血管类器官的构建及鉴定

目的 探讨人诱导多能干细胞(hiPSC)培养血管类器官的方法并进行鉴定。方法 体外培养hiPSC,先诱导hiPSC形成拟胚体,再诱导分化中胚层和血管祖细胞后,在Ⅰ型胶原蛋白-基质胶介质中分化形成血管网,分离单个血管网,形成血管类器官;倒置显微镜观察分化过程中拟胚体,血管网及血管类器官的生长情况;免疫荧光染色检测血管类器官的内皮细胞特异性抗体CD31,平滑肌细胞特异性抗体α平滑肌肌动蛋白(α-SMA),血小板衍生生长因子受体β(PDGF-β)蛋白表达。结果 构建血管类器官3D细胞模型,免疫荧光染色显示,CD31阳性,α-SMA阳性,PDGF-β阳性,与人体血管组织结构特征相似。结论 初步构建了血管类器官3D细胞模型,将为心血管疾病的基础研究和靶向药物的筛选提供新的模型。     

bFGF和PDGF-BB对内皮祖细胞分化的内皮样细胞和平滑肌样细胞增殖和迁移的影响

目的探讨内皮祖细胞分化的内皮样和平滑肌样细胞在碱性成纤维生长因子和血小板源生长因子BB作用下的增殖和迁移能力。方法将分离、培养及纯化的内皮祖细胞培养5天后进行分组:对照组、碱性成纤维生长因子组和血小板源长因子BB组。对照组使用20%胎牛血清的DMEM培养基;碱性成纤维生长因子组和血小板源生因子BB组在20%胎牛血清的DMEM培养基中分别添加碱性成纤维生长因子(30μg/L)和血小板源生长因子BB(40μg/L)。免疫荧光染色鉴定内皮祖细胞以及检测内皮祖细胞内皮方向分化标记物CD31和vWF,及平滑肌方向分化标记物α-SMA和Calponin。将分化的内皮样细胞和平滑肌样细胞用MTT法和Transwell小室分别检测其增殖活性和迁移能力。结果与对照组比较,内皮祖细胞经碱性成纤维生长因子或血小板源生长因子BB诱导后呈现较强的内皮细胞(CD31,vWF)或平滑肌细胞(α-SMA,Calponin)的荧光染色,被诱导细胞分别称为内皮样细胞和平滑肌样细胞;碱性成纤维生长因子促进内皮样细胞和血小板源生长因子BB促进平滑肌样细胞增殖的作用在一定范围内具有时间(0～48 h)和浓度(碱性成纤维生长因子:0～16μg/L;血小...     

人脐静脉血内皮祖细胞的分离扩增和生物学特征

目的:探索人脐静脉血内皮祖细胞的分离和扩增条件,并观测其生物学特性.方法:采集人脐静脉血,应用密度梯度离心法,分离其中单个核细胞,流式细胞术检测CD133 CD34 阳性率;利用差速贴壁法(48 h内贴壁和48 h后贴壁)联合内皮细胞专用培养基EGM-2培养细胞,接种于预先包埋了明胶培养瓶或培养板,倒置显微镜观察细胞生长形态和形成集落能力,免疫细胞化学法检测其免疫表型,摄取Ac-LDL和连接UEA-1功能,在生长因子培养体系中诱导其向成熟内皮细胞分化.结果:所获单个核细胞中CD133 CD34 百分比为1.06%;在EGM-2培养体系下可获得2种亚型的内皮祖细胞,即早期内皮祖细胞和晚期增殖性内皮祖细胞.其中48h后贴壁细胞属于早期内皮祖细胞,增殖能力较弱,免疫荧光检测,显示CD14和CD34KDR胞浆呈阳性表达,Ac-LDL UEA-1 功能特征;而48 h内贴壁细胞在10～17 d时可见由单个细胞增殖形成的克隆,呈铺路石样单层排列,增殖力旺盛形成融合状态,形成次集集落;经免疫荧光检测,显示CD133CD34和CD34KDR细胞质呈阳性表达,Ae-LDL UEA-1 功能特征,传代后vWF,CD31呈强阳性表达,是晚期增殖性内皮祖细胞.结论:经人脐静脉血可分离培养获得2种亚型的内皮祖细胞,在特定的培养体系中细胞可由祖细胞表型向成熟内皮细胞分化.     

