肝细胞移植治疗急性肝衰竭

“20世纪是药物治疗的年代，21世纪却是细胞治疗的年代”。细胞治疗是指将干细胞或由其分化产生的功能细胞植入病变部位代偿病变细胞丧失的功能，或将细胞经体外遗传操作后用于疾病治疗的方法。急性肝衰竭（acute liver failure，ALF）常规药物治疗疗效差，是一种病死率极高的临床综合征，在我国主要由病毒性肝炎引起。近几年开展的较为有效的治疗方法主要有原位肝移植、人工肝支持系统和肝细胞移植（hepatocyte transplantation，HTx）。肝移植被认为是目前治疗ALF的最有效方法，但供肝短缺、免疫排斥和高额费用限制了受益者范围。     

肝细胞移植治疗肝功能衰竭的研究进展

肝细胞移植经过近30年的发展,正由动物实验向临床过渡,初步结果显示,肝细胞移植在肝功能衰竭和遗传代谢性肝病方面具有很好的应用价值。现就肝细胞移植在治疗肝功能衰竭中的研究现状作一综述。一、肝细胞移植的概念和理论基础肝细胞移植(hepatocyte transplantation,HCT)是将正     

微囊化猪肝细胞移植对大鼠急性肝功能衰竭的治疗作用

国内外文献报道异种和同种肝细胞移植对急性药物性肝功能衰竭有相似的疗效。随着免疫隔离技术的发展，国内外已有学者将这一技术应用于同种肝细胞中，并取得了令人鼓舞的成果，本研究将免疫隔离技术之一的微囊包裹技术应用于猪肝细胞，制备微囊包裹肝细胞，移植于肝衰竭大鼠体内，观察其逆转药物性肝衰竭的效果，以期为今后过渡到临床应用打下一定基础。     

微载体粘附培养的肝细胞腹腔内移植治疗急性肝功能衰竭大鼠的实验研究

目的　评价微载体粘附培养的肝细胞腹腔内移植对D 氨基半乳糖盐酸盐诱导的大鼠急性肝功能衰竭的治疗作用。方法　胶原酶灌注分离大鼠肝细胞 ,行Cytodex 3粘附培养 ,移植 1× 10 7个微载体粘附肝细胞至D 氨基半乳糖盐酸盐诱导的急性肝功能衰竭大鼠腹腔内 ,比较各组间生存率、肝功能及肝脏病理改变情况以评估疗效。结果　微载体移植组大鼠存活率明显高于空载体组 ,移植 5d后两组比较差异显著 (P <0 .0 5 ) ;空载体组大鼠肝功能及肝脏修复明显迟于微载体组。结论　微载体粘附培养的肝细胞腹腔内移植可对急性肝衰竭大鼠提供代谢支持作用 ,提高急性肝衰竭大鼠存活率。     

肝细胞移植治疗肝衰竭研究进展

约有17.5%的肝衰竭患者需行肝移植治疗。虽然肝移植已经广泛开展,并且术后一年生存率可达80%,但限于供体肝脏来源困难和昂贵的治疗费用,难以普及使用。肝细胞移植则是治疗暴发性肝衰竭与遗传性肝病的另一治疗手段,已经有数名患者为恢复肝功能而接受了这种细胞移植。肝细胞移植与原位肝移植相比具有以下几方面优势:1.同一来源的肝细胞可以用于多个受体;2.不需要手术,也没有整肝移植的高死亡率;3.可以临时应用起过渡作用,以等待肝脏复原或移植。     

微囊化肝细胞移植治疗暴发性肝衰竭大鼠的疗效观察

目的观察微囊化肝细胞腹腔移植对暴发性肝衰竭大鼠的疗效。方法气流法制备含肝细胞的微囊。D-氨基半乳糖诱导大鼠急性肝功能衰竭模型,设模型组、裸肝细胞腹腔移植组、微囊化肝细胞移植组,每组8只测定ALT、AST、TBil水平。样本比较采用重复测量的多元方差分析或单因素方差分析,组间比较采用t检验。结果肝衰竭模型建立后ALT、AST、TBil迅速升高（P〈0．05）,并于24h～72h达高峰。同一时间点两两比较发现,Ⅱ、Ⅲ组在48h、72h、120h均明显低于Ⅰ组（P〈0．05）,Ⅲ组在48h、72h均低于Ⅱ组（P〈0．05）。Ⅲ组峰值较Ⅰ组前移。模型组、裸肝组和微囊组大鼠生存率分别是26．7％（4／15）、40．0％（6／15）、73．3％（11／15）,微囊组较模型组、裸肝组大鼠生存率有显著性提高（P〈0．05）。结论HCT能降低FHF大鼠ALT、AST和TBil水平,减轻肝损伤程度,可能改善FHF预后。     

