C3A永生化肝细胞系的部分功能评价

全面检测肝永生化细胞系的功能,对评价其功能状态、比较不同肝细胞系的差别、研究生物人工肝的疗效、判断药物对肝细胞功能的影响,以及改建与优化更好的肝细胞系等方面具有重要的意义。C3A细胞是目前用于临床研究的生物人工肝永生化肝细胞系,它从肝纤维母细胞瘤细胞转化获得。本研究以C3A细胞为对象,对反映肝细胞生长增殖、蛋白合成、代谢、分泌、抗氧化的部分指标进行检测,以期建立较全面反映其功能状态的检测方法，为其细胞改建与功能优化提供依据，为新一代生物反应器及生物人工肝发展服务。[第一段]     

中空纤维型人工肝反应器研究和应用最新进展

据医学空间网1月22日报道，生物人工肝（BAL）系统正在成为急性肝衰竭患体外有效的肝支持治疗手段。生物反应器是BAL系统的核心部件，承担着大量培养和维持肝细胞功能的重任，以及实现肝细胞与肝衰竭患血液之间的双向物质交换与作用。目前设计和研究的生物反应器包括平板式反应器、中空纤维型反应器、灌流床式／支架型反应器、肝细胞包裹与悬浮型反应器等，尤以中空纤维型反应器研究和应用得最为广泛。[第一段]     

生物人工肝体外支持系统合成功能的评价

目的 对人肝细胞型体外生物人工肝进行生物合成功能的评价,藉以探讨其用于肝衰竭治疗的可能性。方法 用2×10~8个混合聚集球形培养的人肝细胞、肝非实质细胞与中空纤维型生物反应器组成体外生物人工肝支持系统(EBLSS),通过循环液(RPMI 1640)中肝细胞合成分泌产物的分析,评价其生物功能。结果 经过6小时的体外循环,循环液中总蛋白、白蛋白和甲胎蛋白含量均显著增加,尿素和葡萄糖水平也有明显改变,该循环液使体外培养大鼠肝细胞DNA合成率显著增加(与对照组比较P<0.01),实验后球形体肝细胞仍保持良好的活动。结论 由培养人肝细胞相中空纤维型生物反应器组成的体外生物人工肝具备肝脏生物合成功能,并有刺激肝细胞增殖的能力,对肝衰竭可能有一定的支持治疗作用。     

体外生物人工肝辅助装置生物转化功能的初步研究

为评价培养人肝细胞与中空纤维生物反应器组成的人工肝辅助装置(LAD)的生物功能，采用荧光偏振分析方法，进行了LAD转化安定和利多卡目的体外实验。结果显示；使用2×10^8个混合球形聚集培养的人肝细胞、肝非实质细胞，体外实验5小时后，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LAD循环1小时内。实验前后，肝细胞乳酸脱氢酶释放不明显、肝细胞活力和再贴壁能力良好。结果表明，LAD具有肝脏P450系统生物转化功能，该装置对化学药物性肝衰竭可能具有解毒代谢与支持治疗作用。     

体外生物人工肝支持作用的实验研究

目的 构建理想的体外人工肝支持系统，探讨其对暴发性肝衰竭的支持作用。方法 用经体外两步灌流法分离，限制贴壁技术球形聚集混合培养的人肝细胞－肝非实质细胞与中空纤维型生物反应器及由血液透析仪改装的辅助循环装置共同构建的ＥＢＬＳＳ，对门腔静脉分流加入肝血液阻断法建立的ＦＨＦ模型犬进行人工肝支持实验。结果 在平均约１０＾８肝细胞－肝非实质细胞ＥＢＬＳＳ的支持下，治疗组犬存活时间较无细胞空白循环对照组延长近     

培养猪肝细胞中空纤维型生物反应器的制备

目的 探讨培养猪肝细胞中空纤维型生物反应器的制备方法。方法 将两步法分离的新生实验小型猪肝细胞接种于中空纤维器内,用培养液循环式人工毛细管培养系统进行培养,观察生物反应器的蛋白分泌、利多卡因转换能力;检测培养猪肝细胞的乳酸脱氢酶(LDH)漏出量及细胞形态和活力。结果在反应器制备后第2、4、6天可检测出猪肝细胞分泌的白蛋白,生物反应器对利多卡因的转换率为89.6％～96.1％。培养结束时LDH漏出量由(23.7±4.6)U/L升高至(127.8±17.4)U/L(F=39.582,P<0.01),细胞活力由95.8％±0.3%降低至83.8％±4.7％(t=5.135,P<0.01)。结论 可用人工毛细管培养系统制备出1周内性能较好的培养猪肝细胞中空纤维型生物反应器。     

