免疫隔离技术在异种肝细胞移植中的应用

目的探讨微囊化猪肝细胞腹腔内移植对药物性肝衰大鼠的治疗作用，观察移植大鼠存活率、肝功能的变化。方法以海藻酸钠体外包裹经胶原酶灌注法分离的乳猪肝细胞，以SD大鼠为受体，D-氨基半乳糖腹腔内注射诱导大鼠急性肝衰竭，48h后将微囊化的猪肝细胞移植于大鼠腹腔内，观察移植大鼠存活率、绘制生存曲线，并测定肝功能的变化。结果腹腔内肝细胞移植可显著改善大鼠肝功能，转氨酶及胆红素均较对照组明显下降（P〈0．05）。与裸肝细胞移植组相比．微囊化肝细胞移植组1周存活率（78．6％ vs 66．7％）及2周存活率（42．9％ vs 25．0％）均显著提高（P〈0．01）。结论对异种肝细胞经微囊化处理后移植治疗急性肝衰大鼠，可给予肝功能代谢支持，提高移植治疗效果。     

肝移植治疗暴发性肝衰肝性脑病的临床研究

目的 总结肝移植治疗暴发性肝衰肝性脑病的临床经验。方法 回顾性分析４例暴发性肝衰肝性病病人行肝移植手术治疗的临床资料。结果 肝移植治疗暴发性肝衰肝性脑病１个月存活率７５％（３／４）,超过３个月存活率为５０％（２／４）。结论 肝移植是治疗暴发性肝衰肝性脑病的一种有效方法,暴发性肝衰肝性脑病不是肝移植手术的禁忌证。     

大鼠肝细胞肾脏内移植后组织形态与肝功能衰竭治疗作用研究

本实验通过同种异体大鼠肝细胞肾脏内移植，尝试在受体体内建立第二肝脏。对移植肝细胞进行长期组织形态与功能观察，并利用该方法进行急性肝功能衰竭肝功能辅助支持治疗研究。结果表明肝细胞肾脏内移植后可长期存活、增殖而且具有肝组织结构重建及保持肝脏功能的能力。４×１０７个肝细胞肾脏内移植后可显著提高受体大鼠对肝功能衰竭的承受力，移植组术后３天行９０％肝切除诱发急性外科型肝功能衰竭后其存活率显著高于对照组。以上结果显示肾脏可以成为肝细胞移植又一适宜部位，肝细胞肾脏内移植有可能成为临床上肝脏代谢及功能障碍疾病的一种治疗手段。     

原位肝移植治疗急性肝功能衰竭21例临床分析

目的探讨原位肝移植治疗急性肝功能衰竭的临床疗效。方法回顾性分析天津市第一中心医院21例因急性肝功能衰竭行原位肝脏移植术病人的临床资料，总结急性肝衰竭实施肝移植的经验。结果21例病人随访时间为3～60个月，中位随访时间为25个月。生存时间为1—1530d，中位生存时间为517d。病人的围手术期存活率为66．7％，1年存活率为66．7％，2年存活率为66．7％。死亡原因包括多脏器功能衰竭、颅内出血、上消化道出血、急性成人呼吸窘迫综合征、移植物原发性无功能。14例的远期性生存病人中，13例移植前合并乙型肝炎（乙肝），其中有1例（1／13）在术后1年出现乙肝复发。结论肝移植术是治疗急性肝功能衰竭最有效的方法，严格把握适应证、合理选择手术时机是提高疗效的关键。     

