大鼠实验性肝癌发生中卵圆细胞的变化

目的：探讨卵圆细胞在大鼠肝癌发生、发展过程中的作用．方法：60只SD大鼠随机分为对照组(n=12)和实验组(n=48)．用化学致癌剂3’-甲基-4-二甲基氨偶氮苯(3’-Me-DAB)诱发大鼠肝癌,通过免疫组织化学、RT-PCR,Western blot技术对大鼠诱癌过程(4,8,12,16,20,24 wk)中肝组织内卵圆细胞及p53基因表达的变化进行动态的检测．结果：诱癌4wk,大鼠肝门管区及坏死区内均见大量卵圆细胞,这些细胞呈OV-6染色阳性．诱癌16wk,肝组织内可见癌结节形成,癌结节内外均可见有卵圆细胞聚集,部分增生的卵圆细胞P53阳性反应,二者的分布区域基本一致．诱癌20wk后大鼠肝癌组织内的p53 mRNA(F =4．78,P＜0．05),P53蛋白水平均显著升高(F= 2．46．P＜0．05)．结论：卵圆细胞贯穿了大鼠受化学诱癌剂作用后发生肝癌的全过程,与肝癌的发生有着密切的联系；其机制可能与p53基因的突变有关．     

microRNA在两种不同来源的肝卵圆细胞中的差异表达

目的分离纯化两种不同模型来源的卵圆细胞(HOCs)并初步筛选差异表达的microRNA。方法采用2-乙酰氨基芴(2-AAF)和2/3肝切除(PH)建立SD大鼠HOCs增殖模型,用化学致癌剂3'-甲基-4-二甲基偶氮苯(3'-Me-DAB)诱发SD大鼠肝卵圆细胞增生模型;分离纯化并鉴定两种不同模型来源的大鼠肝卵圆细胞;用microRNA芯片技术检测HOCs/AAF与HOCs/DAB中microRNA的表达谱,筛选差异表达的microRNA;用实时荧光定量PCR方法对筛选出的部分差异microRNA进行验证。结果 1激光扫描共聚焦显微镜显示,两种肝卵圆细胞胞质和胞膜均表达干细胞标志Thy-1及C-kit。2microRNA芯片的结果显示,HOCs/DAB与HOCs/AAF比较,有10个microRNA表达上调,8个microRNA表达下调。3实时荧光定量PCR结果进一步证实mir-210、mir92b、mir-1281在HOCs/DAB中显著上调,而mir-181、mir-1228显著下调。结论两种不同模型来源的肝卵圆细胞存在着显著差异表达的microRNA,为研究肝癌细胞可能来源于肝卵圆细胞HOCs/DAB提供线索。     

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。     

大鼠肝卵圆细胞增殖模型的建立与优化

目的 建立大鼠肝卵圆细胞增殖模型，并观察2-乙酰氨基芴（2-acetaminofluorene,AAF)剂量与模型动物肝卵圆细胞增殖程度间的关系。方法 选择体重150g左右的雄性Wistar大鼠，通过胃管灌喂AAF，每日1次，连续4d，第5日行三分之二肝切除（手术当日不灌喂AAF），第6日继续灌喂，连续1周。AAF剂量分别按2.5、5、10、20mg/kg给予，对照组给予生理盐水灌喂。各组手术后复隔2-3d分别取3只大鼠肝组织作常规组织学观察及免疫组织化学染色。结果 对照组大鼠肝组织内未见到肝卵圆细胞，2.5mg/kg剂量组及5mg/kg剂量组仅见少量肝卵圆细胞增生，而10、15、20mg/kg三个剂量组肝组织内均见明显的肝卵圆细胞增殖反应；肝卵圆细胞胞浆细胞角蛋白19（CK19）、OV6及波形蛋白染色均呈阳性，胞核增殖细胞抗源染色呈阳性。结论 AAF剂量为10-20mg/kg时可获得满意的大鼠肝卵圆细胞增殖模型。     

