肝干细胞的来源与作用

卵圆细胞(oval cells)是肝脏内体积小、细胞质清澈、核卵圆形、不易定性的细胞。它们可能来源于骨髓或与骨髓有关的细胞群,是活化形式的肝干细胞。肝干细胞的形态特征与卵圆细胞相似,生化标记为c-kit／CD^45／TER19。本文总结了人们对肝干细胞的认识和肝干细胞在肝脏发育、肝再生、肝癌发生中的作用及与其它细胞来源的联系。     

胚胎干细胞向肝实质细胞体外定向诱导分化过程中的肝卵圆细胞

如果肝脏严重受损致使肝细胞大部分坏死,或由于某些原因 ( 肝毒性物质、致癌物质的作用 ) 抑制残存肝细胞增殖时,肝内前体／干细胞———肝卵圆细胞便被激活并分化生成肝细胞和胆管细胞等以参与肝修复 . 基于此理论,人们建立了啮齿类动物肝卵圆细胞诱导实验模型 . 但显然上述模型不适用于人类,所以有必要开发一种适用于人类的、高效的肝卵圆细胞的新诱导模型 . 选用小鼠胚胎干细胞,转成拟胚体分化 3 天后分组,诱导组添加肝细胞生长因子 (HGF) 、表皮生长因子 (EGF) 作定向诱导分化 . 其间用免疫细胞化学 (ICC) 检测肝卵圆细胞标志物 A6 等的表达,用流式细胞仪筛选肝卵圆细胞并行 RT-PCR 、透射电镜检测 . 所筛选的肝卵圆细胞进一步体外培养并进行 ICC 和 RT-PCR ,检测其分化生成成熟的肝细胞和胆管细胞的能力 . 研究证实胚胎干细胞体外定向诱导生成肝实质细胞的过程中,存在着有双向分化能力的肝卵圆细胞这个中间分化阶段 . 诱导组肝卵圆细胞分化率均显著地高于对照组,最高时可达 6.11% 左右 . HGF 和 EGF 能显著性诱导胚胎干细胞源性卵圆细胞的生成 . 流式细胞仪筛选 Sca-1+/CD34+ 细胞占总细胞数的 4.59% ,其中 A6 阳性肝卵圆细胞占 90.81% 左右 . 使用流式细胞仪可获得高富集的 A6+/Sca-1+/CD34+ 肝卵圆细胞 . 提供了一种可适用于人类的肝卵圆细胞的新诱导模型 .     

胎肝中肝干细胞的免疫组织化学研究

目的采用免疫组织化学方法显示不同时期人胚胎肝脏的干细胞,分析肝干细胞的形态与分布特点及发育过程中干细胞在肝脏中的迁徙,探讨肝脏的发生发育及肝内干细胞的来源。方法不同发育时期胎儿肝脏,取材、固定、制成石蜡切片,ABC法检测肝干细胞特异性的表面标记物CD34、CK19、C-11和OV6。结果胎肝内汇管区周边界板处有卵圆样细胞表达CD34、C-11、CK19和OV6,阳性细胞紧密排列成管,呈鞘样包绕着早期汇管区,部分包绕着初级汇管区,随着次级汇管区的成熟,卵圆样干细胞逐渐局限于赫令氏管周围;此外,胚胎发育的不同阶段均可见CD34、OV6阳性的单核样细胞分散在肝索、肝血窦之内,多见于汇管区的问充质组织之内,肝血管内鲜见。结论胚胎发育早期汇管区周边界板处含有丰富的干细胞,可能是肝脏发育的起点,这些干细胞逐渐分化为胆管上皮样细胞,然后分化为肝细胞和胆管上皮细胞;造血干细胞是肝内的另一干细胞来源,造血干细胞在肝内受到诱导作用分化为小部分的肝实质细胞。     

成体干细胞向肝细胞分化

成体干细胞跨越胚层限制分化为其他胚层来源的细胞,对揭示不同胚层细胞间相互分化的生物学意义和机制具有重要学术价值,并可以为临床细胞移植治疗开辟新的途径,从而成为当前研究的热点之一。综述了近年来肝源性卵圆细胞、成肝细胞、骨髓源干细胞和其他成体干细胞跨越分化为肝细胞的研究现状与进展,以及卵圆细胞、成肝细胞等的分离鉴定,表面标志、生物学特征和跨越分化机制,并对成体干细胞在肝脏疾病细胞治疗上的应用前景作了展望。     

骨髓源性肝干细胞的研究

近年来全球掀起了有关干细胞的研究热潮 ,除血液干细胞、胚胎干细胞和神经干细胞外 ,肝干细胞也是引人注目的内容之一。肝干细胞的存在与否曾是一个争议性问题 ,目前已趋于肯定。自从Ｐｅ ｔｅｒｓｅｎｅｔａｌ[1] 有关骨髓细胞能分化为肝上皮细胞系的论文发表后 ,关于血源性干细胞能转化为肝实质细胞的事实引起了广泛的关注[2～ 4 ] ,本文就有关研究进展综述如下。1.肝内干细胞成体动物肝脏内有干细胞吗 ?这个问题已经争论了半个多世纪 ,目前通过体内、外实验证实了肝干细胞的存在 ,肝的卵圆细胞和小肝细胞作为肝干细胞或肝前体细胞的观点…     