两种人工肝生物反应器内乳猪肝细胞培养的比较研究

目的 采用普通型中空纤维及编织型中空纤维两种人工肝生物反应器,比较其内培养的肝细胞生存时间及功能,为生物反应器的设计提供新的方法和思路.方法 将新生中国实验小型猪的肝细胞与微载体共同培养,待肝细胞与微载体充分粘附形成微载体-球形聚集体后,将其注入生物反应器的外腔,在培养液内加入氯化氨、醋胺酚检测生物反应器的生物转化和代谢功能,行无血清培养检测肝细胞白蛋白的合成功能,并检测肝细胞酶的漏出量.锥虫蓝染色测定肝细胞的活力,透射电镜观察其超微结构.结果 肝细胞与微载体相互聚集,形成了具有一定结构层次的肝细胞聚集体,重构了肝细胞之间的相互连接;接种入生物反应器后,在编织型生物反应器中肝细胞1周内保持较高的活力,至第7天肝细胞活力仍高达(75±6.3)%,对醋氨酚和氯化铵有良好的代谢、生物转化功能,白蛋白合成在第7天为(0.57±0.13)mg/106个肝细胞,酶的漏出量也少,各项指标均明显优于普通型生物反应器组(P＜0.05).结论 编织型生物反应器为肝细胞的生长、增殖提供一个类似体内的三维立体环境,是一种性能良好的生物反应器.     

生物人工肝在肝衰竭治疗中的应用

生物人工肝一般是指以培养肝细胞为基础的体外生物人工肝支持系统(EBLSS)。从理论上讲,以肝细胞为材料的生物人工肝最能代替正常肝脏的功能,为暴发性或急性肝衰竭(FIIF/ALF)患者提供可靠的过渡支持治疗,使患者通过自身肝再生或肝移植而恢复。 1.基本原理:生物人工肝是肝细胞培养技术与血液净化技术相结合的产物,其基本原理是将体外培养增殖的肝细胞(人肝细胞、哺乳动物肝细胞或肝细胞系)置于特殊的生物反应器内,利用体外循环装置将肝衰竭患者血液或血浆引入生物反应器,通过反应器内的半透膜与肝细胞进行物质交换与生物作用。由于这一过程如正常机体血液流过肝脏肝窦一样,一方面血液中的毒性物质被培养肝细胞摄取、转化、代谢,另一方面血液中因肝功能衰竭而缺乏的机体必需物质由培养肝细胞合成、补     

肝衰竭患者血浆(血清)对体外培养肝细胞毒性作用的研究现状

生物人工肝是以培养肝细胞为基础的人工肝支持系统,高活性和功能良好的肝细胞是其基本要素之一,然而,由于肝衰竭患者肝脏大量肝细胞坏死,导致机体代谢紊乱和大量毒性物质堆集,这些毒性物质会对反应器中肝细胞产生毒性作用,直接影响生物人工肝的治疗效果.因此,了解肝衰竭患者血浆（liver failure plasma,LFP）或血清（liver failure serum,LFS）对体外培养肝细胞的各种不良作用,对生物人工肝的临床应用有重要意义.本文综述近年来在该领域的研究进展.     

中空纤维反应器管外腔胶原凝胶混合肝细胞培养

早期的生物人工肝是将培养液混悬肝细胞灌人中空纤维反应器管外腔,一般效果不好。后改将培养液混悬肝细胞或微载体肝细胞灌人中空纤维管内腔或先用胶原液在中空纤维管内腔壁涂层,再将培养液混悬肝细胞灌人中空纤维管内腔;使患者血（血浆）流过管外腔,通过中空纤维膜上的微孔,管内腔和管外腔发生物质交换,使血中的毒物被肝细胞解毒。近年来Excorp Medical公司推出将胶原凝胶混合肝细胞灌注中空纤维管外腔的生物人工肝反应器,效果尚待验证。本研究将胶原凝胶混合肝细胞灌注中空纤维反应器管外腔培养（凝胶混合法）,与将培养液混悬肝细胞灌注腔壁涂有胶原层的中空纤维管外腔培养（单层凝胶法）对照,验证肝细胞功能。     

In vitro studies on a bioreactor module containing encapsulated goat hepatocytes for the development of bioartificial liver.

BACKGROUND: A bioartificial liver may act as a temporary metabolic bridge in patients with acute liver failure. We devised a bioreactor module containing encapsulated goat hepatocytes and studied its efficiency in detoxifying ammonia. METHODS: A hollow-glass bioreactor module was designed and fabricated locally. The module was inoculated with alginate poly-L-lysine microcapsules with entrapped goat hepatocytes. Metabolism of ammonia and glucose was assessed. Antibody-mediated cell cytotoxicity was also assessed. RESULTS: The optimum encapsulated goat hepatocyte concentration was 12-18 billion at a perfusate flow rate of 30 mL/min under oxygenated condition. The optimum ammonium chloride concentration for detoxification was 2.5-5.0 mM. There was little or no cytolysis of encapsulated hepatocytes on exposure to complement-inactivated human AB serum. CONCLUSION: Encapsulated goat hepatocytes efficiently detoxified ammonia to urea. Cells were metabolically active up to 48 hours, indicating their feasibility for use in a bioreactor module. Encapsulation protected the hepatocytes from antibody-mediated cell lysis.     