大鼠脐带间充质干细胞对肝细胞及卵圆细胞体外增殖与功能的影响

目的：探讨大鼠脐带间充质干细胞（UMSC）对原代大鼠肝细胞及肝卵圆细胞增殖与功能的影响。方法采用2-乙酰氨基芴加肝脏三分之二切除术建立肝卵圆细胞增殖模型,通过原位二步胶原酶灌流法分离到单个肝脏细胞,再经过 Percoll 密度梯度离心分离到肝卵圆细胞。原代大鼠肝细胞/肝卵圆细胞各分为3组：UMSC 组、原代大鼠肝细胞组/肝卵圆细胞组及 UMSC 与原代大鼠肝细胞共培养组/UMSC 与肝卵圆细胞共培养组,分别于第1、3、6、8天通过MTT 法检测各组细胞增殖能力,于第3天通过 ELISA 法检测各组细胞培养上清液中白蛋白含量。结果UMSC 与原代大鼠肝细胞共培养组在第3、6、8天的 A 值均比相应时间点的 UMSC 组 A 值与原代大鼠肝细胞组 A 值之和大（P ＜0．05）;UMSC 与肝卵圆细胞共培养组在第3、6、8、10天的 A 值均比相应时间点的 UMSC 组 A 值与肝卵圆细胞组 A 值之和大（P ＜0．05）。UMSC 无白蛋白分泌能力,UMSC 与原代大鼠肝细胞共培养组培养上清液中白蛋白水平为（266．21±50．44）ng/mL,较原代大鼠肝细胞组（130．79±22．10）ng/mL 高（P ＝0．013）;UMSC 与肝卵圆细胞共培养组培养上清液中白蛋白水平（（49．64±3．56）ng/mL）较肝卵圆细胞组（13．54±1．53）ng/mL 高（P ＝0．000）。结论UMSC 在体外可以促进原代大鼠肝细胞及肝卵圆细胞的存活和增殖,并可增强其分泌白蛋白的作用。     

人胚胎肝干细胞的形态特点

目的:观察胚胎发育早期肝干细胞的形态特征、时空分布及分化,以探讨肝干细胞的生物学特征．方法:运用发育第3-12 wk人胚标本47例(其中 3．5 wk各8例;6-8 wk各5例,9-12 wk各2例),石蜡切片,连续切片,免疫组化染色,光镜下观察人胚肝及肝干细胞的发育及其AFP、c-Met和 CK19的时空表达．结果:发育第3 wk,肝芽形成,第4 wk形成肝索,第5 wk出现原始肝血窦．第3-5 wk人胚肝芽和肝索细胞排列紧密,较小,形态不规则,核圆形或卵圆形,核质比例大,核深染,胞质颜色较淡,偏蓝色,显示出幼稚细胞的形态学特征,并呈甲胎蛋白(α-Fetoprotein,AFP)、c-Met 阳性反应．第6 wk,肝索内出现了体积大、核大、淡染的细胞,呈AFP、c-Met阴性反应．随胚龄增加,这类细胞数量增加．10-12 wk． AFP、c-Met阳性细胞主要分布于汇管区周围． CK19阳性反应在7 wk时开始出现于一些与 AFP、c-Met阳性反应的细胞形态类似的肝索细胞中．10-11 wk时,CK19阳性反应主要位于汇管区附近的肝索细胞、胆管板细胞及胆管上皮细胞,12 wk时,CK19阳性信号仅见于胆管板和胆管上皮细胞．此时所有的胆管板细胞及胆管上皮细胞均呈AFP、c-Met和CK19阳性．结论:人胚发育3-5 wk肝实质由肝干细胞组成,其表型为AFP /c-Met ．6 wk,肝干细胞开始向肝细胞系分化,7 wk向胆管系分化,10-12 wk,肝干细胞主要局限于汇管区周围的肝索, 与成年肝中卵圆细胞(成年肝干细胞)的分布一致．AFP ／c-Met／ CK19 细胞可能为胆管祖细胞．     

