骨髓间充质干细胞向肝系细胞分化的研究进展

干细胞(stem cell)是指一类具有自我增殖能力和分化潜能的细胞,在特定的条件下可以分化为不同的功能细胞,甚至形成多种组织和器官.因此,干细胞的研究已成为生命科学的一个重要领域.其中,骨髓干细胞由于其取材方便,而成为研究的热点.     

体外诱导入骨髓间充质干细胞向肝系细胞分化过程中的基因表达谱变化

目的 应用微阵列分析骨髓间充质干细胞向肝系细胞分化的基因表达变化.方法 收集健康志愿者胸骨骨髓,分离间充质干细胞体外培养,应用HGF+ FGF4诱导分化并鉴定分化细胞;利用微阵列技术筛选骨髓间充质干细胞向肝系细胞分化的有关基因.结果 121条基因表达发生变化,其中89条上调,32条下调.结论 相关基因涉及信号转导、能量代谢、基因转录、蛋白质翻译与合成等.SGK、ApoB、LDLR和CETP等可能在其分化过程中有非常重要的意义.     

脂肪间充质干细胞向肝细胞横向分化的研究

目的探讨脂肪源性间充质干细胞（AMSC）向肝细胞横向分化的可能性。方法胶原酶消化脂肪组织,贴壁培养,体外扩增后以流式细胞仪鉴定其表面标志。取扩增3代的AMSC分为2组,诱导分化组在含有2％FBS的DMEM-F12培养基中加入肝细胞生长因子20 ng/ml和成纤维细胞生长因子4 10 ng/ml、1×ITS和地塞米松0．1μmol/L,培养14 d；空白对照组则不加任何细胞因子。RT-PCR检测诱导分化过程中肝细胞核因子1、GATA4等基因转录水平的变化。2周后,采用流式细胞术检测AFP和Alb阳性细胞在两组细胞中的比例,检测肝细胞特异性细胞角蛋白（CK） 18、CK19的表达。结果分离、培养的AMSC呈成纤维细胞样生长,可以稳定传代。流式细胞术检测结果显示第3代脂肪间充质干细胞高表达表面CD29、CD44；不表达CD34、CD45。RT-PCR检测诱导5、8、11、14 d的细胞,显示有肝细胞特异性转录因子GATA4和肝细胞核因子1A基因的表达,并随时间延长而逐渐增多。流式细胞术检测诱导14 d的细胞,发现30．0％的细胞表达Alb,17．8％细胞表达AFP,双阳性的细胞为6．9％；免疫荧光检测发现诱导细胞表达CK18、CK19。空白对照组脂肪间充质干细胞则未见上述各项变化。结论在低血清培养体系中,采用细胞因子联合诱导,显示脂肪间充质细胞在体外能定向分化为肝细胞样细胞。     

体外诱导间充质干细胞向心肌细胞分化的研究进展

间充质干细胞是近年来一类倍受人们关注的干细胞,它有望成为心脏组织修复工程最理想的种子细胞。多项研究表明,间充质干细胞能在体外被诱导分化为心肌细胞,但是与其分化的相关因素和分化机制还需要进一步的研究探讨。     

体外培养成人脂肪间充质干细胞及向平滑肌细胞诱导分化

目的 体外培养成人脂肪间充质干细胞,并应用转化生长因子β将脂肪间充质干细胞诱导分化为平滑肌细胞.方法 采用酶消化法和贴壁培养法分离培养脂肪间充质干细胞,流式细胞仪对第3代和第5代细胞进行表面抗原检测,对第5代细胞进行转化生长因子β诱导,于诱导后第10天进行免疫化学鉴定.结果 体外培养的脂肪间充质干细胞呈扁平的长梭形,细胞形态均一,传代稳定.干细胞相关标志CD29和CD44表达阳性,造血干细胞相关标志CD34随传代次数的增加由弱阳性逐渐转为阴性,内皮细胞相关标志CD31表达阴性.流式细胞仪检测结果 发现脂肪间充质干细胞中G0/G1、S和G2/M期的细胞分别占90.14%、3.77%和6.09%.转化生长因子β定向诱导后倒置显微镜下观察细胞呈"峰"、"谷"样形态,免疫荧光化学检测发现诱导组细胞α平滑肌肌动蛋白表达阳性.结论 成人脂肪组织中含有间充质干细胞,且可在转化生长因子β诱导后分化为平滑肌细胞.     

