培养肝细胞应用于人工肝的研究进展

培养肝细胞应用于人工肝的研究进展徐小平综述杨继震高毅审校作者单位：５１０２８２广州第一军医大学珠江医院人工肝的研究始于５０年代，早期的人工肝支持系统主要有：血液透析、体外肝灌注。交叉血循环。活性碳灌流、肝透析、交叉输血及血浆交换。经临床试验表明，对暴...     

异种生物人工肝和猪逆转录病毒

生物型人工肝 (bioartificialliver,BAL)或组合生物型人工肝 (hybridbioartificialliver ,HBL) ,是目前国内外公认最接近自然肝脏 ,功能也最全面的人工肝脏支持系统〔1〕。所谓生物型人工肝 ,就是在整个治疗系统中包含有活的肝细胞或组织 ,因此具有相应生物活性的人工肝支持体系。目前应用最多的生物部分是原代培养的猪肝脏细胞。但作为异种细胞 ,猪肝细胞在人体的应用存在着猪逆转录病毒 (porcineen dogenousretrovirus ,PERV)感染及由此引起人畜共患疾病的危险。本文就猪逆转录病毒的有关问题作一简要回顾。1　猪逆转录病毒 (PERV1)…     

人工肝生物材料人肝细胞系CL-1的微载体培养.

目的使用微载体培养人肝细胞系ＣＬ１,并观察细胞的转化及合成功能．方法使用Ｃｙｏｔｄｅｘ３微载体进行人肝细胞系ＣＬ１的高密度培养,应用光镜及扫描电镜于培养１,３,５,７,９ｄ动态观察细胞的生长情况,并同时检测培养系统的安定转化及清蛋白合成量．结果培养７ｄＣＬ１细胞密度达到最高,为２１６×１０９／Ｌ;安定转化量最高为６１９μｇ;清蛋白合成量最高为７８μｇ．结论使用微载体培养高分化人肝细胞系可达到较高的密度,且具有较好的生物转化及合成功能,可望作为组合性人工肝的生物材料     

生物人工肝之肝细胞分离和培养研究现状

组合型生物人工肝支持系统(Hybrid Bioartificial Liver Support System. HBLSS)具备解毒、分泌、合成与转化等功能。在国外已完成动物试验,并进入了Ⅰ期临床试验,取得良好效果。笔者拟就现代生物人工肝的基础——肝细胞的分离和培养研究作一综述。 1 肝细胞来源 生物人工肝(BLSS)的肝细胞,要求既要有足够的数量,又要有完备的生物学活性。肝细胞来源大概有3条途径:①来自人或动物的肝脏分离的原代肝细胞;②肿瘤肝细胞建株后的     

用于生物人工肝的大量猪肝细胞低温冻存技术

目的：比较大量猪肝细胞程序冻存与常规冻存的效果。方法采用二步法胶原酶经肝静脉逆行灌注的方法分离猪肝细胞,分离的肝细胞根据冻存方法与冻存袋的不同分5组：a 组,50 mL 冻存袋,程序冻存;b 组,100 mL 冻存袋,程序冻存;c 组,50 mL 冻存袋,常规冻存;d 组,100 mL 冻存袋,常规冻存;e 组,新鲜分离的肝细胞。各冻存组解冻后,比较各组的肝细胞活性、贴壁率、LDH 漏出量及白蛋白合成能力。结果新鲜分离的肝细胞产量为（1．8＆#177;0．4）1010/肝,活性高达（90．5＆#177;1．7）％,培养后贴壁率为（70．5＆#177;8．5）％。采用程序冻存的 a、b 组在肝细胞活性、贴壁率、LDH 漏出量及白蛋白合成能力方面明显优于常规冻存的 c、d 组（P＜0．05）,但与 e 组新鲜分离的肝细胞相比,其解冻后肝细胞的活性、贴壁率及蛋白合成能力均降低（P＜0．05）,LDH 漏出量增加（P＜0．05）。相同冻存方法条件下,50 mL 袋装与100 mL 袋装冻存效果差异无统计学意义（P＞0．05）。结论采用袋装程序冻存法大量冻存猪肝细胞可满足生物人工肝对肝细胞活性及数量的要求。     