载肝细胞生长因子的纳米粒子治疗急性肝衰竭大鼠

目的 观察载肝细胞生长因子(HGF)的聚乳酸-O-羧甲基壳聚糖(PLA-O-CMC)纳米粒子培养的大鼠肝细胞腹腔内移植对急性肝衰竭(ALF)大鼠的治疗效果.方法 分别将5 ml培养24 h的大鼠肝细胞(5.0×107个)(Ⅰ组)、PLA-O-CMC纳米粒子培养的大鼠肝细胞(Ⅱ组)、载HGF的PLA-O-CMC纳米粒子培养的大鼠肝细胞(Ⅲ组)移植到ALF大鼠腹腔内,以PLA-O-CMC纳米粒子培养的大鼠肝细胞腹腔移植加每天静脉注射HGF 10 μg/kg×7 d(Ⅳ组)和5 ml RPMI 1640培养基腹腔内注射(Ⅴ组)作为对照.观察受体大鼠存活率、肝功能、肝组织光镜和电镜变化.结果 移植后14d大鼠的存活率Ⅰ～Ⅴ组分别为50.00％、68.75％、81.25％、75.00％和18.75％,各移植组高于对照组,Ⅲ组最高.移植后24 h,Ⅱ～Ⅳ组各项肝功能指标开始好转,至移植后7d,各组间除谷丙转氨酶(ALT)外,差异无统计学意义(P＞0.05).Ⅲ组的肝功能和肝脏病理损害恢复最快,其次为Ⅳ、Ⅱ、Ⅰ组,Ⅴ组恢复最慢、最差.结论 应用载HGF的PLA-O-CMC纳米粒子培养的大鼠肝细胞腹腔内移植治疗ALF大鼠能逆转肝功能,提高生存率,较静脉途径给予HGF的治疗效果更好.     

急性肝衰竭大鼠载肝细胞生长因子纳米粒子肝细胞移植后有丝分裂指数和Ki-67表达变化及意义

目的 探讨急性肝衰竭(ALF)大鼠载肝细胞生长因子(HGF)的聚乳酸-氧-羧甲基壳聚糖(PLA-O-CMC)纳米粒子肝细胞移植后有丝分裂指数和Ki-67抗原的表达变化及意义.方法 D-氨基半乳糖腹腔内注射制作大鼠ALF模型,48 h后分别将Ⅰ型胶原(Ⅰ组)、PLA-O-CMC纳米粒子(Ⅱ、Ⅳ组)、载HGF的PLA-O-CMC纳米粒子(Ⅲ组)培养的大鼠肝细胞(均为5×107个,5 ml)移植到ALF大鼠腹腔内.以每日静脉注射HGF 10μg/kg×7 d(Ⅳ组)、5 Ml RPMI 1640腹腔内注射(Ⅴ组)作为对照.观察其14 d存活率、血清及肝组织HGF浓度、肝组织病理及有丝分裂指数(MI)、Ki-67变化.结果移植后14 d Ⅰ～Ⅴ组ALF大鼠的存活率分别为50.00％、68.75％、81.25％、75.00％、18.75％.各纳米移植组高于V组.Ⅲ组3 d时肝组织HGF浓度最高,平均为5882.91 μG／L.Ⅲ组肝组织结构恢复更快,5 d时平均MI 10.20％0、7 d时平均MI 10.20‰、7 d时Ki-67平均标记指数16.8‰,均高于Ⅴ组.结论 应用载HGF的PLA-O-CMC纳米粒子培养的大鼠肝细胞腹腔内移植治疗ALF大鼠能促进肝再生相关抗原表达.

