地黄低聚糖对人脂肪组织源性间充质干细胞分泌血管内皮细胞生长因子的影响

目的 探讨地黄低聚糖(RGO)对分离、培养的人脂肪组织源性间充质干细胞(ADMSCs)分泌血管内皮细胞生长因子(VEGF)的影响。方法 人脂肪组织用2.5 g/L胶原蛋白酶Ⅰ消化、分离ADMSCs。取沉淀的细胞进行培养,用免疫细胞化学染色法鉴定其表面CD44、CD105、CD45和CD34分子的表达。以IMDM培养基作为空白对照,加入RGO配成不同浓度（0、1、10、100及400 mg/L）的IMDM分别培养ADMSCs,用MTT比色法检测ADMSCs的增殖。培养72 h后,用ELISA法测定不同培养基中VEGF的含量。结果 免疫细胞化学染色显示,分离培养的细胞表面CD44+、CD105+、CD34-、CD45-。MTT比色法的结果显示,与空白对照组相比,1和10 mg/L组无明显差别,100及400mg/L组明显升高（均P<0.01）,且100mg/L组明显高于400mg/L组(P<0.05)。0、1、10、100、400mg/L5个组VEGF的含量,分别为(627.88±33.86)、(655.50±40.29)、(666.50±43.20)、（843.50±53.05）和（722.88±51.34）ng/L。结论 RGO对ADMSCs 的增殖有促进作用,且呈一定的浓度依赖性,并导致其分泌VEGF的作用增强。     

5-氮胞苷体外诱导成人脂肪间充质干细胞分化为心肌样细胞的初步研究

目的:探索不同浓度5-氮胞苷(5-aza)在不同诱导时间下对成人脂肪间充质干细胞(ADMSCs)分化为心肌细胞的诱导作用,确定最佳诱导条件。方法:体外分离成人脂肪组织来源的间充质干细胞并进行传代培养,流式细胞仪鉴定细胞CD44、CD34的表达,用含不同浓度5-aza(3、5、10、15、20μmol／L)培养基分别诱导12小时、24小时、48小时、72小时,倒置相差显微镜下逐日观察细胞形态变化;于诱导后14、28天时免疫细胞化学染色鉴定心肌特异性肌钙蛋白I(cTn-I)的表达,分析细胞转化率。结果:分离培养的ADMSCs表达CD44,不表达CD34;5-aza诱导后14天时免疫细胞化学未见有心肌细胞特异性cTn- I表达,诱导28天细胞免疫细胞化学显示cTn-I表达阳性,以10μmol/L 5-aza诱导24小时为最佳体外诱导条件。结论:成人脂肪组织间充质干细胞可在体外5-aza诱导下向心肌细胞分化。     

兔脂肪源性间充质干细胞的扩增及其基本生物学特性

目的探讨兔脂肪间充质干细胞在体外的基本生物学特性。方法选取清洁级新西兰大白兔4只，采用Ⅱ型胶原酶消化兔腹股沟区皮下脂肪组织，贴壁法培养脂肪间充质干细胞。检测细胞生长动力学，测定生长因子EGF对细胞生长的影响，采用免疫细胞荧光法检测基质细胞表面抗原CD44、CD45。结果所培养细胞由P0传代至P8，每代倍增数目未发现有下降趋势；EGF具有显著的促进ADSCs增殖的作用，所培养细胞表达CD44抗原，荧光显微镜下呈红色荧光，CD45近似于阴性表达。结论由兔脂肪组织分离扩增脂肪间充质干细胞，不仅具备干细胞的基本生物学特性，而且增殖稳定，可获得充足的细胞数量，有望成为组织工程的种子细胞来源。     

糖尿病患者脂肪间充质干细胞的分离培养与鉴定

目的：探讨体外分离培养糖尿病患者脂肪间充质干细胞（ADSCs）的方法并分析其生物学特性。方法采用胶原酶消化法从糖尿病患者的脂肪组织中分离出ADSCs。对ADSCs进行体外培养,倒置相差显微镜下观察细胞形态;绘制第3、6、8代传代细胞的生长曲线;流式细胞仪检测第3代细胞表面标记物（ CD34、CD45、CD29、CD44、CD105、CD90）;通过成脂诱导实验鉴定细胞的分化潜能（油红染色阳性判定为脂肪细胞,证实细胞可向脂肪细胞分化）。结果分离出的ADSCs,能稳定传代;其细胞表面标记物CD34、CD45呈阴性表达,CD29、CD90、CD44、CD105呈阳性表达。成脂诱导后油红染色阳性。结论在糖尿病患者脂肪组织中成功分离到ADSCs,在体外适宜条件下ADSCs可向脂肪细胞分化,具有分化潜能。     