D-氨基半乳糖/脂多糖诱导急性肝衰竭大鼠肝细胞凋亡的电镜观察

目的通过透射电镜观察急性肝衰竭大鼠肝脏超微结构,以了解肝细胞凋亡的形态学变化。方法给予雌性Wistar大鼠D-氨基半乳糖/脂多糖联合腹腔注射,计算其死亡率及生存时间,动态观察给药后4、8、12小时肝功能和组织病理学变化,并以TUNEL法检测和电镜观察原位细胞凋亡。结果实验组80%大鼠死于急性肝衰竭,平均生存时间为15.6±1.8小时。给药后4小时电镜观察,发现肝细胞线粒体肿胀、内质网扩张,胞浆包含体形成,部分细胞核呈早期凋亡表现,细胞内凋亡小体形成;8小时时,肝细胞凋亡明显增多并呈不同阶段表现,凋亡肝细胞内线粒体病变程度不一;TUNEL检测给药后8小时肝细胞,凋亡指数达高峰,与电镜观察结果一致。结论肝细胞凋亡为D-氨基半乳糖/脂多糖致急性肝衰竭大鼠肝细胞的主要病理形态学表现,这可能为该类型肝衰竭的主要病理学变化基础。     

肝细胞体内移植治疗重型肝炎初步临床研究

目的探讨人肝细胞体内移植治疗肝衰竭患者的有效性与安全性.方法分离健康志愿者捐献的肝脏,获得人原代肝细胞并冷冻保存,复苏后经股动脉插管移植到脾脏,观察治疗前后肝衰竭患者临床症状、血液生化指标及脾脏核磁共振信号的改变.结果人肝脏可获取2×1010肝细胞,复苏后肝细胞活率在70%以上,移植的肝细胞数为2×109个.移植后1个月,患者临床症状明显改善,血胆红素、NH3、转氨酶明显降低,凝血酶原活动度水平明显升高.出院50 d后随访各项血生化指标均恢复正常,脾脏内可见肝细胞信号.结论肝细胞体内移植是一项安全有效的治疗方法,移植的肝细胞能在脾脏内增殖、分化,替代或部分恢复肝脏合成、解毒和代谢功能,将为终末期肝病治疗开辟一项新的治疗措施.     

非人灵长类动物急性肝功能衰竭模型的建立

目的:构建一种稳定的非人灵长类大动物急性肝衰竭模型.方法:将15只食蟹猴随机分成3组,经颈外静脉分别注射0.45、0.3和0.15 g/kg D-氨基半乳糖胺,构建食蟹猴肝衰竭动物模型.动态检测给药前(0 h)、给药后12、24、36、48、60、72、96 h生命体征、颅内压(ICP)及血清丙氨酸转氨酶(ALT)、凝血酶原时间(PT)、总胆红素(TBIL)、血氨(NH3)的变化.观察动物临床表现及病程进展情况,记录动物生存时间,动物死亡后行组织病理学检查.结果:0.45 g/Kg和0.3 g/Kg组动物全部死亡,生存时间分别为56.1 h±8.1 h和109.8 h±11.2 h,0.15 g/Kg实验猴除1只98 h死亡外其余全部存活.0.45 g/Kg和0.3 g/Kg组动物各时点的ALT、TBIL、PT、NH3及ICP均明显高于基线值(0h),差异有统计学意义(P＜0.05),0.15 g/Kg实验猴各指标在60 h后渐恢复正常.死亡动物组织病理检查见肝细胞坏死,肝内炎性细胞浸润.结论:以0.3 g/Kg的D-氨基半乳糖胺诱发食蟹猴急性肝功能衰竭动物模型,符合急性肝功能衰竭的临床表现、病理变化,可用于急性肝功能衰竭的动物实验研究.     