脂肪源性间充质干细胞与肝病

脂肪源性间充质干细胞是近年来发现的一种成体干细胞,具有自我更新和多向分化潜能。由于其广泛存在于脂肪组织中,取材容易,获得率高而有望成为组织工程和基因工程中新的干细胞来源,极具临床应用及基础研究前景。本文对脂肪源性间充质干细胞的生物学特性、多向分化潜能及其在肝脏疾病治疗方面的最新研究进展进行了阐述。     

低密度脂蛋白诱导骨髓间充质干细胞向平滑肌样细胞分化的作用

目的 探讨低密度脂蛋白诱导骨髓间充质干细胞向平滑肌样细胞分化及myocardin在其分化过程中的作用.方法 不同浓度血清培养骨髓间充质干细胞条件下加以低密度脂蛋白刺激.细胞分为常规培养组,常规培养+低密度脂蛋白刺激组(25 mg/L), 20%胎牛血清培养组, 20%胎牛血清+低密度脂蛋白刺激组.免疫细胞化学检测myocardin、平滑肌肌动蛋白及平滑肌肌球蛋白重链的表达、RT-PCR法检测myocardin、平滑肌肌动蛋白及平滑肌钙结合相关蛋白mRNA的表达.结果 免疫细胞化学结果显示常规培养+低密度脂蛋白刺激组(25 mg/L),20%胎牛血清培养组, 20%胎牛血清+低密度脂蛋白刺激组分别与常规培养组比较myocardin、平滑肌肌动蛋白及平滑肌肌球蛋白重链的表达增强(P<0.05);20%胎牛血清+低密度脂蛋白刺激组与20%胎牛血清组比较上述三种蛋白表达增强(P<0.05);平滑肌肌动蛋白及平滑肌肌球蛋白重链蛋白的表达与myocardin表达呈正相关(r=0.9018,P<0.05;r=0.7969,P<0.05).RT-PCR结果显示常规培养+低密度脂蛋白刺激组(25 mg/L), 20%胎牛血清培养组, 20%胎牛血清+低密度脂蛋白刺激组分别与常规培养组比较myocardin、平滑肌肌动蛋白及平滑肌钙结合相关蛋白mRNA表达明显增强(P<0.05);20%胎牛血清培养组, 20%胎牛血清+低密度脂蛋白刺激组比较上述三个指标的表达也增强(P<0.05).平滑肌肌动蛋白、平滑肌钙结合相关蛋白mRNA表达与myocardin正相关(r=0.9295,P<0.05;r=0.8940,P<0.05).结论 高浓度血清,低密度脂蛋白均可诱导骨髓间充质干细胞向平滑肌样细胞分化,低密度脂蛋白联合20%胎牛血清诱导骨髓间充质干细胞向平滑肌样细胞分化的作用最强.myocardin可能在此过程中起重要作用.     

骨髓间充质干细胞治疗肝纤维化的研究进展

骨髓间充质干细胞是一种多能干细胞,具有分化成肝细胞的潜力.近年研究表明,骨髓间充质干细胞还具有很强的抗肝纤维化的作用,并且不发生同种异体移植排斥反应,这为肝纤维化、肝硬化的治疗带来了新的策略.     

大鼠脂肪间充质干细胞在部分肝切除模型中向肝细胞分化的研究

目的探讨脂肪间充质干细胞（ADMSCs）在部分肝切除模型中向肝细胞的分化。方法从大鼠脂肪组织中分离出干细胞，并进行体外扩增、传代，取第2代ADMSCs用PKH26标记，制作部分肝切除模型，将标记细胞经门静脉自体植入体内。2周后切下肝脏制成冰冻切片，荧光显微镜下观察标记细胞在肝脏的定位，进行免疫荧光染色检测标记细胞白蛋白的表达。结果从脂肪组织中分离出的ADMSCs能在体外大量扩增，PKH26标记后细胞在荧光显微镜下发红色荧光，细胞标记率约95％；荧光显微镜下可见肝脏冰冻切片中散在分布红色标记细胞，免疫荧光染色显示大多数标记细胞白蛋白染色阳性。结论 大鼠脂肪间充质干细胞在肝再生环境中能向肝细胞分化，有可能在肝部分切除后参与肝再生。     

