肝干细胞的研究进展

肝干细胞是成体千细胞的一种，由于其具有强大的增殖能力，并能分化为具有功能的成熟肝细胞，为肝病的细胞治疗和组织工程提供了重要的细胞来源。现就肝干细胞的来源、标志物、诱导与示踪、体外培养与扩增、分化及其与肝细胞癌的关系作一综述。     

小鼠胚胎与成体肝脏中干细胞变化的探讨

目的 依据干细胞生物学特性,探讨从胚胎到成体肝脏内具干细胞特性细胞的变化状况.方法 分离不同胎龄与日龄小鼠肝脏细胞进行体外培养,观察分离获得的细胞数、培养形成的集落数及生长状态,并对集落进行干细胞表面标记物(c-kit、c-Met、Nestin)、肝细胞表面标记物(AFP、ALB)及胆管上皮细胞表面标记物(CK19)的标记.结果 各组分离的细胞经体外培养均有集落形成,肝干细胞的表面标记提示形成的集落具有肝干细胞特性.其中以第15天胚胎肝脏分离所获的细胞数和培养形成的集落数最多、生长状态最好.15 d后观察的各项指标渐减,细胞生长状态渐差.结论 早期胚胎肝脏中大部分是具有干细胞特性的细胞,随胎龄增加,有干细胞特性的细胞逐渐减少,直至出生第5天仅有少数保持未分化状态的细胞存留在肝脏内.     

人胎肝干细胞的体外培养及诱导分化

为体外分离培养和诱导分化人胎肝干细胞 ,从原代分离培养人胎肝干细胞集落 ,免疫细胞化学鉴定细胞集落分子标志物的表达 ;在体外特定细胞因子作用下诱导干细胞定向分化为成熟肝脏细胞 ,对其生物学特性进行初步鉴定。结果 ,从人胎肝组织中成功分离表达AFP、Albumin、Cytokeratin等标志物的胎肝干细胞集落 ,在体外特定细胞因子作用下肝干细胞可定向分化为成熟肝脏细胞。因此人胎肝中同样存在具有干细胞特性的原始细胞 ,体外可定向分化为成熟肝细胞。人胎肝干细胞的分离培养对于生物型人工肝的制备及其深入研究奠定了良好的基础     

三氯化镧对CBRH-7919细胞CDK4和P21蛋白表达的影响

目的 研究三氯化镧(LaCl3)对大鼠肝癌细胞CBRH-7919 CDK4和P21蛋白表达的影响.方法 实验分为3组,2个实验组:CBRH-7919细胞分别培养在含0.10和1.00 mmol/L LaCl3的DMEM培养液中,对照组:CBRH-7919培养在不含LaCl3的DMEM培养液中.培养时间分别为1、3、5d.采用MTT法观察细胞生长变化,同时观察集落形成情况,运用流式细胞术检测细胞周期变化,采用免疫细胞化学方法检测CDK4和P21的变化情况.结果 与对照组比较,0.10和1.00 mmol/L LaCl3组培养后3、5 d CBRH-7919细胞的OD值均明显下降(P＜0.01);集落形成数量明显减少(P＜0.01);G0/G1期细胞百分数显著增加,S期细胞百分数显著减少(P ＜0.01);CDK4阳性率明显下降,P21阳性率明显增加(P＜0.01).结论 LaCl3可通过下调CDK4及上调P21,使肿瘤细胞从G1期进入S期受阻,从而抑制CBRH-7919细胞生长.     

肝再生大鼠血清诱导骨髓干细胞向肝细胞分化的实验研究

目的观察肝大部切除大鼠再生血清和肝细胞生长因子(HGF)诱导骨髓干细胞向肝实质细胞分化的作用；探讨骨髓干细胞向肝细胞分化和增殖的体外培养条件和方法，为患者应用自身骨髓细胞修复损伤肝脏提供实验依据。方法制作肝大部切除大鼠肝再生动物模型，收集术后24h血清；分别应用鼠肝再生血清和HGF对大鼠骨髓细胞进行定向诱导分化培养；以免疫组化、RTPCR和western blot等方法对分化不同阶段细胞进行检测。将分化细胞经尾静脉输入同系大鼠，观察输注细胞在肝、脾内的生长情况。结果活体移植分化细胞能定向迁移至肝和脾；体外及体内分化细胞在mRNA水平和蛋白质水平均表达白蛋白，并在一定时期内表达甲胎蛋白。结论大鼠骨髓干细胞在肝大部切除再生血清或HGF的诱导下能横向分化为肝实质样细胞。     

