【作者】 胡还章；

【导师】 高毅；

【作者基本信息】 第一军医大学 ， 外科学（普通外科）， 2000， 硕士

【摘要】 急性肝衰（ALF）的治疗是当代临床医学的一大难题，常规治疗病死率高达80％。虽然肝移植（LT）可将ALF的病死率降低到30-50％，但是，LT存在急诊手术时供体来源困难、手术复杂、患者需要终生接受免疫抑制治疗及费用昂贵等缺点。作为肝脏替代疗法的另一个方向，人工肝的研究在近40年来取得了长足的进展。目前，国外以培养的肝细胞与人工辅助装置相结合的生物人工肝（BAL）所进行的动物实验及临床初步应用结果已表明，BAL不仅可能代偿ALF患者肝脏解毒、生物合成功能及稳定内环境、阻断恶性循环，而且为具有强大再生能力的肝细胞提供了再生的条件和时间。经过逐渐成熟和完善后，BAL可望将不会仅停留为等待LT的一种过渡治疗手段，而极有可能象肾透析曾给肾功能衰竭的治疗带来革命性变化一样，为ALF的现代治疗提供最大的希望。结合本课题的前期工作，本研究用微载体粘附悬浮培养的原代正常猪肝细胞作为生物材料构建BAL，进行治疗猪ALF的实验，旨在为BAL早期进入临床Ⅰ期试验提供依据。 第一部分：猪肝细胞的分离与微载体粘附悬浮培养 目的 获得一个具有丰富来源的肝细胞生产技术。 方法 取本地杂种小猪作为肝细胞供体。用Seglen改良二步胶原酶原位循环灌注法分离获取肝细胞悬液，台盼蓝（TB）染色法计算细胞数及细胞活率。将Cytodex-3微载体按5mg/mL的浓度加入经5％甲基硅树脂乙酸乙酯溶液硅化的STUART搅拌培养瓶中。猪肝细胞按5×10~6/mL的密度全部接种于培养瓶，随后加入培养液及附加因子至1/3最终体积，搅拌培养6小时后加入培养液及附加因子至最终体积200mL，然后持续搅拌培养。光镜及电镜下观察细胞生长情况，动态检测培养上清中白蛋白及尿素浓度。 结果 每只猪肝平均获取肝细胞数为N．26土 0．75）XIO‘＼活率为o91．22士0．83）％。猪肝细胞在接种后即呈明显的Cytodex刁聚集趋势，在培养48－72小时后可见典型的多细胞聚球形成，该形态特点及白蛋白、尿素合成能力可保持约4周左右。 结论 采用IV型胶原酶原位门腔循环灌注可分离获取大量。高活性的原代正常猪肝细胞，经微载体粘附悬浮培养后可达到高密度。高活性、长期离体培养的目的，可满足BAL对生物材料的要求。更多还原

【Abstract】 The treatment of acute liver failure (ALF) is extremely difficult in the current medicine. With a routine treatment, the mortality of ALF is high about 80%. Although liver transplantation (LT) can reduce that to 30-50%, LT has the shortcomings such as the shortage of donor liver so not to be able to meet the urgent need of treating ALF with emergency LT, complex operation, the life-long immunosuppressant treatment on sufferer and high expenses. As another way of liver-substituting therapy, the study on artificial liver has made great achievements in the past forty years. Nowadays the animal experiments and primary clinical applications with bioartificial liver (BAL) based on cultured hepatocytes and artificial assistant device at abroad have shown that BAL can not only be possible to compenste the functions of detoxification and biosynthesis of liver, stabilization of inner condition and interdiction of vicious circle, but also provide conditions and time for regeneration of hepatocytes which possess great ability to regenerate. With gradually maturation and perfection BAL can be promise to bring the lightest hope for the treatment of ALF just as the revolutionary change of the treatment of kidney failure with kidney dialysis instead of only being a bridging therapy for waiting LT. Combining with the prophase work on this subject, we cultured the normal primary porcine hepatocytes on microcarriers suspendingly as biomaterial to develop BAL to experiment on treating ALF pigs with aiming to provide evidences for BAL to enter phase I clinical examination earlier. ?? Part I Seperation and culture on microcarriers suspendingly of porcine hepatocytes Aim To establish a technique of harvesting hepatocytes of abundant sources. Methods Hepatocytes were from the donors of native hybrid pigs and isolated on the way of two-step orthotopic collagenase circulating perfusing method as described by Seglen. The yield and viability were assessed by trypan blue exclusion test. Microcarriers Cytodex-3 were added at the concentration of Smg/L into the STUART stirring culture vessel siliconized by 5% methyl silicon resin ethyl acetate. Porcine hepatocytes were entirely inoculated into the vessel at the concentration of 5 X 106/mL. Together with the supplemental factors, the culture matrix was added in according to one third of the final volume. After being stirred for 6 hours the culture matrix and supplemental factors were added to the 200mL final volume and from then on stirring were kept on. Cell growth was observed on light and electron microscopes. Concentrations of albumin and urea were examined dynamically in the supemation. Results Each liver provided (4.26?.75) X 1010 hepatocytes averagely and their vivid rate was (91.22 ?0.83)%. Most hepatocytes tended to conglomerate obviously after being inoculated and 48-72 hours later typical multicellular aggregate spheroids could be observed. Their morphological characteristics and abilities to synthesize albumin and urea could be maintained for about 4 weeks. Conclusion large-amount and high-activity normal primary porcine hepatocytes could be isolated on the way of two-step orthotopic tyep IV collagenase circulating perfusing method. When being cultured on microcarriers they could be achieved to the objects of high更多还原