Abstract：

Objective To evaluate the change and significance of Ki-67 antigen expression follow ing hepatocyte transplantation using hepatocyte growth factor (HGF) loaded polylactic acid-O-carboxymethylchitosan (PLA-O-CMC) nanoparticles in rats with acute liver failure (ALF). Methods ALF models of rats were established by D-galactosamine intraperitoneal injection. Rat hepatocytes type Ⅰ collagen suspension (group Ⅰ ),rat hepatocytes co-cultured with PLA-O-CMC nanoparticles ( groups Ⅱ ,Ⅳ ) and rat hepatocytes co-cultured with HGF loaded PLA-O-CMC nanoparticles ( group Ⅱ ) (5 × 107 cells each group,5 ml) were transplanted into the abdominal cavity of SD rats at 48 h after D-Gal injection. Intravenous injection of HGF ( 10 μg/kg for 7 days) ( group Ⅳ ) and RPMI 1640 injection (group Ⅴ ) were used as the negative groups. The 14th-day survival rate of rats,pathological change and mitotic index of liver,Ki-67 antigen labeling index of ALF rat livers were observed. Results The 14th-day survival rate in groups Ⅰ -Ⅴ was 50.00％,68. 75％,81.25％,75.00％,18.75％,and that in nano-transplanted groups was higher than in group Ⅴ. At the 3rd day,the concentration of HGF in liver tissue of group Ⅲ,average 5882. 91 μg/L was the highest. The hepatic lobules structure in group Ⅲ recovered faster. The average mitotic index in group Ⅱ at the 5th day was 10. 20‰ and the average Ki-67 labeling index at the 7th day was 16. 8‰,which were all higher than in group Ⅴ. Conclusion Intraperitoneal transplantation of HGF loaded PLA-O-CMC nanoparticle-attached hepatocytes for treatment of ALF can promote the liver regenerationassociated antigen expression.     

自体脾内移植的肝细胞凋亡及与其功能的关系

目的探讨肝细胞脾内移植后发生非免疫排斥性细胞凋亡及其对总体功能发挥的影响。方法应用TUNEL法原位观察80例大鼠肝细胞脾内移植后移植肝细胞的凋亡;分析移植肝细胞凋亡指数与肝化脾ALT含量及同位素99mTC-HIDA摄取量的相关性。结果应用了肝细胞生长因子的大鼠肝细胞脾内移植后移植肝细胞的凋亡指数为（2．76±1．08）,而未应用者则为（5．26±2．14）,两者差异有显著性（P＜0.01）。两组中移植肝细胞凋亡指数与肝化脾ALT含量和99mTc-HIDA摄取量均呈负相关。结论肝细胞脾内移植后较易发生非免疫排斥性细胞凋亡,其凋亡指数与肝细胞生存量及总体功能呈负相关,肝细胞生长因子可抑制移植肝细胞凋亡,从而提高其长期存活量。     

肝移植治疗药物性急性肝衰竭的体会（附8例报告）

目的 探讨肝移植治疗药物性急性肝衰竭的疗效.方法 药物性急性肝衰竭患者8例,术前肝功能Child-Pugh分级均为C级,均合并肝性脑病（Ⅲ～Ⅳ期）.所有患者均行经典原位肝移植,供体均为尸体供肝,均未行静脉转流.术后予免疫诱导、免疫抑制治疗,并予抗感染及支持治疗.结果 8例患者均顺利完成肝移植手术.术后1例女性患者并发原发性移植肝无功能,死亡.余7例患者于术后16～72 h苏醒.1例发生胆道铸型结石,手术取石效果不佳,予再次肝移植,行胆总管空肠吻合术,术后发生吻合口漏死亡.其余6例存活患者均痊愈,生活状况良好.其中2例曾发生急性呼吸窘迫综合征,行气管切开、呼吸机辅助呼吸,最后康复出院.结论 对于保守治疗无效的药物性急性肝衰竭,肝移植是唯一有效的治疗措施.     

肝移植术后急性肝动脉闭塞的介入治疗

目的评价介入医学在诊断和治疗肝移植术后急性肝动脉闭塞中的作用.方法对7例原位肝移植术后急性肝动脉闭塞病例的诊断和介入治疗过程进行了回顾性分析.结果 59例肝移植术后血管造影证实存在肝动脉闭塞7例,其中多普勒超声发现5例;经导管溶栓治疗,7例肝动脉闭塞中5例部分或全部开通,对其中4例进行了血管内支架植入治疗,经1～13个月随访,肝动脉血流通常,避免了再次肝移植.结论血管造影对肝移植后肝动脉并发症的诊断具有重要价值,介入手段是处理肝移植术后急性肝动脉闭塞的有效方法.     