主动脉瓣体外钙化模型的建立

目的:探讨一种体外分离、扩增瓣膜间质细胞并诱导钙化的方法,建立体外瓣膜细胞钙化模型。方法:采用胶原酶消化法从新鲜猪主动脉瓣膜上分离并体外扩增瓣膜间质细胞,免疫荧光染色行细胞鉴定。4～8代间质细胞以含β-甘油磷酸的钙化培养基钙化诱导培养2周。钙化结节计数,茜素红S染色观察并检测钙沉积。实时定量逆转录聚合酶链反应(Real Time rt-PCR)检测α-平滑肌波动蛋白(α-SMA)及钙化相关因子骨钙素、骨桥蛋白、核心结合因子a1(Cbfa1)表达。结果:胶原酶消化法可从猪主动脉瓣膜上成功分离并体外扩增瓣膜间质细胞,α-SMA和波形蛋白(Vimentin)免疫荧光染色阳性,血管性血友病因子(vWF)染色阴性。钙化培养基体外钙化诱导培养1～2周可成功诱导钙化,间质细胞自发形成钙化结节,茜素红S染色阳性,钙沉积明显增加(P0.05)。实时定量逆转录PCR提示钙化间质细胞α-SMA表达上调,并相对高表达钙化相关因子(P0.05)。结论:胶原酶法联合钙化培养基可成功体外构建主动脉瓣膜细胞钙化模型。     

基质细胞衍生因子1α对大鼠骨髓源内皮祖细胞迁移的影响

目的观察基质细胞衍生因子1α对体外培养的大鼠骨髓源内皮祖细胞迁移的影响。方法微孔法从大鼠骨髓提取内皮祖细胞。免疫荧光鉴定血管内皮生长因子受体2和CD133,不同浓度基质细胞衍生因子1α处理内皮祖细胞后,采用Transwell迁移系统检测内皮祖细胞迁移能力。结果分离出的大鼠骨髓内皮祖细胞免疫荧光下血管内皮生长因子受体2 和CD133 双阳性,基质细胞衍生因子1α呈浓度依赖性促进内皮祖细胞迁移,对照组与基质细胞衍生因子1α各处理组比较差异均具显著性(P<0.05或P<0.01)。结论基质细胞衍生因子1α呈浓度依赖性促大鼠骨髓源内皮祖细胞迁移。     

人脐血内皮祖细胞的体外分离和培养

目的建立一种稳定的体外分离和培养人脐血来源内皮祖细胞的方法。方法采用密度梯度离心法从人脐带血中分离单个核细胞,将其接种至人纤维连接蛋白包被的六孔板中,用EGM-2培养基诱导培养。通过形态学观察、细胞表面特异性抗原、摄取功能和体外血管形成能力对内皮祖细胞进行鉴定。结果细胞形态学观察发现,刚分离的单个核细胞较小,呈圆形,4 d后可见少量的圆形和梭形贴壁细胞,8 d后有明显集落形成,14 d后相邻集落相互融合,呈现出典型铺路石样改变。内皮祖细胞能摄取乙酰化低密度脂蛋白,结合荆豆凝集素1,表达CD34、CD133和血管内皮细胞生长因子受体2,并且具有体外血管生成能力。结论采用密度梯度离心法可从人脐带血中成功分离和培养出内皮祖细胞,以用于相关实验研究。     

冠心病干细胞移植疗法种子细胞体外诱导分化的实验研究

目的　为探讨冠心病的干细胞移植疗法提供理想的种子细胞来源。方法　收集健康成人骨髓 ,通过Ficoll离心法获得骨髓单个核细胞 ,置于纤维连接蛋白预衬的DMEM培养基中 ,用血管内皮生长因子 (vascularendothelialgrowthfactor,VEGF)和碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)加以诱导 ,并通过荧光显微镜和免疫组织化学方法对诱导后的细胞进行形态学观察和鉴定。结果　在VEGF、bFGF等诱导因子存在的条件下 ,骨髓单个核细胞分化成为内皮祖细胞 ,倒置荧光微镜下呈典型的“纺锤样”梭形细胞 ,单层细胞贴壁生长融合时呈铺路石样排列 ,免疫组织化学示vWF抗体染色阳性 ,CD34抗体染色阳性。结论　成人骨髓单个核细胞在特定条件下可诱导分化成为内皮祖细胞 ,此细胞可作为冠心病干细胞移植疗法的种子细胞。     