实验用小型猪相关生物人工肝猪逆转录病毒感染的初步研究

猪肝细胞型生物人工肝治疗肝功能衰竭在我国刚起步．而影响猪肝细胞用于临床治疗的主要问题之一是猪内源性逆转录病毒（PERV）感染。我们建立PCR、RT—PCR方法，检测了猪肝细胞、肝细胞悬液及经生物人工肝治疗患者治疗前后的血清，旨在初步说明PERV在上述样本中的存在和表达及生物人工肝治疗后PERV的感染情况。     

人体可移植肝细胞来源的研究现状

肝细胞移植和生物人工肝装置的核心问题是具有生物活性的足够数量肝细胞来源，肝细胞来源是解决肝衰竭患者的关键。     

生物人工肝的研究进展

肝移植是治疗急慢性肝功能衰竭最有效的方法,但是,由于供肝缺乏,必需寻求终未性肝病替代疗法。其中主要方法之一是体外生物人工肝支持系统,简称生物人工肝(bioartificialliver ,BAL) ,发展至今已有40多年。临床试验证明BAL能促进肝功能衰竭病人的恢复,或过渡到肝移植。生物反应器是BAL的核心部件,生物反应器设计的主要目的在于保持肝细胞活力和功能,且不妨碍肝细胞营养及代谢产物的交换,同时还能起到治疗作用。细胞材料是BAL治疗基础,维持细胞活率和功能对BAL功效有决定性作用。本文就生物人工肝装置在实验和临床试验中的研究进展作一…     

An improved ex vivo method of primary porcine hepatocyte isolation for use in bioartificial liver systems

INTRODUCTION: Primary porcine hepatocytes are commonly, used in bioartificial liver devices and for in vitro studies of hepatocyte function. Although in vivo isolation of porcine hepatocytes can give high yield and viability, such methods are time-consuming and expensive, requiring specialist surgical facilities. AIM: To develop a simple, low-cost, high viability, high yield, reproducible ex vivo method for obtaining functional porcine hepatocytes for use in bioartificial liver systems. METHODS: Weanling piglets (12 kg) were killed with pentobarbitone sodium, the infra-hepatic inferior vena cava was clamped and the supra-hepatic inferior vena cava cannulated. The whole liver was retrogradely perfused in situ with cold saline and excised, followed by an ex vivo open-loop and re-circulating perfusion method (at 37 degrees C) in five steps. The liver was disrupted, sequentially filtered in washing buffer, purified by centrifugation and resuspended in Williams E medium. Viability and cell number were assessed using trypan blue exclusion. The cells were subsequently cultured in serum-free chemically-defined medium and function was assessed. RESULTS: The time interval from when the animals were killed to the final cell wash was 105+/-5 min (n = 20). Cell viability was 85+/-6% with a yield of (2.4+/-0.5) x 10(10) from 12+/-1 kg piglets using 0.03% (w/v) collagenase (n = 20). Hepatocytes from all isolations were successfully plated and grown in monolayer culture. In freshly isolated hepatocytes (day 0) total protein content (TP) was 1.2+/-0.1 mg/10(6) cells (n = 5) and 1.2+/-0.3 mg/10(6) cells (n = 5) for day 2 monolayer cultures, corresponding to approximately 9x10(6) hepatocytes per dish. The percentage of total LDH released into the medium was 13+/-4% for day 0 and 8+/-4% at day 2; conversely, intracellular LDH activities were 87+/-4% and 92+/-4% of the total, respectively. The urea synthesis rate was 196+/-36 nmol/h/mg total protein at day 0 (n = 5) and 292+/-62 nmol/h/mg protein (n = 9) at day 2. The total P450 content was 99+/-11 pmol/mg total protein for fresh cells (n = 5) and maintained at 89+/-35 pmol/mg total protein in day 2 cultures. CONCLUSIONS: This ex vivo method provides a high viability, high yield, cost-effective and rapid technique for isolating functional porcine hepatocytes with high plating efficiency, which compares favourably with results obtained using complex in vivo techniques.     

