肝细胞移植的研究进展

肝细胞移植已初步完成了从基础研究向临床应用的转化,目前认为,肝细胞移植在治疗终末期肝病和代谢性肝病中发挥重要作用。本文综述了肝细胞移植过程中所遇到的关键问题及其研究进展。尽管面临肝细胞来源缺乏及一些关键技术有待改进等问题,但越来越多的临床试验数据表明肝细胞移植具有十分广阔的应用前景。     

药物性重型肝炎1例肝细胞体内移植治疗的效果

目的探讨人肝细胞体内移植治疗药物性重型肝炎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BIL、NH3、ALT、AST明显降低,PA水平明显升高。出院50天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,移植的肝细胞能够在脾脏内增殖、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途径。     

骨髓干细胞应用于肝脏疾病的治疗

最近许多研究已经报道了骨髓干细胞在正常肝脏及一些肝脏病理条件下能够分化为有功能的肝细胞和胆管细胞且可修复受损肝脏。综述骨髓源性肝脏干细胞动物模型的证据、可能的机制和条件、目前争论的问题以及未来临床应用细胞移植治疗肝病的方式等。     

骨髓干细胞应用于肝脏疾病的治疗

最近许多研究已经报道了骨髓干细胞在正常肝脏及一些肝脏病理条件下能够分化为有功能的肝细胞和胆管细胞且可修复受损肝脏.综述骨髓源性肝脏干细胞动物模型的证据、可能的机制和条件、目前争论的问题以及未来临床应用细胞移植治疗肝病的方式等.     

磁标记干细胞肝移植的MR活体示踪研究进展

肝脏干细胞移植不但可以替代坏死的组织,还可刺激受体组织再生以达自身修复;此外,通过体外基因修饰肝脏干细胞,再移植给相应基因缺陷的受体肝脏以分化为具有正常功能的肝细胞,可以治疗肝脏代谢疾病.目前人骨髓干细胞自体移植技术已应用于临床终末期肝病的治疗,而且取得了较好的疗效.     

肝细胞移植临床应用的研究现状

尽管移植和免疫学的不断发展 ,美国每年仍有2 6 0 0 0人死于终末期肝脏疾病。原位肝移植可以治疗各种原因 (包括先天遗传因素或后天获得疾病 )所致的急、慢性肝功能衰竭或代谢紊乱。但受肝脏来源限制、移植费用巨大、病死率高以及长期需应用免疫抑制剂等影响 ,使临床应用受到限制。肝细胞移植是将游离的、有活性的肝细胞大量移植到受体内 ,移植入的肝细胞在受体内生长、增殖并发挥正常肝细胞的作用 ,可代替部分肝功能并介导基因治疗。目前肝细胞移植在各类动物模型中被广泛运用 ,以研究其临床应用的有效性和安全性[1 3] 。近年来的临床资料…     

肝细胞移植治疗重型肝炎临床观察

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 从健康志愿者捐献的肝脏中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,观察治疗后肝衰竭患者临床症状、生化指标、免疫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75%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BIL、NH3、ALT、AST明显降低,IgG、IgA、IgM、C3和CD4/CD3在细胞移植后两周降至最低,4周后恢复移植前水平;PTA水平明显升高.出院80天和270天后,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,移植的肝细胞能够在脾脏内增殖、分化,替代或部分恢复肝脏合成、解毒和代谢功能;应用肝细胞增强剂做MIR扫描可成为一项新的肝细胞移植无创随访检测手段.     

胚肝细胞移植后照射小鼠造血与淋巴组织的重建

胚肝细胞移植已用于儿童免疫缺陷病等疾患的治疗,亦曾试用于急性辐射事故的病例。移植胚肝细胞可减轻同种骨髓移植时发生的GVH病,但迄至目前为止,胚肝细胞移植在临床上的应用,无论在病例的数量上及病种的广度上都远不及骨髓移植。对胚肝造血细胞性能的了解,也不及骨髓。目     

肝细胞再生因子的研究近况

肝细胞再生因子的动物实验研究在国外已进行多年,多数学者肯定了用于治疗肝病的可能性。国内是先从人胎肝治疗重型肝炎开始的。1989年9月全国人胎肝临床研讨会上张宜俊介绍了由乳猪肝提取的小分子肝细胞生长素(Hepatocyte growth factor)的实验研究和临床应用后,引起关注。本文就肝细胞再生因子研究的进展作一摘要介绍。     

肝细胞移植解决α1抗胰蛋白酶缺乏

[动态]遗传性的α 1抗胰蛋白酶缺乏会引发肝病和肺气肿.肝脏合成的该蛋白的正常功能是抑制降解肺部结缔组织的中性粒细胞弹性蛋白酶.该病的典型形式是突变的α1抗胰蛋白酶低效分泌并积累在肝细胞中,因而降低了蛋白酶抑制剂的活性,导致肝损伤和肺气肿.鉴于变异蛋白的积累增加了肝细胞的压力,美国科学家最近研究了向表达该变异蛋白的转基因鼠模型移植表达野生型α1抗胰蛋白酶的肝细胞能否有效.     