大鼠肝卵圆细胞的分离培养及脾内移植研究

目的 观察肝卵圆细胞在同种异体大鼠脾内移植的演变结果,为肝干细胞移植治疗临床肝功能衰竭提供实验依据。方法 采用改进的梯度离心法分离肝卵圆细胞,体外培养鉴定后移植入2/3肝切除的同种异体大鼠脾脏内。结果 每只模型大鼠肝脏中可分离获得约1.69×10~5/ml肝卵圆细胞。体外培养的肝卵圆细胞呈现上皮细胞的生长特点,对OV6、细胞角蛋白19及甲胎蛋白染色呈阳性反应,对白细胞共同抗原染色呈阴性反应。肝卵圆细胞植入异体大鼠脾脏内可形成岛屿状肝组织结构,形成“肝化脾”。结论 大鼠肝卵圆细胞具有肝干细胞的生物学特征,在一定条件下可分化为肝细胞及胆管上皮细胞。     

p16真核表达载体的构建及其对肝癌细胞的作用

研究p16基因对肝癌细胞的作用;构建pcDNA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中,对其p16基因的表达、细胞的生长抑制及机制进行分析;转染细胞p16蛋白免疫组化阳性,MTY法结果显示,50×103/cm2细胞经培养24h～96h后,每组细胞数均有不同程度的增加,但经pcDNA3.0/p16转染的CBRH-7919细胞数比对照明显减少。与空白对照比,细胞在24h,48h,72h和96h的生长抑制率分别为29％,29％,40％和52％,流式细胞仪检测细胞周期显示经pcDNA3.0/p16转染的CBRH-7919细胞有显著的细胞凋亡现象和G0/G1期阻滞。pcD-NA3.0/p16真核表达质粒转导到大鼠肝癌细胞CBRH-7919中能够抑制细胞的增殖活性,p16基因可能通过诱导肿瘤细胞凋亡及G1期阻滞在肿瘤的基因治疗方面发挥作用。     

肝干细胞的生物学作用

干细胞是具有自我复制能力和多分化功能的一组细胞。近年来 ,除了造血系统、皮肤等再生力强的组织外 ,在神经系统、骨、肝脏等组织也相继发现了干细胞。本文主要对肝干细胞及其生物学作用进行综述。1　肝干细胞肝干细胞首先是由Ｗｉｌｏｓｎ及Ｌｅｄｎｃ在 195 8年提出的。 2 0世纪 80年代中期Ｚａｊｉｃｅｋ提出了“流肝学说” ,并得到认证。该学说认为肝细胞在肝小叶门脉区生成 ,推向中心静脉区域 ,逐渐成熟至凋亡。他们用3Ｈ -胸腺嘧啶标记分裂期细胞 ,被标记的肝细胞从门脉区向中心静脉区域迁移 ,速度 1 4 4μｍ/天 ,历时 2 0 1天。这提…     