大鼠肝脏前体细胞——小圆细胞的分离与特性的初步研究

通过与大鼠肝卵圆细胞的对比,对肝脏中一种新的前体细胞——小圆细胞进行分离和初步特性分析。以分步消化法制备小圆细胞,MTT法观察其增殖特点,以白蛋白、细胞角蛋白19、甲胎蛋白为标志物,应用免疫细胞化学染色的方法,对其进行鉴定。同时用DMSO刺激小圆细胞分化,检测分化结果,并与卵圆细胞加以比较。结果显示,小圆细胞增殖不明显。小圆细胞与卵圆细胞均表达白蛋白和角蛋白19。DMSO刺激后小圆细胞向卵圆细胞分化,分化前细胞AFP呈阳性,分化后细胞形状、标志物表达与卵圆细胞一致。本实验提示,小圆细胞很可能是早于卵圆细胞的一种肝脏前体细胞。     

肝干细胞的几个生物学问题

对肝干细胞的认识,自卵圆细胞发现以来已经经历了五十多年。期间许多的研究提示和证明肝干细胞具有很好的生物医学应用前景。近年来,肝干细胞的概念已被广泛接受和认可,肝干细胞的生物学特性及其医学应用的探索也成为了研究的热点。目前在该领域中,主要以肝干细胞存在的位置、与其所处微环境的相互作用、肝干细胞的分子标记以及其分化特性等方面备受关注。肝干细胞的研究为肝脏疾病及其他相关疾病的治疗带来了新希望。然而,肝干细胞研究的过程中仍存在很多未解的问题,因此将肝干细胞研究引入临床应用则还需要更加深入的探索。     

肝干细胞分析研究进展

近年来干细胞研究成为生物医学领域研究的热点。肝干细胞是一种具有双向分化能力和自我更新能力的细胞,参与肝脏生长、发育与再生。肝干细胞分化的过程和机理较为复杂,利用肝干细胞进行细胞移植与生物人工肝治疗各种终末期肝病被认为是最有前途的方法之一。本文综述肝干细胞领域近年来研究进展。     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

大鼠肝脏前体细胞--小圆细胞的分离与特性的初步研究

通过与大鼠肝卵圆细胞的对比,对肝脏中一种新的前体细胞--小圆细胞进行分离和初步特性分析.以分步消化法制备小圆细胞,MTI法观察其增殖特点,以白蛋白、细胞角蛋白19、甲胎蛋白为标志物,应用免疫细胞化学染色的方法,对其进行鉴定.同时用DMSO刺激小圆细胞分化,检测分化结果,并与卵圆细胞加以比较.结果显示,小圆细胞增殖不明显.小圆细胞与卵圆细胞均表达白蛋白和角蛋白19.DMSO刺激后小圆细胞向卵圆细胞分化,分化前细胞AFP呈阳性,分化后细胞形状、标志物表达与卵圆细胞一致.本实验提示,小圆细胞很可能是早于卵圆细胞的一种肝脏前体细胞.     

Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats

The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the mechanisms that control liver regeneration after bone marrow cell transplantation are still controversial. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of extracellular matrix proteins with adhesive and chemotactic functions, expressed in the portal and centrolobular veins of the normal liver. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation. Rat BMMNCs were isolated by Ficoll-gradient centrifugation, stained with DAPI and injected into recently hepatectomyzed rats via the portal vein. Liver sections obtained 15 min, 1 day and 3 days after the surgery were immunolabeled with anti-rat CD34 and/or laminin primary antibodies and observed under a laser scanning confocal microscope. Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrolobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.     

骨髓干细胞移植治疗肝硬化的基础与临床研究现状

肝硬化是一种临床常见的肝病良性终末期表现。目前临床上尚缺乏有效的治疗措施。肝脏移植是最理想的治疗方法,但受供体肝脏来源限制,且费用昂贵。近年来开展的自体骨髓干细胞(BMSCs)移植治疗,为肝硬化的治疗带来了新的希望。BMSCs主要包括造型血干细胞和间充质干细胞,其具有可塑性,体外通过生长因子,体内利用特定微环境均可诱导BMSCs分化为肝前体细胞和成熟肝细胞,并明显改善肝功能。从动物实验到临床研究亦表明,BMSCs具有来源丰富、费用低廉、损伤小、自体移植不栓塞、无排斥反应等优点,为治疗肝病带来了新思路,有望成为生物人工肝的细胞来源。本文就BMSCs移植治疗肝硬化的研究现状,尤其是移植途径以及在肝脏内定居、迁移和分化机制的示踪观察方法和存在的问题作一综述,以期为从事肝病研究的同仁提供参考依据。通过对BMSCs移植从基础研究及临床应用的最新进展的描述,展示BMSCs在肝硬化治疗方面良好的治疗前景。     