丁酸钠诱导体外培养的大鼠肝卵圆细胞分化为成熟肝细胞

目的探讨分化刺激剂丁酸钠对体外培养的大鼠肝卵圆细胞分化的影响。方法从喂养含0．1%乙硫氨酸的胆碱缺乏性饮食4～6周的人鼠肝脏中分离出盱卵圆细胞,用免疫细胞化学和逆转录聚合酶链反应(RT-PCR)等方法对其进行鉴定。用0．75mmol/L酸钠处理大鼠肝卵圆细咆后．姬姆萨染色观察细胞表型改变,western blot检测细胞白蛋白的表达水平。结果免疫细胞化学结果显示分离出的细胞既表达成熟肝细咆的标志物白蛋白,也表达胆管细胞的标志物细胞角蛋白19,RT-PCR结果显示这些细胞还表达干细胞的标志物c-kit,但不表达造血干细胞的标志物CD34,表明这些细胞是大鼠肝前体细胞——肝卵圆细胞。0．75mmol/L丁酸钠能诱导大鼠肝卵圆细胞出现明显的表型改变,细胞变大,变圆,核浆比减小,且双核细胞数增多,约占总细胞数的50%左右,同时western blot的结果显示0．75mmol/L丁酸钠能够提高大鼠肝卵圆细胞白蛋白的表达水平。。结论分化刺激剂丁酸钠能诱导体外培养的大鼠肝卵圆细胞向成熟肝细胞分化。     

二甲基奶油黄诱发大鼠肝癌病理形态观察

目的 研究二甲基奶油黄诱发的大鼠肝癌的组织学改变。方法 设对照组（饲料中无ＤＡＢ）和实验组（饲料中含ＤＡＢ）,４周至１６周间分批处死动物,光镜和电镜下观察肝脏病变情况。结果 整个过程分为非特民划性炎症、肝硬化、肝癌３个阶段。非特异性炎症阶段光镜下改变为肝细胞气球样变性、炎性细胞浸润。肝硬化阶段表现为肝细胞和胆管腺瘤样增生,假小叶形成,肝组织炎性细胞浸润。肝癌有肝细胞癌、胆管细胞癌和混合细胞癌３种组     

胞外基质Matrilin-2在肝再生中与大鼠卵圆细胞的关系

目的:探讨肝脏胞外基质Matrilin-2在肝再生中与卵圆细胞的关系及作用.方法:采用改良的Soft-Farber建立大鼠肝脏卵圆细胞增殖模型,对照组灌喂生理盐水.分别取术后2、4、6、9、12、15 d大鼠肝组织,采用免疫组织化学以及Western blot的方法动态观察大鼠卵圆细胞增殖模型中肝脏胞外基质成分Matrilin-2的变化与卵圆细胞的关系.结果:肝脏部分切除术(partial hepatectomy,PH)后第2天,卵圆细胞开始向门静脉周围区域增殖,Matrilin-2主要出现在门静脉周围的肝窦状隙内;术后第9天,卵圆细胞进一步向肝实质内增殖,Matrilin-2表达增加;术后第12天,随着卵圆细胞分化为小肝细胞结节,大多数Matrilin-2位于结节周边,少数出现在结节内.Matrilin-2的含量自肝切除后第2天开始升高,第9天达到高峰,第12天后逐步恢复生理水平.结论:肝脏胞外基质成分Matrilin-2与卵圆细胞介导的肝脏再生存在紧密联系并发挥重要的调控作用.     

肝卵圆细胞——肝干细胞研究进展

<正>近年来对干细胞的研究不断深入,人们发现成年肝组织内存在具有干细胞特性的细胞,在特定环境因素作用下可向肝细胞和胆管细胞分化,甚至可向肝癌细胞方向分化,该细胞被称为肝卵圆细胞或肝前体细胞,是具有一定共性的干细胞群体,与其分化的上下游细胞难以区分,本文就     

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.