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王英杰
摘 要 生物人工肝支持系统的出现是人工肝研究领域的重大进展，由于该系统以培养肝细胞为生物材料，兼有生物合成和转化代谢两大功能，并可为损伤肝组织的再生修复提供必要的营养物质和细胞因子，故人们期待着生物人工肝研究能够不断取得进展，使之成为治疗暴发性肝衰竭（FHF）的一条新的途径。 本实验在对人肝细胞分离、培养方法研究的基础上，构建了人肝细胞－中空纤维型体外生物人工肝支持系统（EBLSS），并对该系统进行了生物功能的体外评价和肝衰竭动物的支持实验。 材料与方法 一、人肝细胞的分离、常规培养及其分泌上清生物活性的观察 1、肝细胞的分离与培养 体外两步灌流法分离胎肝细胞，条件培养液培养。收集培养肝细胞分泌上清（HCS）超滤浓缩备用，用倒置相差显微镜（PCM）和生化分析法观察肝细胞形态与功能。 2、HCS对培养大鼠肝细胞的影响 同上方法分离大鼠肝细胞，普通培养，采用PCM、3H-TdR掺入法观察不同剂量HCS作用大鼠肝细胞的形态和DNA合成率。 3、HCS对FHF小鼠的保护作用 D－氨基半乳糖/脂多糖（Ｄ-Gal／LPS）腹腔注射制备FHF模型，观察HCS作用小鼠的存活率、转氨酶（ALT）和肝脏病理改变。 二、高密度悬浮培养人肝细胞系统的建立 1、肝细胞－肝非实质细胞的分离 按前述体外两步灌流法消化分离细胞，差速离心获取肝细胞和肝非实质细胞。 2、肝细胞－肝非实质细胞的微载体粘附培养 采用微载体cytodex3进行培养。 3、肝细胞-肝非实质细胞的球形聚集培养 采用综合限制条件进行培养。 4、聚集肝细胞的中空纤维管培养 采用循环培养液法进行培养。 5、培养肝细胞的功能与形态观察 应用生化分析方法检测肝细胞的合成功能，PCM、透射电镜观察肝细胞形态及贴壁能力。 三、生物人工肝支持系统的构建及其功能评价 1、EBLSS的组成 以混合培养的球形聚集肝细胞、肝非实质细胞（约2×108）及HCS为材料，中空纤维管为生物反应器，与蠕动泵、循环液贮存池相接共同组成EBLSS。 2、EBLSS生物合成产物的分析 循环EBLSS反应器内腔和贮存池内的1640培养液，定时吸取少量循环液，观测EBLSS的生物合成功能。 3、EBLSS药物转化实验 循环含有安定、利多卡因的1640培养液，定时取样，置自动荧光偏振分析仪动态分析药物浓度变化。 4、EBLSS循环重型肝炎病人血清实验 取死亡重型肝炎病人血清，置EBLSS进行体外循环实验，检测体外循环前后血清中部分生化指标的变化。 5、EBLSS肝细胞的活力观察 用台盼蓝拒染实验鉴定肝细胞存活率，PCM观察肝细胞的形态变化，自然沉降法观察聚集肝细胞贴壁能力，并用生化分析法检测循环液中肝细胞释放的乳酸脱氨酶（LDH）量。 四、生物人工肝支持系统对肝衰竭动物的支持作用 1、FHF兔的生物人工肝支持 以致死性剂量D-Gal耳缘一次性注射制备兔FHF模型，股动、静脉插管，连接EBLSS建立体外闭路循环通道，循环肝素抗凝血液。定时采兔血检测ALT、TB和肌酐变化，并取肝组织行病理检查。 2、“无肝”犬的生物人工肝支持 采用门-腔静脉侧侧吻合、门静脉和肝动脉结扎法建立犬“无肝”模型，同上法进行人肝细胞型EBLSS支持实验，观察犬生命征变化，存活时间，同时采用酶动力法动态观察犬血氨、乳酸改变。 结果与讨论 一、人胎肝细胞的分离、培养及其在肝衰竭治疗中的意义 1、体外两步灌流法分离胎肝细胞的存活率在95%以上，产量达到108数量级以上，已足以满足一般实验要求。由于该法胶原酶消耗量少，不需特殊装置，操作简便，是一种简便、实用、有效的肝细胞分离法，有助于肝细胞培养的广泛开展。通过添加糖皮质激素、胰高血糖素等多种成分构成条件培养液，使胎肝细胞的培养存活时间达到2周，可保持正常形态和良好的蛋白合成分泌功能。显示分离、培养的人胎肝细胞本身具有旺盛的生命力和极强的增殖能力，具有较高的应用价值。 2、肝细胞受损或人为分离时，肝细胞之间存在的正常接触抑制因失去表面调节因子的作用而被解除，通过一系列反馈效应产生出HGF等，促进肝细胞再生与修复，本研究发现，HCS能使培养大鼠肝细胞DNA合成率增加，显著提高Ｄ-Gal／LPS所致FHF小鼠的存活率，降低其血清转氨酶水平，减轻肝细胞坏死程度，并促进肝细胞的再生。提示分离、培养的胎肝细胞分泌上清中富含促肝细胞生长因子类物质，从而对FHF起更好的保护和治疗作用。 二、高密度悬浮培养人肝细胞系统的建立 1、分离肝细胞、肝非实质细胞在孵育时即呈明显的聚集倾向，预振荡30min后，肝细胞易与载体相粘附。在被覆聚羟乙基异丁烯酸（poly-HEMA）的培养瓶中继续间歇性振荡24h，约90%的肝细胞附着于微载体之上。在使用激素限定条件培养液培养的1月内，培养细胞保持多细胞粘附聚集体形态特征，并维持白蛋白、葡萄糖合成功能。大量培养肝细胞拟或用于提取大量细胞因子，或用于以培养肝细胞为材料的新型体外生物人工肝支持系统，或直接用于细胞移植治疗FHF等严重肝病，已成为人们关注的热门研究课题。生物人工肝的肝细胞需要量往往要达到108(109以上，更需超产细胞培养技术。本文人胎肝细胞的微载体培养，为上述研究提供了较完善的高密度、高活性肝细胞培养系统。 2、在被覆贴壁抑制剂与激素限定条件下，24h后肝细胞呈多细胞聚集状态，3(4天后形成完整的多细胞球体，悬浮生长于培养液中，亦保持较好的形态特征和蛋白合成能力，终止培养时球形聚集体在被覆胶原培养板上的重新贴壁率为47.2%，可继续生长数天。 由于在分离胎肝细胞后，先置37℃水浴中旋转振荡1h，以促进肝细胞初步形成粘连聚集状态，接种于poly-HEMA被覆的培养瓶进行培养后，继续定期振荡以防止贴壁并促进球体形成，同时使用成本较低的含血清激素限定培养液，培养的肝细胞球体形成满意，细胞活性良好，从而综合形成一种简便的肝细胞球体培养法。由于不需微载体也能达到高密度、高活性培养肝细胞的目的，故认为，人胎肝细胞的球形体培养更具实用价值。 3、本实验首先采用综合限制肝细胞贴壁法，促使肝细胞相互聚集悬浮生长，进一步将这种本身已具高密度特点的聚集肝细胞移入中空纤维管，辅以定期循环培养液法加以培养，结果不但使培养肝细胞的球形体特征得以长期保存，并可增加培养肝细胞的数量与密度，而且能使肝细胞保持较好的生物学功能长达6周，表明本文所设计的中空纤维管对肝细胞的球体培养也是一种较长期的高密度、高活性原代肝细胞培养系统。该方法与生物人工肝的设计原理相吻合，并首次同时采用了肝细胞聚集球形体和中空纤维培养技术，在某种程度上为生物人工肝的实验研究和临床应用提供了更为开拓和实用的研究领域。 三、生物人工肝支持系统的构建及其功能 1、EBLSS经过6h的体外循环，循环液中总蛋白、白蛋白、甲胎蛋白、尿素含量均显著增加，该循环液使体外培养大鼠肝细胞DNA合成率显著增加，实验后球形体肝细胞仍保持良好的活力。