胎肝来源间充质干细胞的分离、培养与多向分化

目的从胎肝中分离培养间充质下细胞，并研究其牛物学特性。方法用优化的方法从胎肝中分离获得间充质干细胞。利用流式细胞仪分析细胞表型和细胞周期分布，并体外诱导成骨、成脂肪和成肝组织细胞分化。并用染色方法鉴定成骨、成脂肪分化结合形态学方法和RT-PCR方法鉴定成肝组织分化结果。结果从胎肝中分离培养的细胞为成纤维样，贴壁生长．表型相对均一，表面标志为CD90，CD44，CD147，而CD34，CD45，HLA-DR，具有向肝组织分化的潜能，并可向成骨和成脂肪分化。结论从胎肝中可分离培养得到具有多向分化潜能的间充质干细胞。     

人脂肪间充质干细胞体外分离、培养的方法研究

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。     

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。     

人脐带间充质干细胞的分离培养及成脂成骨分化

目的建立小胎龄人脐带间充质干细胞(hUCMSCs)的分离和培养方法,探讨hUCMSCs的成脂、成骨分化潜能。方法采用Ⅱ型胶原酶和透明质酸酶消化法从胎龄12～18w的流产胎儿脐带中分离hUCMSCs;流式细胞仪检测其免疫表型;应用不同因子诱导hUCMSCs向脂肪细胞及成骨细胞分化并进行鉴定。结果体外培养的hUCMSCs呈长梭形,细胞形态均一;表达CD29、CD44、CD73、CD105、CD166;不表达CD34、CD45、CD40、CD40L、CD80、CD86、HLA-DR;油红O、茜素红染色及RT-PCR证实hUCMSCs可分化为脂肪细胞和成骨细胞。结论建立了小胎龄hUCMSCs分离培养方法,证实其具有成脂、成骨分化潜能,有望成为细胞治疗及组织工程更为理想的种子细胞。     

脑组织匀浆诱导人脐带间充质干细胞分化为神经样细胞

目的模拟体内微环境,研究人脐带间充质干细胞(hUCMSCs)在脑组织匀浆诱导下向神经样细胞的分化程度。方法体外分离、培养、扩增hUCMSCs,流式细胞仪鉴定其表面抗原CD29、CD44、CD45、CD105、CD34、HLA-DR;制备大鼠脑组织匀浆与hUCMSCs共培养,在倒置显微镜下观察细胞形态变化,并应用免疫细胞化学技术检测共培养3 d后细胞内神经干细胞表面标志物巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)及胶质纤维酸性蛋白(GFAP)的表达。结果脑组织匀浆培养hUCMSCs后,细胞表达nestin、NSE及GFAP,而正常培养的hUCMSCs不表达。结论脑组织匀浆可以诱导hUCMSCs向神经样细胞分化。     

人胎肺间充质干细胞的分离

目的 探索分离人胎肺间充质干细胞（MSCs）的有效方法.方法 在无菌条件下采集人工流产胎儿的肺组织,分别以组织块培养法和酶消化法分离细胞;通过相差显微镜观察细胞形态,流式细胞学方法检测细胞表型、成脂和成骨分化的多向分化潜能并证实其MSCs特征.结果 两种分离方法所得细胞,在原代培养初期的形态有所不同,但培养后期和传代后均为长梭形的贴壁细胞.流式细胞学检测显示,两种方法分离的细胞均表达MSCs标志物CD13、CD29、CD44、CD90、CD105、CD166及HLA-ABC,但不表达造血细胞系的标志物CD45、CD34、CD14、CD38、CD133和内皮相关抗原CD31,也不表达CD41a、CD42b、CD49d、CD106、CD61和HLA-DR.成脂诱导3～5d后,部分细胞转变为肥大、扁平的多角形细胞;1周后可见细胞内有囊泡状脂滴积聚;2周后脂滴增多、变大,并可被红O着色.成骨诱导者细胞内逐渐出现钙盐沉着,经诱导2周后大部分细胞内可见因钙盐沉着被茜素红S着色.结论 组织块培养法和胶原酶消化法均为获得人胎肺MSCs的有效方法.     