大鼠骨髓单个核细胞体外诱导向内皮分化的研究

目的 :为组织工程研究作准备 ,分离大鼠骨髓单个核细胞并诱导培养向内皮分化。方法 :成年SD大鼠 ,应用Ficoll淋巴细胞分离液 (密度1 .0 77g/ml) ,将长骨骨髓经非连续密度梯度离心 ,收集中层的单个核细胞 ,加入诱导培养基并置于纤维连接素包被的培养板上进行诱导分化 ,观察细胞生长状况 ,以透射电镜及Ⅷ因子、CD3 1、Lectin免疫组化以及流式细胞仪方法对培养细胞进行鉴定。结果 :细胞圆形、纺锤形单层融合贴壁生长 ;Ⅷ因子、CD3 1、Lectin免疫组化染色阳性 ;透射电镜显示细胞具有内皮特征性的Weibel Palade小体。结论 :采用Ficoll密度梯度离心可获得较高纯度的骨髓单个核细胞 ,经体外培养并诱导分化 ,具有血管内皮细胞的特征     

小鼠骨髓源性内皮祖细胞的分离培养与鉴定

目的 建立一种高效、稳定的从小鼠骨髓中分离培养与定向诱导分化内皮祖细胞的方法.方法 通过密度梯度离心法从小鼠骨髓中分离单个核细胞,经差速贴壁结合特殊培养基扩增,诱导分化为内皮祖细胞.应用流式细胞技术鉴定内皮细胞系列标志:CD34、CD31、Flk-1和祖细胞标志CD133.结果 经密度梯度离心和差速贴壁法分离所得的细胞经EBM-2专用培养基培养后,第4天可见集落形成,培养第12天流式细胞仪检测其CD34、CD133、Flk-1、CD31的阳性率分别为65%±4%、48%±3%、37%±3%和51%±4%.结论 从小鼠骨髓中分离培养与定向诱导分化内皮祖细胞的方法效率高,稳定性和重复性好.     

大鼠外周血晚期内皮前体细胞体外培养研究

目的体外培养大鼠外周血内皮前体细胞（EPCs）,观察细胞克隆形态并进行鉴定。方法密度梯度离心法分离SD大鼠外周血单个核细胞,EGM-2培养基离体培养。免疫荧光染色鉴定细胞CD34、CD31、CD133、FLk-1、vwF细胞表面标志。Real—TimePCR检测细胞CD34、CD133、FLk-1、eNOSmRNA表达。结果细胞培养10天后可见“铺路石样细胞”和少数“紊乱生长细胞”,传代培养后经免疫荧光及Real—TimePCR鉴定“铺路石样细胞”符合晚期EPCs特点,紊乱生长细胞不表达相应标志。结论通过体外分离长时培养可从大鼠外周血获得晚期内皮前体细胞,具有内皮细胞的特征。     

侧群细胞与肝癌干细胞

肝癌是严重威胁人类生命和健康的一种疾病.其病因和发病机制尚不完全清楚,治疗缺少有效靶点.对肝癌恶性生长、转移及复发机制的研究正在逐渐深入.近年来的研究认为,肿瘤中存在一小群具有自我更新和分化潜能的细胞,即肿瘤干细胞,可能是肿瘤转移和复发的根源.肝癌中应同样存在这样的一群细胞.侧群(side population,SP)细胞是肿瘤细胞中一小部分,具备干细胞的多种特性且易于分离.肝癌组织中SP细胞的鉴定和分离有可能找到肝癌干细胞,有助于肝癌的转移和复发机制的研究,并为肝癌治疗提供有效治疗靶点.     

大鼠胰腺导管来源干细胞向胰岛素分泌细胞分化的体外培养

目的:研究在体外条件下从大鼠胰腺导管分离的干细胞向胰岛素分泌细胞分化的产物细胞的形态、表型及功能.方法:采用胶原酶原位消化法消化大鼠胰腺,差异贴壁法培养出胰腺导管来源千细胞(PDSCs),对其进行形态学与表型鉴定.采用无血清培养基,添加Matrigel、exendin-4诱导干细胞向胰岛素分泌细胞分化,鉴定产物细胞的形...     