表明混合球形聚集培养的人肝细胞－肝非实质细胞及HCS为主要材料的EBLSS符合生物人工肝的要求。本实验虽因技术条件所限，未得到该培养系统产生HGF及类似物质的直接证据，但用3H-TdR掺入法检测发现，EBLSS循环液能明显增强体外培养大鼠肝细胞的DNA合成，间接证明混合培养系统及HCS可能提供具有刺激肝细胞再生的物质，从而使EBLSS的功能更加完善。 2、本文荧光偏振分析发现，循环液中99.1%的安定和91.1%的利多卡因被转化代谢，其中86.3%的利多卡因和97.3%的安定转化于EBLSS循环１h内。实验前后，肝细胞LDH释放不明显、细胞活力和再贴壁能力良好。表明EBLSS具备P450系统的生物转化活性，能够代偿肝脏的解毒代谢功能。此外，由LDH检测和细胞活力、贴壁能力检查可见，较大剂量的安定和利多卡因对EBLSS中的肝细胞并无太大影响，高密度球形聚集培养肝细胞仍保持良好的活性。而药物的高效率清除同时也表明，中空纤维管作为生物反应器，为循环液中药物与外腔肝细胞之间的相互作用提供了理想的场所。故推测该型EBLSS对化学药物引起的肝衰竭可能具有解毒代谢与支持治疗作用。 3、取重型肝炎病人血清进行体外循环实验，所用重型肝炎病人血清总胆红素（TB）显著降低，甲胎蛋白明显增加，总蛋白与白蛋白先降低随后逐渐回升，氨基酸紊乱较前有不同程度的改善。在６h的实验过程中，肝细胞不仅显示了良好的生物功能，而且始终保持了较高的细胞活力。一方面表明，生物反应器能为培养肝细胞提供满意的生存环境和物质交换条件，另一方面表明，实验采用的高密度混合肝细胞培养方法可靠有效，重型肝炎血清中复杂的毒性物质并未对其构成大的威胁，这从另一个角度佐证了人肝细胞型EBLSS用于支持治疗重型肝炎的可能与可行性。 四、生物人工肝支持系统对肝衰竭动物的支持 1、使用EBLSS对D-Gal诱导的FHF兔进行人工肝支持，结果显示：尽管两组FHF兔的存活时间没有明显差别，但在实验25小时内，支持治疗组兔的血清ALT、TB和肌酐水平均低于对照组，肝组织病理检查见肝细胞坏死程度明显轻于对照组。提示EBLSS对D-Gal诱导的FHF动物有一定的支持作用。结合EBLSS体外功能评价结果分析，该保护作用既包括了肝细胞对D-Gal及诱导肝损害后所产生毒性代谢物质的解毒作用，又有肝细胞及HCS提供肝细胞再生物质、促进肝细胞再生的作用。 2、人工肝支持 “无肝”犬的存活时间较无细胞空白循环对照组延长近一倍，其血压、心率、ECG等改变较迟缓，血氨、乳酸升高亦无对照组明显（3h和5h的血氨、乳酸变化有显著差异），实验所用肝细胞保持较好的活力和贴壁能力。结果表明所建EBLSS已发挥出培养肝细胞的生物合成与解毒代谢作用，能够部分代偿FHF的肝脏功能，有可能为肝衰竭患者提供有效的人工肝支持治疗。该结果明显优于Kelly等人用C3A细胞株对无肝（肝切除）模型犬所进行的人工肝支持实验，推测除选用正常肝细胞外，本研究所用的肝细胞－肝非实质细胞混合多细胞球形聚集培养方法，较好地维持了正常肝细胞的立体排列结构，保持了肝细胞良好的生物功能。而用0.2μm纤维型生物反应器，有助于FHF犬血液与培养肝细胞进行更有效的物质交换，从而发挥生物人工肝的生物合成与解毒功能。 自1987年Matsumura等用EBLSS治疗首例FHF获得成功以来，生物人工肝正逐步进入临床试用阶段。大量的研究表明，由培养肝细胞组成的生物人工肝不仅为肝衰竭患者代偿肝脏的解毒、生物合成功能以及稳定内环境、阻断恶性循环提供了可能，而且为具有强大再生能力的肝细胞再生提供了条件和时间。经过逐渐成熟和完善后，生物人工肝将不会仅仅停留为等待肝移植的一种过渡治疗手段，而极有可能像肾透析曾给肾功能衰竭的治疗带来革命性变化一样，为急性肝衰竭的现代治疗提供最大的希望。 我国是病毒性肝炎的高发区，与国外FHF相近似的重型肝炎的治疗尚无重大突破。目前，在我国肝移植的技术条件和经济基础均不成熟，仅有的少数人工肝研究依然相当于Ⅰ、Ⅱ型人工肝水平，迄今尚未见生物人工肝的研究报道。本研究为我国生物人工肝研究的开展和进一步用于重型肝炎肝衰竭的治疗奠定了基础。
Experimental study of cultured human hepatocytes in bioartificial liver for treatment of hepatic failure Postgraduate: Wang Yingjie Advisors: Prof. Li Mengdong and Prof. Wang Yuming Department of Infectious Diseases, Southwest Hospital, Third Military Medical College, Chongqing, 400038 Abstract The recent development of bioartificial liver support system containing hepatocytes is an important event in the research of the bioartificial liver. This system possesses the potential metabolic and synthetic functions of the liver and may provide certain nutrients and cytokines necessary for regeneration of the liver tissue. Further development is expected in the research of the bioartificial liver to provide a new therapeutic procedure in the treatment of fulminant hepatic failure (FHF). Based on the studies of isolation and & culture of human liver cells, we established an extracorporeal bioartificial liver support system (EBLSS) with cultured human hepatocytes and hollow fiber bioreactor. The biosynthetic and metabolic functions of EBLSS were analyzed in vitro and its support efficiency was investigated in vivo by animal model of hepatic failure. 