大鼠骨髓基质细胞分离培养和经门静脉途径移植的初步研究

目的 观察骨髓基质细胞(marrow stromal cell，MSC)经门静脉移植后在同种异体大鼠体内的转归，为MSC体内诱导转化及功能发挥的研究提供基础。方法 采用连续传代培养的方法纯化．MSC，DAPI标记后经门静脉移植入受体，移植细胞数量为10^5个／只，20只受体大鼠分为4组，各组分别于移植后2h、1周、2周、3周、4周处死，观察受体肝脏及肺脏内DAPI标记的移植细胞的分布和存活情况。结果 体外培养的MSC具有良好的增殖能力，20只受体大鼠4只死亡，其余均存活至处死前，MSC经门静脉植入异体大鼠后2h、1周、2周、3周、4周受体肝脏内均可见DAPI标记的移植细胞存活，而各期受体肺内未见DAPI：标记的移植细胞存活。结论 经门静脉注入MSC后，细胞主要分布于受体肝脏，在肝脏内分布呈现由门静脉小分支逐渐向肝实质内移行的过程，并与肝细胞紧密结合呈索状排列。     

Biodistribution of adult derived human liver stem cells following intraportal infusion in a 17-year-old patient with glycogenosis type 1A

IntroductionCurrent treatment of inherited liver inborn errors of metabolism in children consists in appropriate diet and drugs and, for unstable patients, final orthotopic liver transplantation. Unfortunately, liver transplantation remains not easily available because of organ shortage and imposes inherent risks and lifelong immunosuppressive therapy. Therefore alternative treatments are required. Hepatocytes transplantation and its limitations led to consider innovative alternative such as transplantation of adult derived human liver stem cells (ADLHSC). These cells present high proliferative capacity, good resistance to cryopreservation and ability to differentiate into hepatocyte-like cells displaying mature hepatocyte functions.AimBiodistribution of ADHLSC had never been assessed after infusion through the portal vein in patients. This information is required to determine the safety of the method.MethodsADHLSC were efficiently labelled with 111-Indium DTPA radiotracer and SPECT imaging was used for the acquisition of whole body imaging to document short term biodistribution of ADHLSC.ResultsFollowing infusion through the portal vein, ADHLSC diffused homogenously throughout the liver and remained strictly within the targeted organ. Images were acquired until 5 days after infusion. At that time, no signal was observed in any other organs except the liver. Urinary excretion of 111-Indium DTPA was also monitored.ConclusionFor the first time, we documented the short term biodistribution of ADHLSC within the liver after infusion through the portal vein.     

In vivo detection of single cells by MRI.

The use of high-relaxivity, intracellular contrast agents has enabled MRI monitoring of cell migration through and homing to various tissues, such as brain, spinal cord, heart, and muscle. Here it is shown that MRI can detect single cells in vivo, homing to tissue, following cell labeling and transplantation. Primary mouse hepatocytes were double-labeled with green fluorescent 1.63-microm iron oxide particles and red fluorescent endosomal labeling dye, and injected into the spleens of recipient mice. This is a common hepatocyte transplantation paradigm in rodents whereby hepatocytes migrate from the spleen to the liver as single cells. One month later the animals underwent in vivo MRI and punctuated, dark contrast regions were detected scattered through the livers. MRI of perfused, fixed samples and labeled hepatocyte phantoms in combination with histological evaluation confirmed the presence of dispersed single hepatocytes grafted into the livers. Appropriate controls were used to determine whether the observed contrast could have been due to dead cells or free particles, and the results confirmed that the contrast was due to disperse, single cells. Detecting single cells in vivo opens the door to a number of experiments, such as monitoring rare cellular events, assessing the kinetics of stem cell homing, and achieving early detection of metastases.     