骨髓干细胞移植治疗急性肝衰竭的实验研究

目的探讨骨髓干细胞移植治疗实验性肝衰竭大鼠的效率及可行性。方法以D-氨基半乳糖及内毒素制备雌性大鼠肝衰竭模型,然后动员和富集雄性大鼠骨髓干细胞,并经门静脉移植至雌性大鼠肝脏;移植后观察受体雌鼠临床表现、生化改变、肝脏病变及肝组织Y染色体阳性率。结果受体肝衰竭雌鼠在细胞移植后,精神、食欲逐渐好转,生化异常及肝脏病变得以恢复,肝脏可检出Y染色体阳性的供体源性肝细胞;未接受骨髓干细胞移植的对照组雌鼠则全部死亡。结论骨髓干细胞移植对实验性肝衰竭大鼠具有明确治疗作用。     

Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children

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骨髓间充质干细胞移植治疗大鼠急性肝功能衰竭

目的探讨骨髓间充质干细胞（BMSCs）移植对急性肝功能衰竭的治疗作用。方法大鼠90%肝脏切除制备急性肝功能衰竭模型,实验组大鼠肝内移植2×106个BMSCs,对照组仅注射生理盐水。观察大鼠的生存情况,RT-PCR检测BMSCs在肝脏的植入,血清检测肝功能确认BMSCs移植对肝脏修复的作用。结果移植BM-SCs的实验组大鼠存活80%,对照组大鼠仅存活20%。RT-PCR显示,BMSCs移植后定植于大鼠肝脏内。肝功能检测显示,实验组大鼠的血清丙氨酸氨基转移酶水平、天冬氨酸氨基转移酶水平显著降低,血清白蛋白水平显著升高。结论 BMSCs移植可以显著提高急性功能衰竭大鼠的生存率,促进其肝功能的恢复,对急性肝功能衰竭的治疗具有积极作用。     

微囊化肝细胞移植对急性肝功能衰竭大鼠细胞因子的影响

目的 观察微囊化肝细胞移植对急性肝功能衰竭(ALF)大鼠脂多糖(LPS)、TNF-α、IL-1β和IL-6的影响.方法 用D-氨基半乳糖诱导大鼠ALF模型.造模后18 h,60只大鼠均分为模型组、裸肝细胞移植组和微囊化肝细胞移植组,72 h取血标本,检测血常规、Cr、肝功能、PT和血氨;鲎试剂检测LPS;ELISA法检测TNF-α、IL-1β和IL-6.多组比较采用单因素方差分析.结果 裸肝细胞移植组和微囊化肝细胞移植组大鼠Cr、肝功能、PT及血氨较模型组有明显改善(P<0.01),且微囊化肝细胞移植组较裸肝细胞移植组改善更明显,差异有统计学意义(P<0.01).裸肝细胞移植组和微囊化肝细胞移植组LPS为(1.2±0.3)和(0.5±0.1)ng/L,TNF-α为(27.7α4.2)和(21.7±3.2)μg/L,IL-1β为(298.7±13.9)和(247.7±14.1)ng/L,IL-6为(275.7±51.8)和(208.7±48.1)ng/L;模型组分别为(2.1±0.6)ng/L、(37.7±5.1)μg/L、(355.5±26.4)ng/L和(424.8±67.8)ng/L,裸肝细胞移植组LPS、TNF-α、IL-1β、IL-6与模型组比较,差异有统计学意义(F=6.24,F=67.3,F=8.38,F=7.59,均P<0.01);微囊化肝细胞移植组与模型组比较,差异亦有统计学意义(F=11.73,F=10.75,F=15.91,F=10.83,均P<0.01);微囊化肝细胞移植组与裸肝细胞移植组比较,差异亦有统计学意义(F=5.49,F=4.01,F=7.53,F=3.35,均P<0.01).结论 微囊化肝细胞移植可通过降低LPS、TNF-α、IL-1β及IL-6而发挥抗ALF的作用.     

Inappropriate pemoline therapy leading to acute liver failure and liver transplantation

A 36-year-old female, presenting with jaundice, developed acute liver failure requiring orthotopic liver transplantation. On admission, none of the known causative factors for acute hepatitis, including use of drugs, were found to be present. Several days after hospitalization, the patient admitted taking therapy prescribed by a "non-traditional" physician, that she had been using for several years due to overweight and which had recently been modified with the introduction of pemoline. A considerable body of evidence exists in the medical literature showing that pemoline, which is a central nervous system stimulant, has variable hepatotoxic effects, ranging from a mild transient increase of serum transaminases to liver failure, including some lethal cases.     

Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes

BACKGROUND/AIMS: Liver failure is a life threatening condition currently treated by palliative measures and, when applicable, organ transplantation. The use of a bioartificial organ capable of fulfilling the main functions of the liver would represent an attractive alternative. However, the shortage of suitable donor cells, and their limited growth ability have impeded the development of this strategy. We investigated whether lentiviral vectors allow for conditional immortalization of human hepatocytes and whether these immortalized hepatocytes could reverse lethal acute liver failure. METHODS: We exposed primary human hepatocytes to Cre-excisable lentiviral vectors coding for SV40T Antigen, telomerase, and/or Bmi-1 and tested the functionality of the resulting cell lines. Therapeutic potential of immortalized hepatocytes were tested in a murine model of acetaminophen-induced hepatic injury. RESULTS: The immortalized hepatocytes grew continuously yet were non-tumorigenic, stopped proliferating when exposed to Cre recombinase, and conserved defining properties of primary hepatocytes, including the ability to secrete liver-specific proteins and to detoxify drugs. The implantation of encapsulated immortalized human hepatocytes rescued mice from lethal doses of acetaminophen. CONCLUSIONS: Lentiviral vectors represent tools of choice for immortalization of non-dividing primary cells, and lentivirally immortalized human hepatocytes are promising reagents for cell-based therapy of acute liver failure.     

Adult-to-adult living donor liver transplantation for acute liver failure in China

AIM:To investigate the long-term outcome of recipients and donors of adult-to-adult living-donor liver transplantation(AALDLT) for acute liver failure(ALF).METHODS:Between January 2005 and March 2010,170 living donor liver transplantations were performed at West China Hospital of Sichuan University.All living liver donor was voluntary and provided informed consent.Twenty ALF patients underwent AALDLT for rapid deterioration of liver function.ALF was defined based on the criteria of the American Association for the Study of Liver Diseases,including evidence of coagulation abnormality [international normalized ratio(INR) ≥ 1.5] and degree of mental alteration without pre-ex-isting cirrhosis and with an illness of 26 wk duration.We reviewed the clinical indications,operative procedure and prognosis of AALDTL performed on patients with ALF and corresponding living donors.The potential factors of recipient with ALF and corresponding donor outcome were respectively investigated using multivariate analysis.Survival rates after operation were analyzed using the Kaplan-Meier method.Receiver operator characteristic(ROC) curve analysis was undertaken to identify the threshold of potential risk factors.RESULTS:The causes of ALF were hepatitis B(n = 18),drug-induced(n = 1) and indeterminate(n = 1).The score of the model for end-stage liver disease was 37.1 ± 8.6,and the waiting duration of recipients was 5 ± 4 d.The graft types included right lobe(n = 17) and dual graft(n = 3).The mean graft weight was 623.3 ± 111.3 g,which corresponded to graft-torecipient weight ratio of 0.95% ± 0.14%.The segment Ⅴor Ⅷ hepatic vein was reconstructed in 11 right-lobe grafts.The 1-year and 3-year recipient's survival and graft survival rates were 65%(13 of 20).Postoperative results of total bilirubin,INR and creatinine showed obvious improvements in the survived patients.However,the creatinine level of the deaths was increased postoperatively and became more aggravated compared with the level of the survived recipients.Multivariate analysis showed that waiting duration was independently correlated with increased mortality(P = 0.014).Furthermore,ROC curve revealed the cut-off value of waiting time was 5 d(P = 0.011,area under the curve = 0.791) for determining the mortality.The short-term creatinine level with different recipient's waiting duration was described.The recipients with waiting duration ≥ 5 d showed the worse renal function and higher mortality than those with waiting duration 5 d(66.7% vs 9.1%,P = 0.017).In addition,all donors had no residual morbidity.Furthermore,univariate analysis did not show that short assessment time induced the high morbidity(P = 0.573).CONCLUSION:Timely AALDLT for patients with ALF greatly improves the recipient survival.However,further systemic review is needed to investigate the optimal treatment strategy for ALF.