Hepatoma cells up-regulate expression of programmed cell death-1 on T cells

AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.2.1.5 cells were cocultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL：2, INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Cytotoxic action of T cells was determined by MIF reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS： The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-γ and IL-10 in the culture supernatant were 202.9 ＋ 53.0 pg/mL, 88.6 ± 4.6 pg/mL and 63.7± 13.4 pg/mL respectively, which were significantly higher than those （102.9 ± 53 pg/mL, 39.3 ± 4.2 pg/mL, and 34.6 =E13.7 pg/mL） in the control group （P 〈 0.05）. The OD value for MTT assay in the blocking group （0.29 ± 0.06） was significantly higher than that （0.19 ± 0.09） in the control group （P 〈 0.05）. CONCLUSION： PD-1 expression on Jurkat cells is upregulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.     

胚胎干细胞向心肌细胞分化中内皮细胞的作用

目的:研究胚胎干细胞向心肌细胞分化过程中,内皮细胞与心肌细胞之间的功能性联系. 方法:通过检测CGR8-GFP小鼠胚胎干细胞系心肌α-肌球蛋白重链(α-MHC)荧光蛋白表达情况变化,观察抑制内源性内皮细胞、添加外源性内皮细胞以及两者并存情况下,胚胎干细胞分化所得心肌细胞数量的变化. 结果:(1)在胚胎干细胞分化过程中,加入外源性内皮细胞后,心肌细胞形成明显增多.(2)特异性抑制内源性内皮细胞,心肌细胞形成明显减少.(3)外源性内皮细胞与胚胎干细胞共培养能够部分挽救由于内源性内皮细胞抑制所导致的心肌细胞形成障碍. 结论:在胚胎干细胞分化过程中,内源性内皮细胞对于促进心肌细胞形成起着至关重要的作用,是形成心肌细胞发育微环境的关键因子.在胚胎干细胞向心肌细胞分化过程中,外源性内皮细胞可以刺激心肌细胞形成,从而得到大量心肌细胞,具有潜在的临床应用价值.     

对链脲佐菌素诱导的糖尿病大鼠壁细胞和G细胞的体视学研究

采用ＨＥ染色和免疫组化方法结合生物体视学技术，对链脲佐菌素诱导的糖尿病大鼠在胃底腺的壁细胞和幽门部胃粘膜的Ｇ细胞进行立体计量研究。结果显示：糖尿病状态的早期，壁细胞和Ｇ细胞的体积均明显增大，数量却显著减少。根据正常情况下壁细胞和Ｇ细胞的细胞动力学变化、胃泌素的生物学作用和上述实验结果，认为大鼠胃底腺峡部的干细胞向壁细胞分化成熟的功能及Ｇ细胞的分裂增殖活动，在胰岛素缺乏的情况下受到一定程度的抑制，而这种功能的抑制是糖尿病状态下易出现胃粘膜萎缩、胃酸分泌减少和胃轻瘫的重要原因之一。     