Use of indium-111-labeled hepatocytes to determine the biodistribution of transplanted hepatocytes through portal vein infusion

PURPOSE: Hepatocyte transplantation is useful for ex vivo gene therapy and liver repopulation. Methods for hepatic reconstitution were recently developed, but hepatocyte transplantation systems must be optimized. The authors report their experience with In-111 oxyquinolone labeling of a test dose of hepatocytes (108 cells) for noninvasive assessment of the biodistribution of transplanted hepatocytes in a 5-year-old child with omithine transcarbamoylase deficiency. MATERIALS AND METHODS: Donor hepatocytes (approximately 108) were radiolabeled using a commercially available In-111 oxyquinolone solution (specific activity of 1 mCi/ml). RESULTS: The overall labeling efficiency was 36.4%. A final dose of approximately 290 ,uCi of the In-111-labeled hepatocytes in 10 ml serum-free phosphate-buffered saline was infused percutaneously into the portal vein approximately 2.5 hours after their preparation. The study was performed 3 hours before cell transplantation (109 cells). Quantitative analysis of the biodistribution of In-111-labeled hepatocytes indicated that cells were predominantly localized in the liver immediately after portal vein-infused transplantation. The predominant hepatic distribution was persistent for as long as 7 days after the procedure, with an average liver-to-spleen ratio of 9.5 to 1. No significant pulmonary radiotracer uptake was present. CONCLUSION: These results indicate that In-111 labeling of hepatocytes is useful for the short-term noninvasive analysis of the biodistribution of transplanted hepatocytes.     

经兔肝动脉自体骨髓干细胞移植治疗肝硬化的研究

目的 评价自体骨髓干细胞经肝动脉移植对兔肝硬化模型的治疗作用,了解促肝细胞生长素(pHGF)在干细胞移植治疗中的作用,为干细胞移植的临床应用提供实验研究基础.方法 用皮下注射四氯化碳(CCl4)橄榄油溶液的方法复制兔肝硬化模型.25只肝硬化模型兔根据治疗方法不同分为对照组(5只)、干细胞移植组(10只)和移植+pHGF组(10只).无菌条件下从兔胫骨上端抽取骨髓4～5 ml,采用密度梯度离心法分离骨髓干细胞,在透视下将分离的骨髓干细胞经肝动脉移植于肝脏.术后移植+pHGF组隔日静脉内注射pHGF 2 mg/kg共20 d.然后在移植后4、8、12周分别抽血检测肝功能,12周时取肝脏行病理学检查.分别对3组12周时肝功能指标进行比较,各组间比较用方差分析.结果 在移植骨髓干细胞后,动物肝功能逐渐改善,至移植治疗后12周,各组肝功能与术前比较均有明显改善.干细胞移植组术后12周,兔血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)逐渐降低,由术前(73.0±10.6)、(152.4±22.8)U/L分别降至移植12周后的(48.0 ±1.0)、(86.7±2.1)U/L(F值分别为72.38、78.86,P值均<0.05);白蛋白(ALB)和凝血酶原活动度(PTA)逐渐上升,分别由(27.5±1.8)g/L和28.3%上升至干细胞移植后12周的(33.2±0.5)g/L和44.1%(F值分别为56.37、126.47,P值均<0.05);但12周时干细胞移植组除PTA外,其他上述血清酶改变与对照组相比差异无统计学意义(P值均>0.05).而干细胞移植+pHGF组肝功能指标改变较移植组迅速,术后12周时ALT、AST分别降至(43.3±0.6)、(78.7±4.0)U/L,而ALB、PTA上升至(35.7±0.4)g/L和50.5%,这些指标改变与对照组相比均差异均有统计学意义(F值分别为47.38、23.52、52.27、174.45,P值均<0.05).病理学检查显示干细胞移植组肝细胞形态较规整,无明显细胞坏死和变性,假小叶结构仍存在,但肝组织纤维化程度较对照组明显减轻;干细胞移植+pHGF组肝结构的改善更明显.免疫组织化学染色发现肝组织内散在分布有CD34<'+>肝样细胞,并且这些肝样细胞在不同组数量差异较大,干细胞移植组较对照组多,移植+pHGF组最多.结论 自体骨髓干细胞移植对肝硬化有一定的治疗作用;促肝细胞生长素可促进干细胞在肝脏的归巢与分化,与骨髓干细胞移植有协同治疗作用.     

In vivo imaging of transplanted hepatocytes with a 1.5-T clinical MRI system—initial experience in mice

The feasibility of in vitro mature mouse hepatocyte labeling with a novel iron oxide particle was assessed and the ability of 1.5-T magnetic resonance imaging (MRI) to track labeled mouse hepatocytes in syngenic recipient livers following intraportal cell transplantation was tested. Mouse hepatocytes were incubated with anionic iron oxide nanoparticles at various iron concentrations. Cell viability was assessed and iron oxide particle uptake quantified. Labeled hepatocytes were intraportally injected into 20 mice, while unlabeled hepatocytes were injected into two mice. Liver T2 values, spleen-to-muscle relative signal intensity (RI _{spleen/muscle} ), and liver-to-muscle relative signal intensity (RI _liver/muscle ) on gradient-echo T2-weighted imaging after injection of either labeled or unlabeled hepatocytes were compared with an ANOVA test followed by Fisher’s a posteriori PLSD test. Livers, spleens and lungs were collected for histological analysis. Iron oxide particle uptake was saturable with a maximum iron content of 20 pg per cell and without viability alteration after 3 days of culture. Following labeled-cell transplantation, recipient livers showed well-defined nodular foci of low signal intensity on MRI—consistent with clusters of labeled hepatocytes on pathological analysis—combined with a significant decrease in both liver T2 values and liver-to-muscle RI _liver/muscle (P = 0.01) with minimal T2 values demonstrated 8 days after transplantation. Conventional MRI can demonstrate the presence of transplanted iron-labeled mature hepatocytes in mouse liver.     