Hepatocyte transplantation for total liver repopulation

Hepatocyte transplantation (HT) is an attractive therapeutic alternative to liver transplantation. A number of experiments have shown the feasibility of total liver parenchymal cell replacement by transplanted hepatocytes. In this review, we would like to highlight researches and clinical reports of HT for liver repopulation. Cellular source of clinical HT should be safety. Immortalized cells, hepatic stem cells, and other stem cells have been used for an experimental model for HT. The exact mechanism of the cell engraftment after HT has not been completely understood, although there were some markers to detect and investigate transplanted cells. In order to achieve liver repopulation following HT, a mild hepatic damage may need to facilitate cell engraftment and replace the host liver by transplanted cells. Hormonal factor may use for the same purpose. Despite the results of preclinical studies promising clinical benefits for cell therapy, the clinical experience of HT has been disappointing, except in a few cases. HT may become an alternative for liver transplantation in the future; however, many efforts should made before establishing an effective method for HT and liver replacement therapy.     

原位肝移植肝动脉重建的外科技巧

目的 探讨肝脏移植肝动脉重建的外科技巧和影响肝动脉重建效果的因素.方法 对2004-2005年施行的368例同种原位肝移植病人的临床资料进行分析总结.结果 368例肝移植病人中与动脉相关的并发症共22例,发生率为5.9%(22/368);围手术期发生的15例动脉相关并发症中,12例为动脉血栓形成,3例为误伤右副肝动脉而继发的大面积肝坏死;术后远期发生的7例动脉相关并发症中,动脉血栓形成3例和动脉广泛狭窄4例.所有22例动脉相关的并发症中,接受再次肝脏移植者13例.因肝动脉并发症导致病人死亡11例,占该组肝移植病人死亡的30.5%(11/36).结论 保护供体肝动脉的完整性、正确选择受体动脉的吻合部位、显微外科血管重建技术的应用以及预防或及时纠正血液高凝状态等是提高肝动脉重建质量,降低肝动脉并发症发生率的重要因素.     

肝功能衰竭患者的肝移植治疗

目的 报告３例危重肝功能衰竭患者的肝移植治疗经验。探讨影响该类患者肝移植效果的诸多因素及其解决办法。方法 ３例患者均同种异体背驮式原位肝移植术治疗。结果 例１，２分别已存活３２个月，１１个月余，生活质量良好；例３术后亦已１月余；例１，２术后曾发生胆道梗阻并发症，均经再次手术将胆管端－端吻合改为胆肠Ｒｏｕｘ－ｅｎ－Ｙ吻合后痊愈。结论 （１）采用有效的方法愉改善患者的术前状况，及时采取手术治疗是保证肝     

异种肝细胞移植治疗大鼠药物性肝衰的疗效观察

目的　观察异种肝细胞移植治疗大鼠药物性肝衰的疗效。方法　杂种豚鼠为供体 ,胶原酶消化法制备肝细胞。SD大鼠为受体 ,氨基半乳糖 (D GI)腹腔内 1次注射制作肝衰模型。 4 8h后将豚鼠肝细胞(1.5× 10 7个 )移植于大鼠脾内。同种移植及生理盐水为对照。移植后观察受体 2周存活率 ,在移植不同时间作移植物病理及组织化学检查。结果　受体 2周存活率 :异种移植组为 71% ,同种组为69 % (P >0 .0 5 ) ,生理盐水对照组为 2 5 % (P>0 .0 1)。豚鼠肝细胞移植后 12～ 2 4h其结构和功能基本保存完好。结论　与同种移植一样 ,异种肝细胞移植能逆转大鼠药物性肝衰。     

