首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 218 毫秒
1.
人脐带间充质干细胞生物学特性及向类肝细胞的分化   总被引:14,自引:0,他引:14  
目的: 研究脐带间充质干细胞(umbilical cord-mesenchymal stem cells, UC-MSCs)生物学的特性及向肝细胞分化的可能性.方法:从脐带中分离间充质干细胞, 体外行传代培养, 检测脐带间充质干细胞表面免疫标志、细胞周期和生长活性等, 利用肝细胞生长因子、成纤维生长因子4和抑瘤素等细胞因子诱导脐带间充质干细胞向肝细胞分化, 用免疫细胞方法对诱导和未诱导的细胞进行免疫学检测, 糖原染色进行功能鉴定.结果: 从人脐带中可分离到贴壁生长的间充质干细胞, 细胞形态类似成纤维细胞,可在体外进行长期稳定培养; CD29、CD105和Vimentin表达阳性, 基本不表达CD34、CD31, 经加入细胞因子可成功将间充质干细胞向肝细胞诱导分化, 分化的细胞表达肝细胞表面标志物ALB、AFP、CK18和CK19, 糖原染色呈现阳性.结论:人脐带中可成功分离到间充质干细胞, 细胞可实现体外长期培养, 表达脐带间充质干细胞的表面标志, 在体外脐带间充质干细胞诱导分化为肝细胞, 有望成为细胞替代治疗的理想来源之一.  相似文献   

2.
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。  相似文献   

3.
目的 体外研究人骨髓间充质干细胞(hMSCs)分化为血管内皮细胞的潜能,探讨作为血管组织工程种子细胞来源的应用前景.方法 用人淋巴细胞分离液通过密度梯度离心法分离出成人骨髓单个核细胞,经贴壁法获取 MSCs ,体外扩增培养并进行鉴定.在内皮细胞生长培养基(DMEM)中以血管内皮细胞生长因子(VEGF) 、碱性成纤维细胞生长因子(bFGF)定向诱导 hMSCs 向血管内皮细胞分化,观察细胞形态学变化,扩增培养后经流式细胞仪进行细胞表型鉴定和超微结构观察.结果 形态学观察显示,培养的人骨髓间充质干细胞经 bFGF 、VEGF 诱导后的 hMSCs 可见类似内皮细胞样改变,向血管内皮诱导分化24 d后,经流式细胞仪检测, CD34 表达转为阳性,而CD106、HLA-DR 表达明显上调.结论 在血管内皮细胞生长因子及碱性成纤维细胞生长因子诱导作用下, hMSCs 具有向内皮细胞分化潜能,产生功能性内皮细胞.  相似文献   

4.
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.  相似文献   

5.
目的:探讨人脂肪间充质干细胞体外分离及培养方法。方法用消化离心的方法获得人脂肪间充质干细胞,以1×104/cm2接种于体积分数为0.1%胎牛血清的LG-DMEM培养基中,并进行细胞形态学观察,绘制细胞生长曲线,流式细胞仪检测细胞表面抗原,行成脂及成骨诱导检测其多向分化潜能。结果获取的脂肪间充质干细胞大小较为均匀,呈梭形的成纤维细胞样,细胞增殖良好;细胞生长曲线测定表明接种后第4天细胞进入指数增长期,第8天后生长进入平台期,体外能长期培养存活,并保持不分化状态;流式细胞仪检测表明细胞高表达 CD13、CD73,低表达 CD34、CD45及 HLA-DR;能诱导成脂及成骨细胞。结论利用消化离心法在体外能分离得到脂肪间充质干细胞,细胞生长良好,可作为组织工程的种子细胞。  相似文献   

6.
本实验用免疫磁性分离(MACS)方法分选骨髓间充质干细胞(MSCs)亚群MAPCs,用成纤维细胞生长因子-4(FGF-4)及肝细胞生长因子(HGF)诱导其向肝细胞方向分化,并采用不同方法鉴定了细胞分化能力。  相似文献   

7.
肝细胞移植治疗、肝组织工程以及生物人工肝等新兴技术的发展,为肝病患者带来了新的治疗方案;不过,这些新技术应用时会遇到一个共同的瓶颈--细胞来源不足.虽然肝细胞可以有多种来源,但是以干细胞的发展最为迅猛,随着近年来干细胞分离、培养、鉴定及其增殖技术的发展和成熟,利用干细胞解决肝细胞的来源问题已经引起了广泛关注.此文总结了骨髓间充质干细胞诱导为肝细胞的研究现状、可能影响因素以及分化机制.  相似文献   

8.
脐带间充质干细胞体外诱导分化为肝细胞样细胞的研究   总被引:2,自引:0,他引:2  
目的探讨人脐带间充质干细胞(UC-MSCs)体外诱导分化为肝细胞的可行性和方法,观察分化后的细胞白细胞表面抗原表达的改变。方法采用酶学法从脐带全层组织中分离UC-MSCs,采用改良的肝分化培养体系体外诱导UC-MSCs向肝细胞分化。诱导后2、4周,观察细胞形态,免疫荧光细胞化学染色法检测甲胎蛋白(AFP)、白蛋白(ALB);RT-PCR检测肝细胞标志性基因AFP、ALB和CK19 mRNA。ELISA法检测诱导后的细胞培养液中ALB水平。流式细胞仪检测诱导后细胞白细胞表面抗原HLA-Ⅰ和HLA-Ⅱ。结果从人脐带基质中分离培养的UC-MSCs在向肝细胞诱导分化培养体系的培养下,细胞形态由长梭形逐渐转化成为多角形或类圆形,并且表达AFP、ALB mRNA和蛋白,诱导后的UC-MSCs以时间依赖方式产生ALB,诱导后的细胞不表达HLA-DR。结论UC-MSCs具有向肝细胞分化的潜能,分化后的M细胞仍具有低免疫原性。  相似文献   