两种含胶原凝胶的人工肝生物反应器的功能比较

目的改进中空纤维型人工肝生物反应器。方法将大鼠肝细胞与胶原溶液混合并注入中空纤维反应器管外腔,待混合物在管外腔中形成胶原凝胶将肝细胞悬浮其中,构成胶原凝胶混悬肝细胞反应器(Ⅰ组);另将大鼠肝细胞混悬液注入涂有胶原层的中空纤维反应器管外腔,构成胶原涂层加肝细胞反应器(Ⅱ组)。两组反应器均由管内腔循环灌注培养液,置孵箱培养9 d,每24 h换液一次,每48 h取灌注液检测白蛋白(Alb)、尿素、乳酸脱氢酶(LDH)浓度,以检验反应器的肝细胞功能。采用统计软件SPSS 13.0进行统计学分析,计量数据以均数±标准差(x±s)表示,两组资料比较用配对t检验。结果Ⅰ组和Ⅱ组的Alb值均在灌注第3天达峰值,分别为(1.41±0.08)和(0.65±0.05)g/L,3~9 d各时间点Alb值Ⅰ组均明显高于Ⅱ组(P值均0.01)。Ⅰ组和Ⅱ组的尿素值分别在灌注第5天和3天达峰值,分别为(1.73±0.14)和(1.56±0.18)mmol/L,5~9 d各时间点尿素值Ⅰ组均明显高于Ⅱ组(P值均0.01)。Ⅰ组和Ⅱ组的LDH值在灌注第9天达峰值,分别为(32.03±9.13)和(70.17±25.28)U/L,1~9 d各时间点LDH值Ⅰ组均明显低于Ⅱ组(P值均0.01)。提示胶原凝胶混悬肝细胞反应器(Ⅰ组)比胶原涂层加肝细胞反应器(Ⅱ组)有更好的肝细胞合成代谢功能,肝细胞酶漏出也更少。结论胶原凝胶混悬固定肝细胞更适用于构建中空纤维型人工肝生物反应器。     

Hybrid artificial liver support system for treatment of severe liver failure

AIM: To construct a novel hybrid artificial liver support system (HALSS) and to evaluate its efficacy in patients with severe liver failure. METHODS: Hepatocytes were isolated from suckling pig by the modified Seglen's method. Isolated hepatocytes were cultured in a spinner flask for 24 h to form spheroids before use and the functions of spheroids were detected. HALSS consisted of a plasma separator, a hemo-adsorba and a bioreactor with hepatocytes spheroids in its extra-fiber space. HALSS was applied to 10 patients with severe liver failure. The general condition and the biochemical indexes of the patients were studied just before and after the treatment. RESULTS: The number of cells per liver was about 2-4×1010 (mean, 3.1±1.5×1010). The cell viabilities were more than 95%. After 24 h of spheroid culture, most hepatocytes formed spheroids. The levels of albumin and urea in the medium of spheroid culture were higher than those in supernatant of petri dish culture (P= 0.0015 and 0.0001, respectively). The capacity of albumin production and urea synthesis remained stable for more than one wk and declined rapidly after two weeks in vitro. In HALSS group, the duration of HALSS treatment was 6-10 h each time. All patients tolerated the treatment well without any fatal adverse reaction. After HALSS treatment, the general condition, psychic state, encephalopathy and hepatic function of the patients were improved. The survival rate of the HALSS group, Plasmapheresis group and control group was 30% (3/10), 20% (2/10) and 0% (0/10), respectively (P = 0.024). Two weeks after treatment, Tbil and ALT decreased and the PTA level elevated in HALSS group and pasmapheresis group (Pvalue: 0.015 vs0.020, 0.009 vs 0.012 and 0.032 vs 0.041, respectively). But there was no significant change of blood albumin concentration before and after treatment in HALSS group and Plasmapheresis group. CONCLUSION: The HALSS established by us is effective in supporting liver function of patients with severe liver failure.     

In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates

Background/Aims: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy. We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli.Methods: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e. a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO₂ removal. Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact. Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion.Results: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo. The biochemical performance of the bioreactor remained stable over the investigated period. The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures. Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size.Conclusions: The novel bioreactor showed encouraging efficiency. The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure.     