Robust measurement of telomere length in single cells

Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.Telomeres are the ribonucleoprotein structures that cap and protect linear chromosome ends from genomic instability and tumorigenesis (1, 2). Intriguingly, telomere shortening protects against tumorigenesis by limiting cell growth (3, 4), but also can impair tissue regenerative capability and cell viability (5, 6).Thus far, most assays of telomere length measure average telomere length from aggregates of many cells derived from dissected tissues, cultured cells, or blood (7). Telomere restriction fragment (TRF) determination (1, 8), a Southern blot-based technique, remains the “gold standard” for determining absolute telomere length, but requires a large amount of starting material (0.5–5 µg DNA) and several days for processing. Moreover, the requirements for gel electrophoresis and hybridization limit the scalability of this assay. Recently, a quantitative PCR (qPCR)-based method for telomere length measurement was developed, providing the convenience and scalability of PCR (9). Although the DNA requirement (35 ng) for qPCR is significantly less than TRF, it still relies on populations of cells to derive sufficient amount of DNA.Quantitative FISH (Q-FISH) allows sensitive visualization of relative telomere length from individual cells and individual telomeres, but this method requires many cells or metaphase arrested cells, which precludes its application to many sample types, including postmitotic cells, senescent cells, and other nondividing cells, and when only one actual cell is required to test. In addition, preparing chromosome spreads requires significant technical skill, and only proliferating cells within a population reach metaphase stage, so this analysis potentially biases the estimates of telomere length for a given cell population (10–12). High-throughput Q-FISH, flow FISH, and single telomere length analysis can be used for telomere measurement of dividing, nondividing, and senescent cells, but these methods also require large cell populations (13–15).The ability to measure telomere length in single cells rather than relying upon average telomere length in cell populations or the entire tissue enables the study of biological heterogeneity on a cell-by-cell basis, an issue of fundamental importance for studies of aging, development, carcinogenesis, and many other diseases. Here, we demonstrate an accurate determination of telomere length in individual cells, with the resolution and scalability of the qPCR telomere length assay.The basis of qPCR is that within a given cell, the ratio of the copy number of telomere repeats to the copy number of a multicopy reference gene is fixed (3), and this method, because of its simplicity, has been widely used to investigate a variety of telomere shortening-associated diseases (7), even sensitive enough to identify mild telomere dysfunction resulting from chronological life stress (16, 17). We adapted qPCR to measure telomere length in individual cells by using a preamplification step that specifically targets both the telomere and multicopy genes, followed by a qPCR assay to obtain telomere to reference gene (T/R) ratio. A single-cell telomere (SCT) length measurement method (SCT-pqPCR) runs robustly, and shows an identical T/R ratio for two sister blastomeres from two-cell–stage mouse embryos. The average result from SCT-qPCR with multiple single cells is linearly correlated to Q-FISH, TRF, and conventional qPCR assays designed for a large number of cells. The heterogeneity of telomere length among several populations of cells by SCT-pqPCR run on multiple single cells is consistent with—and sometimes superior to—results obtained by Q-FISH. Application of SCT-pqPCR to study telomere length during early embryo development, aging, and cancer demonstrate the value of this single-cell telomere length assay method.     

Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry

Left-right (LR) asymmetry (handedness, chirality) is a well-conserved biological property of critical importance to normal development. Changes in orientation of the LR axis due to genetic or environmental factors can lead to malformations and disease. While the LR asymmetry of organs and whole organisms has been extensively studied, little is known about the LR asymmetry at cellular and multicellular levels. Here we show that the cultivation of cell populations on micropatterns with defined boundaries reveals intrinsic cell chirality that can be readily determined by image analysis of cell alignment and directional motion. By patterning 11 different types of cells on ring-shaped micropatterns of various sizes, we found that each cell type exhibited definite LR asymmetry (p value down to 10(-185)) that was different between normal and cancer cells of the same type, and not dependent on surface chemistry, protein coating, or the orientation of the gravitational field. Interestingly, drugs interfering with actin but not microtubule function reversed the LR asymmetry in some cell types. Our results show that micropatterned cell populations exhibit phenotype-specific LR asymmetry that is dependent on the functionality of the actin cytoskeleton. We propose that micropatterning could potentially be used as an effective in vitro tool to study the initiation of LR asymmetry in cell populations, to diagnose disease, and to study factors involved with birth defects in laterality.     

Identification of cells in culture.

Most laboratories using cells cultured in vitro maintain multiple cell lines. Such lines should be monitored for species and intraspecies characteristics to prevent invalidation of research work due to incidents of cell line cross-contamination. This report describes the results obtained when 246 cell cultures were examined for evidence of cross-contamination or mislabeling. Using species-specific antigens, isoenzyme electrophoresis, and chromosomes as markers of identity, 14% of the cultures submitted were found to be contaminated by cells of another species. Of human cell lines submitted 25% were of HeLa cell origin, as determined by 2 intraspecies markers, glucose-6-phosphate dehydrogenase and chromosome analyses. The fact that, overall, nearly 30% of the cell lines examined were incorrectly designated makes the importance of cell line monitoring self-evident.     

肺干细胞研究进展

干细胞研究是现代医学领域研究热点之一.肺部疾病所导致的不同程度呼吸系统病理改变和功能受损,都伴随着肺组织的修复和重塑过程.对于肺部疾病的干细胞研究和应用尚有许多问题有待进一步的明确和探索.肺干细胞包括了肺组织自身的干细胞修复和肺外组织来源的干细胞修复.肺组织内的干细胞包括肺内上皮性干细胞、肺问充质干细胞、肺侧群细胞;其中肺内上皮性干细胞又包括了基底细胞、Clara细胞、Ⅱ型肺泡上皮细胞、"芽孢"样细胞.肺外组织来源的干细胞修复包括骨髓间充质干细胞和造血干细胞.干细胞治疗方法在临床上有巨大的应用前景.