9.
骨髓间充质干细胞体外定向诱导内皮祖细胞的实验研究   总被引:2,自引:1,他引:2  
目的:研究兔骨髓间充质干细胞(mensenchymal stem cell,MSC)在体外定向诱导分化为内皮祖细胞(endothelial progenitor cell,EPC)的状况。方法:取兔髂骨骨髓,经密度梯度离心法分离并培养骨髓间充质干细胞,实验组细胞在内皮细胞生长添加剂、血管内皮生长因子、成纤维细胞生长因子等诱导条件下定向分化为内皮祖细胞。对照组不干预,继续培养。结果:实验组细胞集落接近融合时形态呈现“鹅卵石”样或“铺路石”样外观,流式细胞仪检测发现实验组细胞较对照组高度表达CD133、CD34(P<0.05)。结论:体外诱导可使骨髓间充质干细胞分化为内皮祖细胞,并有效扩增。  相似文献   

10.
人骨髓间充质干细胞体外分化为肝细胞样细胞   总被引:12,自引:3,他引:12  
目的 探讨人骨髓间充质干细胞(MSCs)的体外培养及特异性诱导为肝细胞样细胞的能力。方法 骨髓标本来源于健康志愿者的胸骨,年龄2~35岁,采用淋巴细胞分离液(密度1.077)分离人MSCs,并分别采用HGF、FGF4、HGF FGF4以及无生长因子四种处理因素体外诱导第三代人MSC向肝细胞样细胞分化。通过流式细胞术分析鉴定.MSCs的纯度,于诱导培养的0、7、14、21、28天时留取细胞检测CK18、AFP和白蛋白的表达情况,同时进行糖原染色验证细胞功能。结果 用淋巴细胞分离液分离出的人MSCs纯度可达90%,采用HGF、FGF4及HGF FGF4三种处理因素均可在体外诱导人MSCs特异分化为具有肝细胞样细胞表型和功能的细胞。结论 人MSCs能在体外扩增并定向诱导为肝细胞样细胞。  相似文献   

11.
12.
With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpresed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.  相似文献   

13.
14.
AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like...  相似文献   

15.
BACKGROUND: Differentiation of liver progenitor cells(LPCs) to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS: Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line(HSCLi) we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS: Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS: Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.  相似文献   

16.
BACKGROUND: Differentiation of liver progenitor cells (LPCs) to functional hepatocytes holds great potential to de-velop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efifcient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.
METHODS: Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line (HSC-Li) we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs.
RESULTS: Co-culturing with HSC-Li cells induced differentia-tion of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addi-tion, the differentiated cells expressed liver-speciifc genes and possessed hepatic functions, including glycogen storage, low-density lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.
CONCLUSIONS: Our method, which employs indirect co-culture with HSC-Li cells, can efifciently induce the differentiation of LPCs into functional hepatocytes. This ifnding suggests that this co-culture system can be a useful method for the efifcient generation of functional hepatocytes from LPCs.  相似文献   

17.
18.
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   总被引:24,自引:2,他引:24  
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。  相似文献   

19.
AIM:: It is imperative to explore some ways to gain the functional hepatocytes for hepatocyte transplantation. Bone marrow stem cells can differentiate into hepatocytes in vivo and in vitro. We select fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF) as differentiation factors, and design the appropriate directed differentiation medium in order to gain hepatocytes through directed differentiation of bone marrow stem cells. METHODS:: Bone marrow mononuclear cells (BMMCs) were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocyte were validated and confirmed by RT-PCR. The ALB-, and CK18-expressed cells were gone further step to be confirmed by Western blot analysis, immunofluorescence and flow cytometric analysis. Hepatocyte functional activity, including glycogen synthesis and urea production, were confirmed by periodic acid-Shiff (PAS) staining and urea assay. RESULTS:: Some epithelial-like cells or polygonal cells appeared in the directed differentiation medium within 12 days, and the number and sizes of colonies of epithelial-like cells or polygonal cells increased in the course of the cell directed differentiation. AFP, HNF-3ss, ALB and CK18 mRNA expressions first appeared within day 7, and lasted throughout the later directed differentiation. TTR, G-6-P and TAT mRNA expressions could be detected within day 14, and their expressions lasted in the course of the later directed differentiation. ALB and CK18 were confirmed to exist in the differentiated BMMCs by Western blot analysis. ALB was found in the cytoplasm and cell membrane, while CK18 scattered in the cytoplasm by immunofluorescent staining. On day 21,the ratio of ALB-positive cells was 69.45%, and the ratio of CK18-positive cells was 67.36%. The accumulation of glucogen was detected in the cytoplasm of the differentiated cells. The directed differentiated BMMCs produced urea 3 days later, and they produced urea in a time-dependent manner. CONCLUSIONS:: BMMCs could differentiate into hepatocytes or hepatocyte-like cells in the differentiation media including HGF, FGF-4, EGF, and OSM. These hepatocyte-like cells were identified at the gene level and protein level. Furthermore, these hepatocyte-like cells had some hepatocellular synthesis and metabolism functions.  相似文献   

20.
In vitro hepatic differentiation of human mesenchymal stem cells   总被引:78,自引:0,他引:78  
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.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号