1. Human fetal hepatocytes were isolated by a single two(step perfusion method and cultured in a conditioned medium. The effects of cultured hepatocytes supernatant (HCS) on the proliferation of cultured rat hepatocytes and the mice with fulminant hepatic failure were observed. The results were as the follows: 1)trypan blue exclusion showed that the viability of the hepatocytes was approximately 95% and the hepatocytes could be cultured for 2 weeks preserving normal morphologic appearance as well as the function of producing and secreting alpha(fetoprtein (AFP) and albumin. 2)HCS has a marked stimulating effect on the proliferation of rat hepatocytes in vitro. For example, it activated cell multiplication and growth, prolonged survival time, kept normal appearance and significantly increased the DNA synthesis. 3)after the treatment with HCS, the survival time of mice with FHF was prolonged and the serum alanine aminotransferase (ALT) decreased and the hepatic necrosis was milder and the liver regeneration prominent. These results indicated that the functions of the fetal hepatocytes isolated and cultured by the method mentioned above were satisfactory. Therefore, they can be used as an essential material for bioartifical liver. 2. The findings of the high(density hepatocytes and liver nonparenchymal cells cultured using comprehensive non(adherent methods, microcarriers, hollow fiber tube were follows: 1)human fetal hepatocytes and nonparenchymal cells efficiently adhered to microcarriers after the isolated cells were stirred for 30min. The adherence rate of the liver cells to microcarriers was about 90% on the flasks coated with poly(HEMA 24 hours after incubation and being stirred at regular intervals. In hormone(defined medium, human hepatocytes on microcarriers retained the ability to secrete proteins and characteristics of multicellular adherence and aggregation for one month. 2)in the hormone(defined and coated with adherence inhibitor medium, the cells were found to form multicellular aggregation by stirring at regular intervals for 24 hours. The floating aggregates became spheroidal in shape 3(4 days later. The spheroids maintained the normal morphological characteristics and protein secretion function during the period of aggregating culture. When the culture was stopped, the adherence rate of spheroids was 47.2%, and they can still grow on the collagen-coated substratum. 3)multicellular spheroidal culture was then carried out in a hollow fiber tube with circulating media and the spheroids with intact appearance were formed in the hollow fiber tube 2 days later. The functions of albumin, glucose, urea secretion and adherence rate were maintained for up to 6 weeks at a higher level. Because of the advantages of multicellular aggregation, they could play an important role in the treatment of FHF with liver cell transplantation and bioartificial liver. 