冷冻肝细胞移植治疗大鼠肝硬化和急性肝衰竭效果的比较

目的探讨冷冻肝细胞移植治疗大鼠肝硬化和急性肝衰竭的可行性,并比较其对该两种疾病的治疗效果。方法通过原位两步灌流法分离大鼠肝细胞,采用程序冷冻法冷冻肝细胞。利用四氯化碳(CCl4)及D-氨基半乳糖分别建立大鼠肝硬化及急性肝衰竭模型。实验分组:Ⅰ组为正常对照组;Ⅱa为肝硬化门静脉注射生理盐水组;Ⅱb组为肝硬化肝细胞移植组。Ⅲa组为肝衰竭门静脉注射生理盐水组,Ⅲb组为肝衰竭肝细胞移植组。检测各组门静脉压力、肝功能及病理学变化,并行统计学分析。结果冷冻的肝细胞复苏率为50%～70%。Ⅱb组,移植7 d与未移植0 d比较,肝功能、病理检查、门静脉压力等无显著变化。Ⅲb组,移植7 d与未移植0 d比较,ALT下降,总蛋白及清蛋白升高(P0.05)。结论冷冻肝细胞移植有助于急性肝衰竭大鼠肝功能的恢复,对肝硬化大鼠未发现明显疗效。     

Acute impairment of hepatic microcirculation and recruitment of nonparenchymal cells by intrasplenic hepatocyte transplantation

Background/Purpose

Over the last 20 years, hepatocyte transplantation (HcTx) has advanced from the experimental to the clinical stage. To date, HcTx has been performed in 30 patients in the United States. Regardless whether hepatocytes are transplanted into the spleen and migrate to the liver or are injected directly into the portal vein, transplanted liver cells will, to some extent, congest the recipient liver microcirculation. The potential negative consequences of intrasplenic HcTx were the subject of this study.

Methods

By using intravital microscopy, the authors investigated whether intrasplenic HcTx of 20 × 10⁶ allogenic hepatocytes would influence liver perfusion, excretory liver function, and nonparenchymal cells (Kupffer and Ito cells) in vivo.

Results

The sinusoidal perfusion rate declined significantly from 94% (control) to 84% on day 1 and 76% on day 7. Bile acid excretion decreased in a similar fashion from 0.924 mg/h (control) to 0.669 mg/h on day 7. The authors observed a significant increase of Ito cells from 81.1 cells per microscopic field (control) to 97.1 (day 1) and an increase of Kupffer cells (KC; 6.1 cells per microscopic field on day 1 v 3.8 on control).

Conclusions

This study shows an acute impairment of hepatic microcirculation and hepatucellular function along with an recruitment and activation of nonparenchymal cells in the early posttransplantation period after intrasplenic HcTx. Kupffer cell recruitment indicates an activation of local host defense, and Ito cell activation implies the initiation of liver repair mechanisms owing to ischemia-related cell damage.     

Fulminant hepatic failure post liver transplantation: clinical syndromes,correlations and outcomes