Usefulness of Tc-99m-GSA scintigraphy for liver surgery

Postoperative mortality remains high after hepatectomy compared with other types of surgery in patients who have cirrhosis or chronic hepatitis. Although there are several useful perioperative indicators of liver dysfunction, no standard markers are available to predict postoperative liver failure in patients with hepatocellular carcinoma (HCC) undergoing hepatectomy. The best preoperative method for evaluating the hepatic functional reserve of patients with HCC remains unclear, but technetium-99m diethylenetriamine pentaacetic acid galactosyl human serum albumin (^99mTc-GSA) scintigraphy is a candidate. ^99mTc-GSA is a liver scintigraphy agent that binds to the asialoglycoprotein receptor, and can be used to assess the functional hepatocyte mass and thus determine the hepatic functional reserve in various physiological and pathological states. The maximum removal rate of ^99m Tc-GSA (GSA-Rmax) calculated by using a radiopharmacokinetic model is correlated with the severity of liver disease. There is also a significant difference of GSA-Rmax between patients with chronic hepatitis and persons with normal liver function. Regeneration of the remnant liver and recurrence of hepatitis C virus infection in the donor organ after living donor liver transplantation have also been investigated by ^99mTc-GSA scintigraphy. This review discusses the usefulness of ^99mTc-GSA scintigraphy for liver surgery.     

Scintigraphic evaluation of liver transplant function

Since the initiation of liver transplantation at our institution 9 yr ago, 73 patients ranging in age from 8 mo to 64 yr have undergone this procedure. In the immediate postoperative period and at various times thereafter as deemed necessary, radionuclide studies were performed using one of the iminodiacetic acid (IDA) derivatives labeled with 99mTc. Initially, these studies were performed using labeled PIPIDA with a shift to diisopropyl IDA when this latter agent became available. The IDA agent is administered as a bolus so that the "flow" and "pooling" may be viewed immediately after injection. This is followed by sequential imaging at various times up to 24 hr, with optional graphic tracings of hepatic and bowel patterns of uptake and clearance of radioactivity. An analysis of the initial portion of the IDA scan yields useful information regarding the arterial and portal venous supply of the liver. The rapidity of hepatic concentration and excretion provides a direct measure of hepatocyte function which is particularly helpful when used sequentially to follow the response of the liver to therapy for rejection or infection. The study is also used to assess the biliary system for obstruction or leaks.     

Liver parenchymal enhancement of hepatocyte‐phase images in Gd‐EOB‐DTPA‐enhanced MR imaging: Which biological markers of the liver function affect the enhancement?

Purpose:

To clarify the factors that predict enhancement of the liver parenchyma in hepatocyte‐phase of gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid (Gd‐EOB‐DTPA)‐enhanced MR imaging.

Materials and Methods:

Gd‐EOB‐DTPA–enhanced hepatocyte‐phase MR images of 198 patients with chronic liver diseases (Child‐Pugh class A in 112 patients, class B in 74 patients, and class C in 12 patients) were retrospectively analyzed. The hepatocyte‐phase images were obtained using fat‐suppressed T1‐weighted gradient‐echo images with a 3D acquisition sequence 10 min and 20 min after IV administration of Gd‐EOB‐DTPA (0.025 mmol/kg body weight). The quantitative liver–spleen contrast ratio (Q‐LSC) was calculated using the signal intensities of the liver and spleen. Serum albumin levels, total bilirubin levels, prothrombin activity, and the results of indocyanine green clearance tests (ICGs) were recorded and correlated with the Q‐LSC. Logistic regression analysis was performed to analyze which factors predict sufficient liver enhancement using a Q‐LSC of 1.5 as a cutoff value.

Results:

Only ICGs and Child‐Pugh classifications showed a statistically significant correlation with the Q‐LSC. Logistic regression analysis showed that ICGs were the only factors that accurately predicted liver enhancement on hepatocyte‐phase images.

Conclusion:

ICGs were found to be predictors of sufficient liver enhancement on hepatocyte‐phase images. J. Magn. Reson. Imaging 2009;30:1042–1046. © 2009 Wiley‐Liss, Inc.