骨髓间充质干细胞对肝脏的影响

<正>骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)是成体干细胞主要类型之一,能产生所有的间质谱系细胞,可修复受损的组织,具有干细胞的特性。近年来,随着BMSCs在治疗慢性肝病中的临床研究以及基础研究的不断深入,对肝-骨轴双向调节模式、骨髓细胞及BMSCs对肝脏的影响也有了进一步的了解。     

In vitro hepatic differentiation of human mesenchymal stem cells

This study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from embryonic mesoderm, are able to differentiate into functional hepatocyte-like cells in vitro. MSCs were isolated from human bone marrow and umbilical cord blood, and the surface phenotype and the mesodermal multilineage differentiation potentials of these cells were characterized and tested. To effectively induce hepatic differentiation, we designed a novel 2-step protocol with the use of hepatocyte growth factor and oncostatin M. After 4 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein, and phenobarbital-inducible cytochrome P450 activity. In conclusion, human MSCs from different sources are able to differentiate into functional hepatocyte-like cells and, hence, may serve as a cell source for tissue engineering and cell therapy of hepatic tissues. Furthermore, the broad differentiation potential of MSCs indicates that a revision of the definition may be required.     

Directed differentiation of human embryonic stem cells into functional hepatic cells

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION: We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.     

Hydrogen sulfide increases hepatic differentiation in tooth-pulp stem cells

The toxicity of hydrogen sulfide (H(2)S), an oral malodorous compound, is well reported. We have recently established an experimental model of hepatic differentiation from human tooth-pulp stem cells (HTPC) using serum-free medium. The objective of the present study is to determine the effect of H(2)S on hepatic differentiation. The CD117 positive cell fraction was obtained from deciduous HTPC using magnetic cell sorting. After 3-4 passages, cells were grown in Dulbecco's modified Eagle's medium supplemented with insulin-transferrin-selenium-x (ITS-x), embryotrophic factor (ETF) and hepatocyte growth factor (HGF) for hepatic commitment (five days). For hepatic differentiation the cells were cultured in Iscove's modified Dulbecco's medium supplemented with ITS-x, ETF, oncostatin, HGF and dexamethasone for 15 days in air containing 5% CO(2), with or without H(2)S at 0.05 ng ml(-1). Cells were assayed for the expression of hepatic markers α-fetoprotein, albumin or carbamoyl phosphate synthetase, and urea concentrations and glycogen synthesis were also determined. The panel of hepatic markers was expressed more in the test groups exposed to H(2)S than in the control groups. Urea and glycogen production were also increased, especially glycogen which was approximately five times greater compared to the control (p < 0.01). We concluded that H(2)S at physiological concentrations increased the ability of HTPC to undergo hepatogenic differentiation.     

Characterization of the differentiation capacity of rat-derived hepatic stem cells

The liver in an adult rat maintains a balance between cell gain and cell loss. Although normally proliferatively quiescent, hepatocyte loss such as that caused by partial hepatectomy (PH) invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and "wear and tear" renewal is largely achieved by hepatocyte self-replication. Furthermore, hepatocyte transplants in rats, in which a selective pressure for the transplanted cells can be applied, have shown that a certain proportion of hepatocytes can undergo significant clonal expansion, suggesting that hepatocytes themselves are the functional stem cells of the liver. Fetal liver may also harbor bipotential stem cells capable of sustained clonal expansion. More severe liver injury activates a potential stem cell compartment located within the canals of Hering, giving rise to cords of bipotential oval cells that can differentiate into hepatocytes and biliary epithelial cells. Other cell populations with hepatic potential reside in the bone marrow; whether these hematopoietic cells can function as stem cells for the rat liver remains to be confirmed. Pancreatic cells have also been found to have hepatocytic potential.     