重组腺相关病毒介导人血管内皮生长因子165基因在大鼠骨髓间充质干细胞中的表达

目的探讨以腺相关病毒(rAAV)为血管内皮细胞生长因子165基因(VEGIF165)载体,在体外转染大鼠骨髓间充质干细胞(MSCs),并研究其相关特性。方法 (1)全骨髓培养法提取培养MSCs,利用免疫组化法检测MSCs表面标志CD34、CD44;流式细胞分析法检测CD90。(2)rAAV-VEGF165转染MSCs,采用ELISA及PCR检测VEGF的表达,比较转染前后细胞的变化情况,观察VEGF165基因转染对MSCs的影响。结果 (1)成功培养出MSCs,CD44阳性表达,CD34阴性表达,CD90阳性表达。(2)在转染rAAV-VEGF165后转染组上清中VFGF165分泌水平明显高于未转染组(p<0.05),5 d时达到高峰,此后表达开始下降。琼脂糖凝胶电泳可见高亮度条带。表明rAAv-VEGF165成功转染进MSCs细胞中,绘制生长曲线,显示rAAV-VEGF165基因转染后对MSC生长无影响。结论rAAV-VEGF表达载体可有效感染MSCs,并在体外高效表达,为MSCs联合基因治疗提供了实验依据。     

SD大鼠骨髓间充质干细胞的体外培养及表型、纯度鉴定

目的探索获得高纯度MSCs简单、系统的实验方法。方法采用贴壁培养法体外培养SD大鼠骨髓间充质干细胞（MSCs）,确定最佳接种密度,经数次传代后纯化,以表面抗原CD29、CD44作为标志分子,CD34作为阴性对照,免疫荧光法鉴定MSCs的生物学特性及纯度。结果以0．5—1×10^10个／L的细胞密度接种骨髓细胞原代培养时间明显缩短,免疫荧光法鉴定MSCs生物学特性好,第3代时已达较高纯度。结论本实验建立了较为理想的MSCs体外培养体系,纯化速度快,鉴定方法简单,为进一步研究MSCs生物学行为及临床应用奠定了基础。     

类风湿关节炎骨髓间充质干细胞的体外培养和一般生物学特性的初步研究

目的 建立类风湿关节炎(RA)患者骨髓间充质干细胞(BMSCs)的体外培养、扩增方法,了解其一般生物学特性.方法 采集RA患者的骨髓标本,经密度梯度离心加贴壁法分离、纯化获得其BMSCs,进行体外培养扩增,观察原代和传代细胞的形态和生长状况、免疫表型分析及增殖能力检测,并对BMSCs进行成纤维细胞集落(CFU-F)形成分析.结果 来自RA患者的BMSCs呈典型的成纤维样细胞形态,表型鉴定CIM5阴性,SH2(CD105)、CD71、CD44阳性,其增殖能力和CFU-F形成能力与源于正常供者的BMSCs相当.结论 RA患者骨髓中存在具有多向分化能力的MSCs,来源于RA患者的BMSCs在体外具有良好的细胞增殖能力,在细胞形态、集落形成能力以及细胞免疫表型方面与正常供者的BMSCs差异无统计学意义.     

Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow

Human mesenchymal stem/progenitor cells (MSCs) have been identified in adult bone marrow, but little is known about their presence during fetal life. MSCs were isolated and characterized in first-trimester fetal blood, liver, and bone marrow. When 10(6) fetal blood nucleated cells (median gestational age, 10(+2) weeks [10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the mean number (+/- SEM) of adherent fibroblastlike colonies was 8.2 +/- 0.6/10(6) nucleated cells (69.6 +/- 10/microL fetal blood). Frequency declined with advancing gestation. Fetal blood MSCs could be expanded for at least 20 passages with a mean cumulative population doubling of 50.3 +/- 4.5. In their undifferentiated state, fetal blood MSCs were CD29(+), CD44(+), SH2(+), SH3(+), and SH4(+); produced prolyl-4-hydroxylase, alpha-smooth muscle actin, fibronectin, laminin, and vimentin; and were CD45(-), CD34(-), CD14(-), CD68(-), vWF(-), and HLA-DR(-). Fetal blood MSCs cultured in adipogenic, osteogenic, or chondrogenic media differentiated, respectively, into adipocytes, osteocytes, and chondrocytes. Fetal blood MSCs supported the proliferation and differentiation of cord blood CD34(+) cells in long-term culture. MSCs were also detected in first-trimester fetal liver (11.3 +/- 2.0/10(6) nucleated cells) and bone marrow (12.6 +/- 3.6/10(6) nucleated cells). Their morphology, growth kinetics, and immunophenotype were comparable to those of fetal blood-derived MSCs and similarly differentiated along adipogenic, osteogenic, and chondrogenic lineages, even after sorting and expansion of a single mesenchymal cell. MSCs similar to those derived from adult bone marrow, fetal liver, and fetal bone marrow circulate in first-trimester human blood and may provide novel targets for in utero cellular and gene therapy.     