This paper reports the clinical syndrome of fulminant hepatic failure (FHF) following liver transplantation. FHF was defined as the sudden onset of liver failure [encephalopathy and prolonged International Normalised Ratio (INR)] without arterial thrombosis in the setting of a liver allograft. FHf post-transplant was seen in 8/154 (5.2%) adult patients undergoing transplantation. These eight patients developed a clinical syndrome characterised by: (a) a rapid rise in ALT levels to above 1000 U/l (mean maximum 1600 U/l), (b) a sudden increase in the INR to above 5 (mean maximum 5.6), (c) the development of high fever, (d) the persistence of thrombocytopenia (mean nadir 40×10⁹/dl), (e) a progressive rise in the bilirubin (mean maximum 400 mol/l) and (f) the development of hepatic encephalopathy. In seven cases this syndrome occurred following good initial graft function at day 6 post (mean)-transplant. In one case the above syndrome developed immediately after liver transplantation. Four of the eight patients developed multiorgan failure associated with systemic acidosis (mean pH 6.84). All of these patients died (mean day 11). Four patients developed systemic alkalosis. Two of these four patients underwent successful retransplantation (on days 12 and 13) and remain alive at a mean of 11 months post-transplant. Six of the eight patients received OKT3 therapy without any apparent affect on clinical outcome. Compared to a control group of patients (n=28), 2/8 versus 2/28 had a positive crossmatch with donor lymphocytes (P=NS), 1/8 versus 7/28 were ABO-non-identical (P=NS), 3/8 versus 10/21 had total MHC mismatches (P=NS) and 5/7 versus 6/16 had UW ischemic times above 10 h (P=NS). No patients had main hepatic artery thrombosis on angiography although four patients had evidence of intrahepatic microthrombi or arterial necrosis at autopsy. In all cases the histology showed massive haemorrhagic necrosis. Three cases had evidence of veno-occlusive lesions whilst foam cell arteriopathy was seen in two cases. Immunofluorescence was performed in three cases. In two cases there was evidence of immunoglobulin, complement and fibrin deposition in blood vessels. In conclusion, we describe an uncommon clinical syndrome occurring post liver transplant. This syndrome represents humorally mediated allograft rejection but there seems to be no relationship with tissue matching (antibody, ABO, MHC) or donor ischaemic times. If recognised earlier in the absence of multiorgan failure, urgent retransplantation seems to be the only effective therapy.     

急性肝功能衰竭临床特点和诊疗现状

正1前言虽然急性肝功能衰竭(acute liver failure,ALF)并非急重症中常见的临床疾病,但其具有非常高的死亡率。发达国家的ALF常发生在没有肝病的年轻患者中,药物诱导的肝损伤是主要原因[1];发展中国家的病因学主要是病毒性感染,如乙型     

Auxiliary liver transplantation with arterialization of the portal vein for acute hepatic failure

Six adult patients suffering from acute hepatic failure and with a high urgent status underwent heterotopic auxiliary liver transplantation. In four of these patients, the portal vein of the liver graft was arterialized in order to leave the native liver and the liver hilum untouched and to be able to place the liver graft wherever space was available in the abdomen. The arterial blood flow via the portal vein was tapered by the width of the anastomosis. Two patients died, one of sepsis on postoperative day 17 (POD), the other after 3 months due to a severe CMV pneumonia. There were no technically related deaths. The native liver showed early regeneration in all cases. In one patient, the auxiliary graft was removed 6 weeks after transplantation. Four weeks later, he had to undergo orthotopic retransplantation due to a recurrent fulminant failure of the recovered native liver. This patient is alive more than 1 year after the operation. We conclude that heterotopic auxiliary liver transplantation with portal vein arterialization is a suitable approach to bridging the recovery of the acute failing native liver. Received: 15 September 1997 Received after revision: 4 February 1998 Accepted: 2 March 1998     

急性肝衰竭的治疗进展

急性肝衰竭是多种肝脏疾病发展到终末期的共同病理生理过程.由于病因复杂、诊断困难,因而死亡率很高.目前急性肝衰竭的主要治疗方法有病因治疗和人工肝辅助治疗,而肝移植是治疗的最佳选择.本文就目前急性肝衰竭的治疗方法的最新进展进行综述希望对急性肝衰竭的临床诊治提供参考.     

肝移植治疗肝性脊髓病的临床讨论

肝性脊髓病是继发于终末期肝病的一种特殊类型的神经系统改变,是以脊髓病变为主要症状的综合征,多于自然形成的广泛的门体静脉侧支循环或门体静脉分流术后出现,临床比较罕见。该病预后不良,病情呈慢性进展。目前的治疗方法主要是针对原发肝病进行干预。外科医师为减少毒性物质的吸收行结肠游离手术或回肠直肠吻合术,短期内可降低血氨和改善神经症状,但长期效果不理想。2012年4月大连医科大学附属二医院收治1例肝性脊髓病患者。该例患者行肝移植术后临床症状、体征及生命质量均获得明显改善,双下肢肌力得到有效恢复。肝移植是从病因上治疗肝性脊髓病的有效方法。