3. A total of 2×108 hepatoctyes and liver nonparenchymal cells cultured to be the aggregating spheroids, a hollow fiber bioreactor and a supplementary circulation device were employed to construct EBLSS. Its biologic functions were obtained 5-6 hours after circulating in vitro. The results showed that：1)the total protein, albumin, AFP and urea were markedly increased in circulating medium and the DNA synthesis of rat hepatocytes cultured in this medium was significantly increased. 2)fluorescence polarization demonstrated that 99.1% of diazepam and 91.1% of lidocaine were transformed by EBLSS, during which main metabolism performed within the first hour. After the experiment, no marked release of lactic dehydrogenase was found and the cells still maintained good viability and capacity of attachment. 3)the total bilirubin of circulating sera of patients with severe hepatitis were markedly decreased while AFP levels were increased. Both the total protein and albumin decreased in the early stage of the experiment and increased later. The disturbance of amino acid profile in the sera was improved. No significant difference of the aggregation and adherence rate was after the experiments as compared with that before. These preliminary findings indicated that EBLSS possesses the biosynthesis and biotransformation function of the liver and a stimulating effect on the proliferation of hepatocytes. It might play certain supporting and therapeutic role in hepatic failure. 4. In this experiment, the efficacy of EBLSS consisting of spheriodal human liver cells and HCS was assessed in vivo by the use of galactosamine(induced rabbit model of FHF and dehepatized dog model. There was no difference of survival time between the two groups of rabbits. In the supported rabbits, significantly lower serum ALT, TB, creatinine and markedly milder hepatocyte necrosis were observed. We then compared the two groups of dehepatized dogs treated by EBLSS with or without liver cells. In the artificial liver(supported dogs, the survival time was significantly prolonged, the changes of blood pressure, heart rate and ECG were slowed down and the serum ammonia and lactic acid levels were significantly decreased at the 3rd and 5th h after the treatment. In addition, a good viability of human liver cells was noted 5h after the experiment. These data indicate that this EBLSS with cultured human hepatocytes plays the metabolic role, compensates the function of the liver and provides effective support and therapy for patients with hepatic failure. Key words： Cell culture Human hepatocyte Microcarriers Hollow fiber Multicellular spheroid Bioreactor Bioartificial liver Fulminant hepatic failure Artificial liver support system Rat Mouse Rabbit Dog | ||||||||