人脂肪干细胞向内皮分化最佳诱导体系的建立

目的 探讨人脂肪干细胞(human Adipose-derived stem cells,hADSCs)体外分化为内皮细胞的最佳诱导体系.方法 利用胶原酶消化法和贴壁筛选法从人脂肪组织中分离、培养及扩增hADSCs,分3组分别进行内皮诱导:即纤维连接蛋白(fibronectin,FN)包被组、明胶包被组和未铺组;同时诱导液分为两组:单纯内皮细胞生长培养基(EGM2-MV)组及内皮细胞生长培养基(EGM2-MV)+50 ng/ml血管内皮细胞生长因子(VEGF_(165))组.诱导15 d后,通过流式细胞术检测各组内皮细胞特异性表面抗原CD34的表达;通过相差显微镜观察诱导前后细胞的一般形态学特征.结果 体外分离、培养出高度同源性的hADSCs,CD29、CD44、CD90、CD105及CD166呈阳性表达,而CD34、CD45及HLA-DR呈阴性表达;诱导后细胞流式结果显示单纯EGM-2MV诱导的3组细胞内皮细胞特异性标志CD34阴性表达,而EGM2-MV+50 ng/ml VEGF_(165)诱导的细胞,其中FN包被组CD34表达率为8.4%,明胶包被组为5.4%,未铺组为4.2%;相差显微镜下可见诱导后细胞呈三角形或多边形,融合处呈铺路石样生长.结论 FN作为细胞外基质与EGM2-MV+50 ng/ml VEGF_(165)组成的诱导液协同作用,构成了hADSCs体外分化为内皮细胞的最佳诱导体系,可为临床上缺血性疾病的自体细胞移植治疗提供大量的种子细胞来源.     

Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells

Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, the in vitro directed differentiation of human pluripotent stem cells into mature hepatocytes remains challenging. Little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. In the current study, we developed an improved hepatic differentiation protocol and compared 28 hiPSC lines originated from various somatic cells and derived using retroviruses, Sendai viruses, or episomal plasmids. This comparison indicated that the origins, but not the derivation methods, may be a major determinant of variation in hepatic differentiation. The hiPSC clones derived from peripheral blood cells consistently showed good differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts showed poor differentiation. However, when we compared hiPSCs from peripheral blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. These data underscore the importance of donor differences when comparing the differentiation propensities of hiPSC clones.     

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

INTRODUCTIONMost liver diseases lead to hepatocyte dysfunction with the possibility of eventual organ failure. The replacement of diseased hepatocytes and the stimulation of endogenous or exogenous regeneration by stem cells are the main aims of liver-dir…     

肝星状细胞移行及肝细胞间质变

肝星状细胞(HSC)是合成细胞外基质(ECM)的主要细胞,HSC的活化、增殖是肝纤维化发生的中心环节.新近研究结果显示,HSC移行到损伤部位或正常区域,对正常肝组织的愈合及肝纤维化形成起蕈要作用.     

脂肪组织来源多能干细胞的心肌分化潜能及意义

脂肪组织提取细胞(PLA细胞)已成为干细胞研究的又一热点.PLA细胞不仅具有容易获取、提取量大、无需特殊血清即可稳定增殖、低水平衰减等特点,且具有多向分化潜能,可定向分化为成肌细胞、脂肪细胞、成骨细胞、成软骨细胞、神经细胞等.本文就PLA细胞的概念、提取分离方法、基本特性等进行综述,并探讨其在心脏疾病治疗中的作用及临床应用前景.     

Advantages of adipose tissue stem cells over CD34+ mobilization to decrease hepatic fibrosis in Wistar rats

Introduction and ObjectivesChronic liver inflammation may lead to hepatic cirrhosis, limiting its regenerative capacity. The clinical standard of care is transplantation, although stem cell therapy may be an alternative option. The study aim was to induce endogenous hematopoietic stem cells (HSCs) with granulocyte colony stimulating factor (G-CSF) and/or intravenous administration of adipose tissue-derived mesenchymal stem cells (MSCs) to decrease hepatic fibrosis in an experimental model.Material and methodsA liver fibrosis model was developed with female Wistar rats via multiple intraperitoneal doses of carbon tetrachloride. Three rats were selected to confirm cirrhosis, and the rest were set into experimental groups to evaluate single and combined therapies of G-CSF-stimulated HSC mobilization and intravenous MSC administration.ResultsTreatment with MSCs and G-CSF significantly improved alanine amino transferase levels, while treatment with G-CSF, MSCs, and G-CSF + MSCs decreased aspartate amino transferase levels. Hepatocyte growth factor (HGF) and interleukin 10 levels increased with MSC treatment. Transforming growth factor β levels were lower with MSC treatment. Interleukin 1β and tumor necrosis factor alpha levels decreased in all treated groups. Histopathology showed that MSCs and G-CSF reduced liver fibrosis from F4 to F2.ConclusionsMSC treatment improves liver function, decreases hepatic fibrosis, and plays an anti-inflammatory role; it promotes HGF levels and increased proliferating cell nuclear antigen when followed by MSC treatment mobilization using G-CSF. When these therapies were combined, however, fibrosis improvement was less evident.