Identification of mesenchymal stem cells in aorta-gonad-mesonephros and yolk sac of human embryos

Mesenchymal stem cells (MSCs) are multipotent stem cells that can generate various microenvironment components in bone marrow, ensuring a precise control over self-renewal and multilineage differentiation of hematopoietic stem cells. Nevertheless, their spatiotemporal correlation with embryonic hematopoiesis remains rudimentary, particularly in relation to the human being. Here, we reported that human aorta-gonad-mesonephros (AGM) resided with bona fide MSCs. They were highly proliferative as fibroblastoid population bearing uniform surface markers (CD45(-), CD34(-), CD105(+), CD73(+), CD29(+), and CD44(+)), expressed pluripotential molecules Oct-4 and Nanog, and clonally demonstrated trilineage differentiation capacity (osteocytes, chondrocytes, and adipocytes). The frequency and absolute number of MSCs in aorta plus surrounding mesenchyme (E26-E27) were 0.3% and 164, respectively. Moreover, they were functionally equivalent to MSCs from adult bone marrow, that is, supporting long-term hematopoiesis and suppressing T-lymphocyte proliferation in vitro. In comparison, the matching yolk sac contained bipotent mesenchymal precursors that propagated more slowly and failed to generate chondrocytes in vitro. Together with previous knowledge, we propose that a proportion of MSCs initially develop in human AGM prior to their emergence in embryonic circulation and fetal liver.     

Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton’s jelly explants of human umbilical cord

We isolated mesenchymal stem cells (MSC) from arteries (UCA), veins (UCV), and Wharton’s jelly (UCWJ) of human umbilical cords (UC) and determined their relative capacities for sustained proliferation and multilineage differentiation. Individual UC components were dissected, diced into 1–2 mm3 fragments, and aligned in explant cultures from which migrating cells were isolated using trypsinization. Preparations from 13 UCs produced 13 UCWJ, 11 UCV, and 10 UCA cultures of fibroblast-like, spindle-shaped cells negative for CD31, CD34, CD45, CD271, and HLA-class II, but positive for CD13, CD29, CD44, CD73, CD90, CD105, and HLA-class I. UCV cells exhibited a significantly higher frequency of colony-forming units fibroblasts than did UCWJ and UCA cells. Individual MSCs could be selectively differentiated into osteoblasts, chondrocytes, and adipocytes. When compared for osteogenic potential, UCWJ cells were the least effective precursors, whereas UCA-derived cells developed alkaline phosphatase activity with or without an osteogenic stimulus. UC components, especially blood vessels, could provide a promising source of MSCs with important clinical applications.     

Abnormality of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus

Bone marrow-derived mesenchymal stem cells (MSCs) are key components of the hematopoietic microenvironment and provide support to hematopoiesis and modulate immune system. Several studies suggest that SLE may be seen as stem cell disorders. However, it is unclear that whether MSCs from SLE patients are defective. So in this research, we studied the biological character of bone marrow derived MSCs in patients with SLE, focused on their phenotype (morphology and immunophenotype), karyotype, cytokines expression and hematopoietic support of MSCs. Our results showed that MSCs from SLE patients and normal controls can be successfully culture-expanded, but the MSCs from SLE grew more slowly than those of normal controls (P < 0.05). Cells from both groups were positive for CD29, CD44 and CD105, and negative for CD14, CD34, CD45 and HLA-DR. MSCs from SLE have a normal karyotype. Both groups express IL-6, IL7, IL-11, macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) at mRNA level. While IL-6 and IL-7 were down-regulated in MSCs from SLE patient (P < 0.05) at mRNA level. The MSCs from SLE patients and normal controls were infused into ICR (Tac: Icr: Ha strain) mice after high-dose chemotherapy, with no adverse events in either group. Recovery of white blood cells, hemoglobin and platelet was more rapid (P < 0.05) compared with the group without MSCs infusion. We conclude that MSCs in patient with SLE have abnormalities compared with those in normal control. MSCs in patient with SLE may play an important role in the SLE pathogenesis.