the pharma-cokinetic aspects, the half - life of diazepam ( DZP) may be 5 times longer in patients with cirrhosis than in the controls. Moreover, DZP and other benzodiaz-epines have been advocated as sedatives for agitated patients with hepatic en-cephalopathy, despite the risks of induction of hepatic coma by their use.Bioartificial liver ( BAL) support system is currently expected to be a novel therapeutic device for the end stage of hepatic failure. Researchers are using various devices as their BAL. The authors group has been utilizing the 3 - dimensional bioreactor, Kigunasu? as a BAL because of its advantages of high density of cells, reliability during a long - term operation, capability of efficiently performing the exchanges of nutrient, waste products and gases between cells; and medium. This device has also been used by Dr Enosawa. He applied a re combined HepG2 overexpressing glutamine synthetase (HepG2 -GS) , which is involved in the accessory pathway of ammonia removal, in Kigunasu to examine; on ischemic liver failure pigs.However, HepG2 - GS does not include all the functions of hepatocytes. Many kinds of substances and drugs such as testosterone, lidocaine and erythro-mycin are not metabolized by GS, but by cytochrome P450 3A4 that also has a high diazepam metabolism capacity.ObjectsThe author started the current study to provide a clue to the treatment of hepatic failure using an artificial liver device. Such a treatment may also be usefulin a clinical setting of hepatic failure patients overdosed with sedatives such as DZP. Therefore, HepG2 - GS -3A, which overexpresses CYP 3A4 and GS, was applied to Kigunasu as our artificial liver to metabolize diazepam and ammonia. Finally, this BAL was attempted to treat acute hepatic failure dogs overdosed with DZP, to decrease the concentrations of DZP and ammonia in the dogs ’plasma. Thus, the aims of the present study are summarized as follows:1. to estabolish and investigate the in vitro metabolism capacity of diazepam and ammonia by HepG2 - GS - 3 A4,2. to measure diazepam and ammonia metabolism capacity by Kigunasu inoculated with HepG2 - GS -3A4, HepG2 and without cell,3. to treat a canine model combining acute hepatic failure with diazepam overdosage using our BAL.Mefhods and resultsFirst, after the establishment of HepG2 - GS -3A4, its vitro metabolism capacity of diazepam and ammonia were studied in dishes. DZP and its metabolites were checked by HPLC. We found that 0.1 to 30 g/ml DZP, were metabolized by HepG2 - GS -3A4 time and concentration - dependently, and the metabolites of DZP were produced by HepG2 - GS -3A4 time and concentration -dependently, too, within 4 hours. Reduction rate of 0. 1 - 30jxg/ml DZP by HepG2 - GS - 3A4 was 0. 12 + 0. 006 - 3. 213 + 0. 210u,g/ml/h/mg Protein. Km for DZP N - demethylation and 3 - hydroxylation by HepG2 - GS - 3A4 were 204. 34, and 61.58 M. Vm for DZP N - demethylation and 3 - hydroxila-tion by HepG2 -GS -3A4 were 106.90, and 85.13pmol/min/mg Protein.After the incubation of lmM and 0. lmM ammonia with HepG2 - GS -3A4 for 6 hours, ammonia concentration decreased by 34. 3% and 43. 1% , respectively. lmM and 0.1mM ammonia reduction rate by HepG2 - GS - 3A4 were 18.6,234.0 ug/h/mg protein, respectively.Then, 4. 0 x 108hepatocytes were inoculated in a circulatory flow bioreac-tor, Kigunasu, as our BAL, and cultured for 7 days, until the cells were nearly confluent. Thereafter DZP and ammonia in vitro metabolism capacities of ourBAL were checked. DZP and NH4C1 were added into the medium to make the ultimate concentration at 5 ug/ml (DZP) and 2. 5mM (ammonia). Samples were collected at scheduled point times for 24 hours. DZP and its metabolites were measured by HPLC. Ammonia concentration was checked with Berthelot Reaction.From the Kigunasu in vitro study, it was found that the DZP metabolism capacity of Kigunasu with更多还原