混合生物人工肝治疗肝衰竭临床疗效的初步评价

目的 构建新型混合生物人工肝支持系统,初步评价其治疗肝衰竭的临床疗效。方法 用血浆置换装置、活性炭灌流器和培养人或猪肝细胞生物反应器构成混合型生物人工肝支持系统,对30例重型肝炎肝衰竭患者进行人工肝支持与治疗。结果 30例患者人工肝支持治疗的显效率43.3％(13/30),有效率43.3％(13/30),总有效率86.7％。好转出院11例,成功等到肝移植6例,死亡6例,自动出院7例。结论 所建混合生物人工肝支持系统对肝衰竭有明显的肝支持作用,可作为重型肝炎的有效治疗手段。     

Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell--cell contact and cell membranes.

In primary monolayer cultures of rat mature hepatocytes, many metabolic functions as well as cell growth are regulated by cell density. There are two types of regulatory response of these functions to change of cell density. Growth-related functions, such as DNA synthesis, induction of glucose-6-phosphate dehydrogenase, 2-aminoisobutyric acid transport, synthesis of cellular protein, and cholesterogenesis, are stimulated by low cell density. In contrast, functions related to hepatocyte-specific characters, such as the inductions of tyrosine aminotransferase, serine dehydratase, and malic enzyme and synthesis of triglycerides, are stimulated by high cell density. The reciprocal responses of these cellular activities to cell density were mimicked by addition of plasma membranes purified from adult rat liver to hepatocytes cultured at low cell density. The modulator activity was heat labile and trypsin sensitive. The activity was also found in plasma membranes from kidney, brain, and erythrocytes, although the specific activities of these preparations seemed to be different. These results suggest that the reciprocal regulations of cell growth and hepatocyte-specific functions are mediated by some surface components via cell-cell contact.     

Hepatocyte dynamics in a three-dimensional rotating bioreactor

BACKGROUND AND AIMS: The use of an artificial liver system with extracorporeal circulation or a three-dimensional bioreactor perfused with liquid culture medium inevitably exposes hepatocytes to fluid mechanical stress (MS). The expression of liver-specific hepatocyte functions seems to be modulated by the magnitude of MS. Nonetheless, few studies have focused on the direct effects of MS on hepatocytes. We subjected hepatocytes to MS using an MS loading device and investigated the effects on the cytoskeleton and hepatocyte dynamics inside three-dimensional scaffolds by monitoring the changes in actin fiber, one of the components of the cytoskeleton. We also assessed the influence of MS on specific hepatocyte functions. METHODS: We subjected hepatocytes to MS by a rotating radial flow bioreactor (RRFB) and examined the effects by comparing the MS-loaded culture cells with cells cultured under stationary conditions without MS loading. The hepatocytes (1 x 10(6)/cm(3)) were seeded on gauze without collagen coating and examined to determine morphological changes after 60 h incubation. Actin filaments in samples from the MS-loaded hepatocyte culture were stained by fluorescein isothiocyanate-labeled phalloidin. RESULTS: Hepatocyte aggregation was observed in the MS-loaded culture, but not in the unloaded stationary culture. Better albumin products were observed in the MS-loaded group than in the stationary culture group at all measurement points. Actin filaments extended toward the scaffold after the start of MS loading incubation and polymerized around the hepatocytes. The hepatocyte aggregation eventually advanced to the formation of spheroids. CONCLUSION: These results suggest that MS-induced polymerization of actin filaments stimulate hepatocyte aggregation and thereby improve hepatocyte-specific function.     

Evaluation of a bioartificial liver based on a nonwoven fabric bioreactor with porcine hepatocytes in pigs

BACKGROUND/AIMS: We developed a bioartificial liver (BAL) based on a direct hemoperfusion typed nonwoven fabric bioreactor containing porcine hepatocytes. In this study, the efficacy of our BAL was evaluated with a pig fulminant hepatic failure (FHF) model. METHODS: FHF was induced with intravenous administration of D-galactosamine (1.3 g/kg) in each pig. Twelve hours post D-galactosamine injection, fifteen pigs were divided into: a BAL group (n = 5), in which pigs received the BAL treatment with 1.0 to 1.3 x 10(9) hepatocytes for 6 h, a sham BAL group (n = 5), in which pigs received the BAL treatment without hepatocytes, and a FHF group (n = 5), in which pigs only received intensive care. Parameters related to liver function and animal survival up to 168 h were determined. RESULTS: In the BAL group, blood ammonia and plasma lactate levels were lower, and serum glucose levels and Fischer index were higher than those in the other two groups. Survival time of pigs in the BAL group was significantly prolonged as compared with the sham BAL and the FHF group. CONCLUSIONS: The BAL based on a nonwoven fabric bioreactor containing porcine hepatocytes appears to be effective in the treatment of FHF in pigs.