细胞生长因子体外对大鼠肝干细胞的影响

目的 研究大鼠肝干细胞系WB-F344细胞在体外地生长分化与多种细胞因子的关系。方法 用^3H掺入法检测细胞因子对WB-F344细胞生长增殖作用。用western blot检测WB-F344细胞因子受体的表达。流式细胞仪检测细胞凋亡。结果 发现肝细胞生长因子（HGF）80ng/mlcpm值为982.95，表皮生长因子（EGF）80ng/ml cpm值为906.32，胰岛素80ng/ml cpm值为968.67，成纤维细胞生长因子（FGFα）80ng/ml cpm值为863.98。促进WB-F344细胞的增殖；白细胞介素-6（IL-6）80ng/ml cpm值为368.67，肿瘤坏死因子α80ng/ml cpm值为372.90对WB-F344细胞的生长无明显影响；而转化生长因子（TGFβ）能够诱导WB-F344细胞凋亡（80ng/ml凋亡率为26.89%)并抑制其生长；western blot检测发现WB-F344细胞表面存在HGF，EGF，FGFα，TGFβ等细胞因子受体，提示细胞因子可能通过与其受体结合方式调控WB-F344细胞的生长分化，进一步用HGF，EGF，胰岛素等细胞因子组成并添加地塞米松的分化培养体系，在体外对WB-F344细胞进行定向诱导分化。发现WB-F344细胞在体外能够向肝实质细胞分化，此外HGF还具有刺激肝干细胞离散作用。结论 肝干细胞的生长分化受多种细胞因子的调控。细胞生长因子可能在严重肝损伤后肝干细胞的动员，增殖及分化调控中起重要作用。     

利用共培养法诱导人胎盘间充质干细胞向成骨细胞分化

目的观察体外人足月胎盘间充质干细胞(h PMSCs)和成骨细胞共培养体系条件下成骨细胞对h PMSCs分化的影响。方法采用胶原酶消化法从人足月胎盘中分离纯化间充质干细胞(MSCs),检测细胞表面标志物、生长曲线、细胞超微结构及成骨能力并对h PMSCs进行鉴定。共培养组将成骨细胞接种于Transwell双层培养皿底层,h PMSCs接种于上层;对照组上层与底层均接种h PMSCs。对诱导后细胞进行碱性磷酸酶染色鉴定。结果胎盘分离细胞经形态、生长速度、细胞表面标志物(CD44和CD29阳性表达为99%,CD34和CD106为1%),确定为胎盘间充质干细胞;头盖骨分离细胞经碱性磷酸酶染色确定为成骨细胞。采用Transwell共培养h PMSCs和成骨细胞组碱性磷酸酶活性染色阳性率为(21.7±5.3)%,表现成骨细胞特性,对照组染色呈阴性。结论人足月胎盘含MSCs,与其他来源MSCs生物学特性相似,成骨细胞生长过程提供的微环境对h PMSCs分化为成骨细胞具有诱导促进作用。     

Selective tropism of liver stem cells to hepatocellular carcinoma in vivo

AIM： To investigate the selective tropism of liver stem cells to hepatocellular carcinoma （HCC） in an animal model and its feasibility as a vector to deliver therapeutic genes for targeted therapy of HCC.
METHODS： WB-F344, a kind of rat liver stem cell, was infected with recombinant virus to establish a cell line with stable, high-level expressing enhanced green fluorescent protein （EGFP）. An animal model of HCC in Wistar rats was established by implanting HCC cells （CBRH7919） combined with an immunosuppressive drug. EGFP labeled liver stern cells were injected into caudal veins of the animals and distribution was observed at different time points after injection. SDF-1 and c-kit expression in non-tumor liver and tumor tissue were analysed by immunohistochemistry for the relationshiop between the expression and migration of liver stem cells. Furthermore, hepatic stern cells were injected via the portal vein, hepatic artery, caudal vein, or directly into the pericancerous liver tissue, respectively, and effects on migration, localization, and proliferation of the hepatic stern cells within the tumor tissue were observed and analyzed.
RESULTS： Recombinant adenovirus could deliver the EGFP gene to hepatic stem cells. A new stem cell line, named WB-EGFP, was established that stably expressed EGFP. WB-EGFP cells still showed selective tropism towards HCC and EGFP expression was stable in vivo. According to immunohistochemistry results, SDF-1 may not be related to the mechanisms of tropism of hepatic stem cells. Different application sites affected the distribution of liver stem cells. Injection via the portal vein was superior with regard to selective migration, localization, and proliferation of the hepatic stem cells within the tumor tissue.
CONCLUSION： Liver stem cells have the biological behavior of selective migration to HCC in vivo and they could localize and proliferate within HCC tissue stably expressing the target gene. Liver stem cells are a potential tool for a targeted gene therapy o     

