肝干细胞研究的现状及其临床应用

目前,肝移植已成为治疗终末期肝病的最佳选择.但肝移植面临着肝源短缺、免疫排斥反应等问题,肝脏干细胞移植的出现为肝病的治疗提供了新的思路.近年来,成熟肝细胞移植已应用于肝脏急性损伤、肝脏先天性疾病及为等待供肝的患者提供过渡性治疗等,肝干细胞可应用的领域基本相同,但许多试验尚刚刚起步.虽然肝源的缺少同样影响供移植干细胞的获得,但多种其它来源的干细胞可诱导其分化成为肝细胞,从而较好地解决了来源短缺的问题,为肝细胞移植和生物型人工肝提供了丰富的细胞来源.     

肝干细胞研究进展

肝移植是目前治疗终末期肝病的主要方法，但供肝短缺限制了其应用，导致许多患者未能获得肝移植。近年来，肝干细胞的分离鉴定、纯化培养技术日渐成熟，在动物实验和临床研究中均取得了较大进展。移植到受体的干细胞已被证实可以分化形成正常肝细胞，从而通过提高肝细胞数量来改善肝功能，延长了患者生存期，使其有机会获得肝移植。同时为急慢性肝损伤、肝代谢性疾病的治疗提供了新的细胞移植疗法，成为肝病学和干细胞研究领域的热点。     

骨髓干细胞与肝细胞移植

肝细胞移植为治疗各种肝病提供了一种新的途径，其目的是通过移植细胞的增殖逐步替代或重建受损的肝组织结构。细胞来源是当前阻碍肝细胞移植发展的主要问题。骨髓干细胞可以分化为与其组织发育或再生来源不同的细胞类型，可望成为肝细胞移植的重要细胞来源问题。本文就近年来相关研究进行综述。     

骨髓干细胞与肝细胞移植

肝细胞移植为治疗各种肝病提供了一种新的途径,其目的是通过移植细胞的增殖逐步替代或重建受损的肝组织结构。细胞来源是当前阻碍肝细胞移植发展的主要问题。骨髓干细胞可以分化为与其组织发育或再生来源不同的细胞类型,可望成为肝细胞移植的重要细胞来源问题。本文就近年来相关研究进行综述。     

骨髓基质干细胞分化来源肝细胞移植的细胞准备及组织学观察

目的 探讨大鼠骨髓基质干细胞向肝细胞分化后体外标记方法及移植肝细胞的肝内组织学表现。方法 分离大鼠骨髓基质细胞，在体外诱导分化为成熟肝细胞。将5-溴脱氧尿嘧啶核苷（BrdU）掺入后的肝细胞移植入已行部分肝切除大鼠体内，分别应用免疫组织化学和免疫荧光方法观察受体肝脏内移植细胞的形态和功能。结果分化成熟肝细胞在BrdU掺入培养后细胞核染色可见特异性棕褐色标记；肝细胞移植后肝组织切片BrdU染色可定位移植细胞；白蛋白抗体染色显示移植细胞具有功能活性。结论 骨髓基质干细胞分化来源的肝细胞移植后形态功能稳定，是进行肝细胞移植的理想细胞来源。     

肝干细胞来源、定向分化及其转化机制

本文综述了近年来胚胎、造血、肝内祖细胞和小肝细胞样祖细胞、骨髓、间充质来源干细胞定向向肝细胞分化的研究进展,并阐述了干细胞转化为肝细胞的机制是融合还是转化.对此深入研究可望为肝干细胞移植和基因治疗带来希望,进而为临床肝移植面临的供肝短缺提供可选择的补充治疗方法.     

肝干细胞来源、定向分化及其转化机制

本文综述了近年来胚胎、造血、肝内祖细胞和小肝细胞样祖细胞、骨髓、间充质来源干细胞定向向肝细胞分化的研究进展,并阐述了干细胞转化为肝细胞的机制是融合还是转化.对此深入研究可望为肝干细胞移植和基因治疗带来希望,进而为临床肝移植面临的供肝短缺提供可选择的补充治疗方法.     

肝干细胞来源研究进展

肝干细胞（hepatic stem cell，HSC）正成为世界各国尤其是欧美以及日本的研究热点。HSC研究热的原因在于HSC移植将解决供体肝脏严重缺乏的重要途径之一；另一方面，自体的HSC移植能有效地避免免疫排斥反应。此外，HSC下向诱导分化为肝细胞，这将为肝细胞移植和生物型人工肝提供无限的细胞来源。几十年来，尽管研究人员一直尝试鉴定及描述HSC的特征，但对于肝干细胞是否存在的问题一直存在争议。     

一种肝细胞移植的新来源—骨髓士细胞可塑性的应用研究

逐步替代或重建受损的肝组织结构是肝细胞移植治疗肝病的最终目的，骨髓干细胞可以演变成为与其组织发育或再生来源无关的细胞类型，即骨髓干细胞的可塑性（plasticity)。骨髓干细胞分化为肝实质细胞的发现提示它可能参与了体内肝组织细胞的再生，及损伤的修复与替代。而这一进程与肝细胞移植的作用机制类似，本文就近年来的有关研究进行综述。     