生物人工肝临床应用研究进展

人工肝支持系统(artificial liver support system,ALSS)是借助体外机械、化学或生物性装置,暂时替代肝功能,清除各种有害物质,补充生物活性物质,从而使肝细胞得以再生,直至自体肝功能恢复或等     

The newly established human hepatocyte cell line: application for the bioartificial liver

BACKGROUND/AIMS: Human hepatocyte cell lines are reported to lose many of their biochemical functions in a hybrid artificial liver support system (HALSS). Differentiation therapy is useful to up-regulate liver function. METHODS: The human hepatoblastoma cell line HepG2 was transfected with HSV/tk gene. Albumin synthesis and ammonia removal activity were evaluated when HepG2/tk was cultured with histone deacetylase inhibitor (FR228) and peroxisome proliferator activated receptor-gamma ligand (pioglitazone). To investigate the function of HepG2/tk in vivo, cell transplantation for 90% hepatectonized rats was conducted. RESULTS: We established stable cell lines which expressed HSV/tk and were sensitive to gancyclovir in vitro and in vivo. Both albumin synthesis rate and ammonia removal rate improved for HepG2/tk incubated with FR228 and pioglitazone for 3 days, which induced nuclear transport of p21. Rats with intrasplenic injection of HepG2/tk precultured for 3 days with FR228 and pioglitazone survived significantly longer than the control rats. The ammonia and total bilirubin concentrations were significantly lower in the test group than in the control group. The injection of gancyclovir inhibited the prolonged survival of the rats with precultured HepG2/tk. CONCLUSIONS: HepG2/tk is safe as well as enhancing high levels of liver function. It will be a potential cell source for HALLS in the future.     

Recent advances in the application of cultured hepatocytes into bioartificial liver

INTRODUCTIONOver the past 3 decades,various experimentalliver support systems have been studied.Early artifi-cial liver support systems included hemodialysis,ex-tracorporeal liver perfusion,human cross-circulation,charcoal hemoperfusion,hepatodialysis,fresh blood,plasma exchange transfusion etc.Thesesystems were developed to remove toxic substancesfrom the blood.Clinical trials showed that thesedetoxification systems could promote the recovery of     

Experimental study of bioartificial liver with cultured human liver cells

ＭＥＴＨＯＤＳＴｈｅｌｉｖｅｒｓｕｐｐｏｒｔｅｘｐｅｒｉｍｅｎｔｏｆＥＢＬＳＳｃｏｎｓｉｓｔｉｎｇｏｆａｇｇｒｅｇａｔｅｓｃｕｌｔｕｒｅｄｈｕｍａｎｌｉｖｅｒｃｅｌｓ,ｈｏｌｏｗｆｉｂｅｒｂｉｏｒｅａｃｔｏｒ,ａｎｄｃｉｒｃｕｌａｔｉｏｎｕｎｉｔ...     

生物人工肝用肝细胞低温保存的现状与进展

大量功能好的肝细胞是生物人工肝支持系统(bio-artificial liver support system,BLASS)的核心,是制约BLASS临床广泛应用的瓶颈.探索出一种实用的肝细胞低温保存方法,建立一个随时可用(ready to use)的肝细胞库是BALSS普遍推广的基础.目前肝细胞低温保存分为4℃或零下非结冰保存和-80℃或-196℃深低温冻存两大类,两大类保存方法各有优缺点.影响肝细胞低温保存的因素很多,如保存(冻存)液、(冻存)保护剂等.有关肝细胞在低温保存过程中死亡的机制尚未完全阐明,但大量研究发现细胞凋亡是除了坏死之外低温保存肝细胞死亡的另一个重要的途径.     

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.