Hematopoietic origin of hepatic stellate cells in the adult liver

Hepatic stellate cells are believed to play a key role in the development of liver fibrosis. Several studies have reported that bone marrow cells can give rise to hepatic stellate cells. We hypothesized that hepatic stellate cells are derived from hematopoietic stem cells. To test this hypothesis, we generated chimeric mice by transplantation of clonal populations of cells derived from single enhanced green fluorescent protein (EGFP)-marked Lin(-)Sca-1(+)c-kit(+)CD34(-) cells and examined the histology of liver tissues obtained from the chimeric mice with carbon tetrachloride (CCl(4))-induced injury. After 12 weeks of CCl(4) treatment, we detected EGFP(+) cells in the liver, and some cells contained intracytoplasmic lipid droplets. Immunofluorescence analysis demonstrated that 50% to 60% of the EGFP(+) cells were negative for CD45 and positive for vimentin, glial fibrillary acidic protein, ADAMTS13, and alpha-smooth muscle actin. Moreover, EGFP(+) cells isolated from the liver synthesized collagen I in culture. These phenotypes were consistent with those of hepatic stellate cells. The hematopoietic stem cell-derived hepatic stellate cells seen in male-to-male transplants revealed only one Y chromosome. Our findings suggest that hematopoietic stem cells contribute to the generation of hepatic stellate cells after liver injury and that the process does not involve cell fusion.     

Liver cancer stem cells: implications for a new therapeutic target

Hepatocellular carcinoma (HCC) is an aggressive tumour with a poor prognosis. Current therapeutic strategies against this disease target mostly rapidly growing differentiated tumour cells. However, the result is often dismal due to the chemoresistant nature of this tumour type. Recent research efforts on stem cells and cancer biology have shed light on new directions for the eradication of cancer stem cells (CSCs) in HCC. The liver is a distinctive organ with the ability of tissue renewal in response to injury. Based on the hypothesis that cancer development is derived from the hierarchy of the stem cell system, we will briefly discuss the origin of liver stem cells and its relation to HCC development. We will also summarize the current CSC markers in HCC and discuss their relevance to the treatment of this deadly disease.     

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP-cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.     

Origin of hepatocellular carcinoma: role of stem cells

The question of whether hepatocellular carcinoma (HCC) arises from the differentiation block of stem cells or dedifferentiation of mature cells remains controversial. Recently, researchers suggested that HCC may originate from the transdifferentiation of bone marrow cells. Interestingly, there are four levels of cells in the hepatic stem cell lineage: bone marrow cells, hepato-pancreas stem cells, oval cells and hepatocytes. Hematopoietic stem cells and the liver are known to have a close relationship in early development. Bone marrow stem cells could differentiate into oval cells, which could differentiate into hepatocytes and duct cells. The development of pancreatic and liver buds in embryogenesis suggests the existence of a common progenitor cell to both the pancreas and liver. Cellular events during hepatocarcinogenesis illustrate that HCC may arise from cells at various stages of differentiation in the hepatic stem cell lineage.     

Patient-derived dendritic cells transduced with an a-fetoprotein-encoding adenovirus and co-cultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against hepatocellular carcinoma cells.

BACKGROUND/AIMS: Breaking immunologic tolerance towards the hepatocellular carcinoma (HCC)-associated alpha-fetoprotein (AFP) antigen is possible. The use of this potential for the treatment of immunocompromised HCC patients is limited. In this study, we analyzed whether dendritic cells (DCs) from HCC patients transduced with a human AFP (hAFP)-expressing adenovirus and co-cultured with cytokine-induced killer (CIK) cells can induce a strong specific immune response against HCC-cells. METHODS: An hAFP-encoding adenovirus (Ad-hAFP) was generated. DCs from healthy donors or patients were transduced at a very high efficacy. Afterwards, DCs were co-cultured with autologous CIK-cells, and their ability to lyse HCC-cells was analyzed. RESULTS: AFP-transduced DCs stimulated CIK cells strongly to lyse about 70% of AFP-expressing HCC cells. Cytotoxicity was significantly higher when lymphocytes were co-cultured with Ad-hAFP-transduced DCs than with Ad-mock-transduced DCs, indicating an AFP-specific immune response. More interestingly, CIK cells from patients with AFP-positive HCC could be stimulated to lyse AFP-expressing HCC cells as effectively as CIK cells from healthy individuals and stronger than CIK cells from patients without AFP-expressing HCC. CONCLUSIONS: The data demonstrate that patient-derived DCs that were transduced with an AFP-expressing adenovirus and co-cultured with autologous CIK cells induce an AFP-specific, strong immune response against HCC cells. Therefore, this approach may have a potential for an adoptive and/or DC-based immunotherapy for HCC patients.     