一种肝细胞移植的新来源--骨髓干细胞可塑性的应用研究(文献综述)

逐步替代或重建受损的肝组织结构是肝细胞移植治疗肝病的最终目的.骨髓干细胞可以演变成为与其组织发育或再生来源无关的细胞类型,即骨髓干细胞的可塑性(plasricity).骨髓干细胞分化为肝实质细胞的发现提示它可能参与了体内肝组织细胞的再生、及损伤的修复与替代,而这一进程与肝细胞移植的作用机制类似,本文就近年来的有关研究进行综述.     

胚胎肝干细胞分离与移植治疗裸鼠急性肝损伤

目的 探讨胚胎肝脏原始/干细胞移植治疗急性肝功能损伤裸鼠的可能性与有效性.方法 酶消化法分离孕13.5 d的BALB/C小鼠的胚胎肝脏原始/干细胞,免疫荧光染色法及RT-PCR法鉴定AFP、Albumin、CK19、c-Met等肝系和胆系标志物,给四氯化碳(CCl4)诱发的肝毒性损伤的裸鼠尾静脉注射4×105个干细胞,于注射前和注射后第1、2、4、7天取裸鼠血清,测定肝功能指标,观察肝脏组织学的改善情况.结果 经分离可得到具有干细胞特点的胚肝原始细胞,治疗组裸鼠在干细胞移植后肝功能指标逐渐降低,第1、2、4天均低于对照组,治疗组裸鼠肝脏组织学的病理损伤的恢复比对照组快. 结论酶消化法能够分离得到较多胚胎肝脏原始/干细胞,并且具有一定的改善急性肝损伤裸鼠的肝功能及肝脏病理指标的作用.     

The phenotypic characteristic of liver-derived stem cells from adult human deceased donor liver

Liver transplantation is the only effective treatment for end-stage liver disease. Because of the limited donor availability, attention has been focused on the possibility to restore liver mass and function through cell transplantation. Stem cells are a promising source for liver repopulation after cell transplantation, but whether or not the adult liver contains hepatic stem cells is highly controversial. Several studies have suggested the presence of stem cells in the adult normal human liver. However, a population with stem cell properties has not yet been isolated. The purpose of this study was to identify and characterize progenitor cells in normal adult human liver. We isolated and expanded human liver stem cells (HLSCs) from a donated liver not suitable for liver transplantation or characterizing them by fluorescence-activated cell sorter, polymerase chain reaction, and immunofluorescence assay. HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, CD90, CD105, and CD166 but not the hematopoietic stem cell markers CD34, CD45, and CD117. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed CD26, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. We concluded that HLSCs expressed several mesenchymal but not hematopoietic stem cell markers as well as CD26 and CK18, indicating a partial commitment to hepatic cells.     

Expansion of hepatic and hematopoietic stem cells utilizing mouse embryonic liver explants

Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-beta leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.     

Efficient human fetal liver cell isolation protocol based on vascular perfusion for liver cell-based therapy and case report on cell transplantation

Although hepatic cell transplantation (CT) holds the promise of bridging patients with end-stage chronic liver failure to whole liver transplantation, suitable cell populations are under debate. In addition to hepatic cells, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being considered as alternative cell sources for initial clinical cell work. Fetal liver (FL) tissue contains potential progenitors for all these cell lineages. Based on the collagenase incubation of tissue fragments, traditional isolation techniques yield only a fraction of the number of available cells. We report a 5-step method in which a portal vein in situ perfusion technique is used for tissue from the late second trimester. This method results in the high viabilities known for adult liver vascular perfusion, addresses the low cell yields of conventional digestion methods, and reduces the exposure of the tissue to collagenase 4-fold. We used donated tissue from gestational weeks 18 to 22, which yielded 1.8 ± 0.7 × 10(9) cells with an average viability of 78%. Because HSC transplantation and MSC transplantation are of interest for the treatment of hepatic failure, we phenotypically confirmed that in addition to hepatic progenitors, the resulting cell preparation contained cells expressing typical MSC and HSC markers. The percentage of FL cells expressing proliferation markers was 45 times greater than the percentage of adult hepatocytes expressing these markers and was comparable to the percentage of immortalized HepG2 liver hepatocellular carcinoma cells; this indicated the strong proliferative capacity of fetal cells. We report a case of human FL CT with the described liver cell population for clinical end-stage chronic liver failure. The patient's Model for End-Stage Liver Disease (MELD) score improved from 15 to 10 within the first 18 months of observation. In conclusion, this human FL cell isolation protocol may be of interest for further clinical translation work on the development of liver cell-based therapies.     

