聚砜膜生物反应器实验系统构建及对重型肝炎患者血浆的影响

背景:人工肝反应器性能的评价通常需要构建一个密闭实验装置模拟生物人工肝支持系统来对反应器内肝细胞的生物功能以及生物反应效率等进行综合评价,对聚砜膜中空纤维反应器的性能进行初步评价对优化生物人工肝支持治疗系统具有一定意义.目的:构建聚砜膜生物反应器实验系统,了解该系统对重型肝炎患者血浆的影响,观察聚砜膜中空纤维反应器作为生物人工肝反应器的可行性. 设计:重复测量实验.单位:第三军医大学西南医院全军感染病研究所.材料:选用7只新生1～7 d中国实验小型猪,雌雄不拘,由第三军医大学实验动物中心提供,许可证号码:F99017实验过程中对动物的处置符合动物伦理学标准.肿瘤坏死因子α、转化生长因子β1试剂盒购自中国晶美生物工程公司;聚砜膜中空纤维反应器订制于中国上海德宏生物材料研究所;Cellco培养循环式人工毛细管培养系统购于美国Spectrum公司;7份重型肝炎患者血浆样本均取自住院慢性重型肝炎患者做血浆置换时分离的血浆.患者均对实验项目知情同意,实验经过医院伦理委员会批准.方法:实验于2004-01/2005-07在第三军医大学西南医院全军感染病研究所实验室完成.①实验过程:实验前12 h对实验猪进行断乳,清洁皮毛后分离肝细胞.将聚砜膜中空纤维反应器的内腔通过氧和二氧化碳弥散管与培养液贮池和Cellco培养液循环式人工毛细管培养系统连接成为密闭系统,外腔接口用洁净橡胶塞封闭. 用磁力搅动法将分离的肝细胞进行球形体培养,接种到本系统的聚砜膜中空纤维反应器外腔,然后加重型肝炎患者血浆样本200 mL于贮液池中,以80 mL/min的速度在反应器内腔及贮液池中循环.②实验评估:在循环的0,2,4,6 h从反应器内腔各取出2 mL循环液,用谷氨酸脱氢酶-紫外法检测上清中氨的水平,全自动生化分析仪上检测标本中总胆红素的含量,在全自动血凝仪上检测标本的凝血酶原时间,肿瘤坏死因子α、转化生长因子β1的检测参照试剂盒说明书进行. 主要观察指标:聚砜膜生物反应器系统对重型肝炎患者血浆的血氨、胆红素、凝血酶原时间、肿瘤坏死因子α及转化生长因子β1的影响. 结果:①血氨、总胆红素、凝血酶原时间检测结果:重型肝炎患者血氨水平持续降低, 0～2 h下降最明显,其后血氨水平下降趋势减缓;总胆红素水平持续降低,6 h的总胆红素水平明显低于0 h,差异有显著性意义 (P < 0.05);凝血酶原时间持续降低,6h的凝血酶原时间较0 h降低,差异具显著性意义(P < 0.05).②肿瘤坏死因子α、转化生长因子β1检测结果:重型肝炎患者血浆的肿瘤坏死因子α及转化生长因子β1含量持续降低,其中6 h均明显低于0 h,差异具显著性意义 (P < 0.05). 结论:砜膜生物反应器实验系统能够清除重型肝炎患者血浆中的小分子有毒物质,补充有益成分,降低细胞因子水平,可作为生物人工肝反应器.

Construction of polysulfone membrane bioreactor experimental system and its effects on plasma of patients with severe hepatitis

BACKGROUND: Property of artificial liver reactor is generally evaluated by using an enclosed experimental device, which is constructed through simulating bioartificial liver supporting system. Moreover, the enclosed experimental device is also comprehensively evaluated biological function of hepatocytes and biological response efficiency. In addition, it is significance for primarily evaluating the properties of polysulfone membrane hollow fiber reactor to optimize bioartificial liver supporting system. OBJECTIVE: To construct polysulfone membrane bioreactor experimental system, understand the effects on plasma of patients with severe hepatitis, and observe the feasibility of hollow fiber reactor regarded as bioartificial liver reactor. DESIGN: Repeated measurement.SETTING: General Infectious Disease Institute, Southwest Hospital, the Third Military Medical University of Chinese PLA. MATERIALS: Seven Chinese mini-pigs with 1-7 days old and of either gender were provided by Experimental Animal Center, the Third Military Medical University (certification: F99017). The animal disposal accorded to the ethical standard. Tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) kits were purchased from Jingmei Bioengineering Company, China; polysulfone membrane hollow fiber reactor by Shanghai Dehong Biomaterial Institute, China; Cellco culture-circular artificial capillary culture system by Spectrum Company, USA; seven plasma exponents were taken from admission patients with chronic severe hepatitis during plasma exchange. All patients provided the informed consent, and the animal experiment was conducted with confirmed consent by the local ethics committee.METHODS: This study was performed at the General Infectious Disease Institute, Southwest Hospital, the Third Military Medical University of Chinese PLA from January 2004 to July 2005. ① Experimental procedure: The experimental pigs underwent ablactation at 12 hours before experiment, and then hepatocytes were separated after cleaning their fur. An enclosed system was composed of polysulfone membrane hollow fiber reactor and Cellco culture-circular artificial capillary culture system, which were connected with oxygen-carbon dioxide diffusion tube and media holding pond. Interface of external cavity was blocked with clean rubber tampon. The separated hepatocytes underwent spheroid culture by using magnetic stirring method, and then they were inoculated in the external cavity of polysulfone membrane hollow fiber reactor. Next, 200 mL plasma from severe hepatitis patients was added in media holding pond and circled from internal cavity of reactor to media holding pond at the speed of 80 mL/min. ② Experimental evaluation: 2 mL liquid circulation separately collected from internal cavity of reactor at 0, 2, 4 and 6 hours after circulation was used to measure level of ammonia in supernatant by using glutamic dehydrogenase-violet method. In addition, content of total bilirubin was detected by using automatic biochemistry analyzer, prothrombin time by using automatic blood coagulometer, and TNF-α and TGF-β1 were detected according to the kits. MAIN OUTCOME MEASURES: Effects of polysulfone membrane bioreactor on ammonia, bilirubin, prothrombin time, TNF-α and TGF-β1 in plasma of patients with severe hepatitis.RESULTS: ① Measurement of ammonia, total bilirubin and prothrombin time in patients with severe hepatitis: Level of ammonia decreased persistently, in particularly, the decrease was obvious from 0 to 2 hours, and then, it was decreased slowly. While, level of total bilirubin was also decreased persistently, and it was significantly lower at 6 hours than that at 0 hour (P < 0.05). Moreover, prothrombin time was decreased persistently, and it was significantly lower at 6 hours than that at 0 hour (P < 0.05). ② Measurement of TNF-α and TGF-β1 in patients with severe hepatitis: Contents of both TNF-α and TGF-β1 decreased persistently, and they were significantly lower at 6 hours than those at 0 hour (P < 0.05). CONCLUSION:Polysulfone membrane bioreactor experimental system can clear noxious substance of small molecules in plasma of patients with severe hepatitis, supply beneficial components, and decrease levels of cytokines. Therefore, it can be regarded as the bioartificial liver reactor.