基因修饰胎肝干细胞治疗肝纤维化

目的探讨肝细胞生长因子（HGF）基因修饰的胎肝干细胞治疗肝纤维化大鼠的效果。方法构建大鼠HGF基因和绿色荧光蛋白基因共同表达的融合载体,转染到胎肝母细胞中。将能够表达HGF的细胞移植到CCl4肝纤维化大鼠体内,将增强型绿色荧光蛋白（EGFP）修饰的胎肝干细胞作为对照组,检测大鼠肝功能和血清HGF及TGFβ1的浓度,肝组织中荧光蛋白数量和分布,肝组织内Ⅰ、Ⅲ型胶原蛋白的沉积情况。结果组织学及血清学检测显示,与对照组相比,经HGF基因修饰的胎肝干细胞能够显著改善肝纤维化指标和大鼠肝功能（P〈0.05）。结论 HGF修饰的胎肝干细胞治疗可能成为肝纤维化潜在的治疗手段。     

MT1E在肝癌形成过程中的表达及其在肝癌细胞中的作用

目的: 探讨MT1E基因在肝癌形成过程中不同阶段的表达及其在肝癌细胞中的生物学功能.方法: DEN诱发大鼠肝癌形成模型,分别观察造模4、8、16、20 wk后肝脏组织病理形态学的改变以及MT1E基因表达的差异;针对MT1E基因mRNA序列设计2个siRNA靶点,分别经质粒重组后电转入SMMC-7721肝癌细胞株中,实时荧光定量PCR检测MT1E基因的表达,MTT法检测细胞生长增殖.结果: 大鼠肝癌造模4、8 wk时肝组织主要表现为炎性病变,而到16 wk后呈现典型的增生病变,20 wk后已全部发展为肝细胞癌. MT1E基因在造模16 wk后表达明显增加,与对照组比较差异显著(芯片读数: 11524 vs 5462). 成功筛选到MT1E基因RNA干扰的一个有效靶序列,电转染72 h后该基因的表达量较空白对照组与阴性对照组明显降低(0.38 vs 1.00,0.93,P<0.01). 与阴性对照组比较,有效干扰靶点电转染144 h后细胞的生长增殖得到了明显抑制(0.1700±0.0313 vs 0.5748±0.0480,P<0.01).结论: 成功应用D E N诱发大鼠肝癌形成,MT1E基因在肝癌形成的后期表达明显升高,其高表达与肿瘤细胞的恶性增殖有关.     

Novel therapeutic target for cancer stem cells in hepatocellular carcinoma

Cancer is a disease of genetic and epigenetic alterations, which are emphasized as the central mechanisms of tumor progression in the multi-stepwise model. Discovery of rare subpopulations of cancer stem cells (CSCs) has created a new focus in cancer research. The heterogeneity of tumors can be explained with the help of CSCs supported by anti-apoptotic signaling. CSCs mimic normal adult stem cells by demonstrating unique characteristics of self-renewal and pluripotency, and the critical role for tumor growth and resistance to anti-cancer therapy. We found that CD13 was a surface marker for CSCs in human liver cancer cell lines and clinical samples, and that CD13+ CSCs were associated with a hypoxic marker in clinical hepatocellular carcinoma (HCC) sample, suggesting that CD13+ CSCs have the critical role in tumor growth and resistance to anti-cancer therapy in liver cancers. In this review article, we update recent findings regarding the involvement of CSCs, especially in HCC.