Stem cells in liver regeneration and therapy

The liver has adapted to the inflow of ingested toxins by the evolutionary development of unique regenerative properties and responds to injury or tissue loss by the rapid division of mature cells. Proliferation of the parenchymal cells, i.e. hepatocytes and epithelial cells of the bile duct, is regulated by numerous cytokine/growth-factor-mediated pathways and is synchronised with extracellular matrix degradation and restoration of the vasculature. Resident hepatic stem/progenitor cells have also been identified in small numbers in normal liver and implicated in liver tissue repair. Their putative role in the physiology, pathophysiology and therapy of the liver, however, is not yet precisely known. Hepatic stem/progenitor cells also known as “oval cells” in rodents have been implicated in liver tissue repair, at a time when the capacity for hepatocyte and bile duct replication is exhausted or experimentally inhibited (facultative stem/progenitor cell pool). Although much more has to be learned about the role of stem/progenitor cells in the physiology and pathophysiology of the liver, experimental analysis of the therapeutic value of these cells has been initiated. Transplantation of hepatic stem/progenitor cells or in vivo pharmacological activation of the pool of hepatic stem cells may provide novel modalities for the therapy of liver diseases. In addition, extrahepatic stem cells (e.g. bone marrow cells) are being investigated for their contribution to liver regeneration. Hepatic progenitor cells derived from embryonic stem cells are included in this review, which also discusses future perspectives of stem cell-based therapies for liver diseases.     

Progenitor cells of the biliary epithelial cell lineage

Stem-like cells have been identified in liver that are able to differentiate in vivo and in culture to biliary epithelial cells (BEC), hepatocytes and oval cells. The growth factors/cytokines and signal pathways required for the differentiation processes are beginning to be evaluated. There is increasing evidence to suggest that these stem-like cells may originate from both the bone marrow population or from a precursor remnant from liver embryogenesis, as they share many of the same markers (CD34, c-kit, CD45). Most recently, it has been shown that a population of progenitor cells can copurify with mesenchymal bone marrow cells and differentiate under specific culture conditions to form both hepatic epithelial and also endothelial cells. The interaction of haemopoietic and mesenchymal stem cells needs further evaluation. The close association of ductular reactive cells and neovessels in end-stage cholestatic liver diseases and the relation to Jagged/Notch signalling pathway may be important in the regulation of stem cells to form both biliary epithelial and endothelial cells.     

Bone marrow to liver: the blood of Prometheus

The existence of hepatic stem or progenitor cells has been controversial for decades, though it was presumed that if such cells existed, they would lie within the liver. There is now consensus, however, that not only do facultative hepatic stem cells exist within the liver, but also that cells from extra-hepatic sites, in particular the bone marrow, can contribute to hepatocyte and cholangiocyte regeneration. Despite confidence that engraftment of marrow cells in the liver occurs, the mechanistic details of this process remain poorly understood. Moreover, the physiological importance and therapeutic utility of this phenomenon remains controversial.     

Cellular origin of hepatocellular carcinomas

There are four levels of cells in the hepatic lineage which may respond to different carcinogenic regimens: (1) the mature hepatocyte, which responds to diethylnitrosamine (DEN) hepatocarcinogenesis. (2) The bile duct progenitor cells, which give rise to cholangiocellular carcinomas when the furan model is used or when hamsters infected with liver flukes (Clornorchis sinensis) are exposed to dimethylnitrosamine. (3) The ductular 'bipolar' progenitor cell which gives rise to hepatocellular carcinomas (HCC) in several N-2-acetylaminofluorene (N-2-AAF) based regimens, and (4) the periductular stem cell, which is the cell of origin of HCC induced by the choline deficiency models of hepatocarcinogenesis. 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.     

Purified hematopoietic stem cells can differentiate into hepatocytes in vivo

The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.     

人胎肝中一种35kD成分的造血调控研究

肝脏作为胚胎发育一定阶段的造血中心器官,其造血干细胞增殖十分活跃,已发现的具有正向调节其作用的因子有IL-3,IL-6,GM-CSF,SCF及FLT 3配基等;然而上述因子的作用并不足以解释胎肝如此活跃的造血活动。推测可能还有其他特异的源于胎肝造血微环境细胞的正向调节因子存在,并以旁分泌方式作用于G_0期造血干细胞,使其进入细胞周期。     

应用骨髓干细胞治疗肝硬化研究进展

随着干细胞研究的深入和技术的发展,再生医学的干细胞疗法治疗肝脏疾病已成为研究热点。骨髓来源造血干细胞和间充质干细胞等在肝脏疾病治疗方面有巨大潜力。骨髓干细胞参与肝纤维化与肝硬化修复主要包括迁移、归巢与转化等过程,并需要多种细胞因子和趋化因子的协同作用促进肝细胞再生与减轻肝纤维化。本文拟对骨髓干细胞治疗肝硬化的最新研究进展进行综述。