肝硬化门脉高压症治疗的新途径——干细胞移植研究进展

肝干细胞是多源性的,可分为肝源性肝干细胞和非肝源性肝干细胞两大类。多种成体组织来源的干细胞可在受者(包括鼠和人)肝脏内分化为肝细胞和胆管上皮细胞,为成体干细胞移植治疗肝硬化门脉高压提供了新思路。在临床应用中,肝干细胞移植具有移植技术简单、价格相对低廉,免疫源性小、易于低温保存,具有广泛的扩展性,体外基因转染率高,并能稳定高效地表达外源基因等优点;但肝内干细胞移植是否会引起肝脏肿瘤等问题尚未明确。     

HNF4α在胎肝干细胞促肝组织损伤修复中的作用

目的探讨肝细胞核因子(HNF)4α在胎肝干细胞促大鼠肝组织损伤修复中的作用。方法胶原酶结合机械消化法制备14 d胎龄大鼠胎肝干细胞悬液并进行体外长期培养。采用四氯化碳制作SD大鼠急性化学性肝损伤模型,造模成功后将肝损伤大鼠随机分为移植组(20只)和对照组(20只),移植组将胎肝干细胞经门静脉移植至大鼠肝脏,对照组注射等容计量的生理盐水,分别于注射前6 h,注射后1,3,5周检测大鼠血清谷氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST),并检查肝组织病理学变化,观察肝脏修复情况。采用免疫组化及Western-blot方法检测肝脏治疗前后HNF4α表达情况。结果对照组大鼠肝功能改善缓慢,ALT,AST明显高于移植组(P0.05),移植组大鼠损伤的肝组织逐渐修复,肝功能恢复正常。第5周移植组大鼠术后存活率显著高于对照组(P=0.002)。肝组织损伤后,HNF4α在肝组织的表达明显升高;随着肝组织的逐渐修复,HNF4α表达逐渐下降。western-blot及免疫组化检测亦证实此结果。结论HNF4α在肝脏损伤初期起保护作用。可能系通过促进胎肝干细胞向正常肝细胞分化增殖,并迁移至损坏的肝小叶从而替代损伤的肝实质,从而提高肝损伤后的恢复能力。     

The role of progenitor cells in repair of liver injury and in liver transplantation

There are three levels of cells in the hepatic lineage that respond to injury or carcinogenesis: the mature hepatocyte, the ductular "bipolar" progenitor cell, and a putative periductular stem cell. Hepatocytes are numerous, and respond rapidly to liver cell loss by one or two cell cycles but can only produce other hepatocytes. The ductular progenitor cells are less numerous, may proliferate for more cycles than hepatocytes, and are generally considered "bipolar," i.e., they can give rise to biliary cells or hepatocytes. Periductular stem cells are rare in the liver, have a very long proliferation potential, and may be multipotent. Extrahepatic (bone marrow) origin of the periductular stem cells is supported by recent data showing that hepatocytes may express genetic markers of donor hematopoietic cells after bone marrow transplantation. These different regenerative cells with variations in potential for proliferation and differentiation may provide different sources of cells for liver transplantation: hepatocytes for treatment of acute liver damage, liver progenitor cell lines for liver-directed gene therapy, and bone marrow-derived cells for chronic long-term liver replacement. A limiting factor in the success of liver cell transplantation is the condition of the hepatic microenvironment in which the cells must proliferate and set up housekeeping.     

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.     

Differentiation and major histocompatibility complex antigen expression in human liver-derived stem cells

Stem cells are a promising source for liver repopulation after cell transplantation, but whether the adult liver contains hepatic stem cells is controversial. The purpose of this study was to characterize the properties and expression profile of major histocompatibility complex (MHC) antigens on the surface of human-derived stem cells. Human liver-derived stem cells (HLSC7) were isolated from the nontumorous tissue of a patient who underwent a resection of an hepatic hemangioendothelioma. We characterized HLSC7 using a fluorescence-activated cell sorter, polymerase chain reactions, and immunofluorescence assays. HLSC7 expressed mesenchymal but not hematopoietic stem cell markers. HLSC7 underwent osteogenic, chondrogenic, and hepatogenic differentiation when cultured in appropriate differentiation media. However, HLSC7 did not differentiate into adipocytes. In addition, HLSC7 did not express MHC class II (HLA-DP, -DQ, and -DR) antigens. However, they did express MHC class I antigens. These results suggest that human liver-derived stem cells express MHC class I antigens and thus may be rejected on transplantation. Therefore, in addition to studies on stem cell differentiation, one must overcome immunologic barriers for successful clinical application of this therapy.     

干细胞移植用于肝再生治疗的相关研究进展

目的对干细胞移植应用于肝再生治疗的相关研究进展进行综述。方法广泛查阅近年的相关文献，对移植研究中常见的干细胞类别、修复机制、植入途径以及临床相关问题进行综述。结果修复肝脏的干细胞可以分为肝源性干细胞和非肝源性干细胞；修复机制主要是融合或转分化；植入途径以门脉内植入多见。目前仍有许多临床相关问题限制干细胞移植的应用。结论提高干细胞的临床实用性需要更深入的研究。