碳酸酐酶在红细胞分化发育过程中的功能研究

目前红系分化调控相关的研究主要集中在细胞因子、转录因子、lncRNA及表观遗传方面,为了对红系分化调控机制进行更加深入的解析,研究了碳酸酐酶在红系分化中的功能。碳酸酐酶可以高效催化二氧化碳的水合,但它在红细胞发育过程中的功能尚不清楚。利用脐带血来源的CD34⁺细胞在体外进行红细胞诱导分化,在分化过程中通过慢病毒介导的基因敲降的方法能够降低碳酸酐酶1和碳酸酐酶2的表达,并使用流式细胞仪检测红细胞的生成和分化效率。研究结果表明,与对照组相比,碳酸酐酶1的表达缺陷使红细胞的晚期分化明显受阻,而碳酸酐酶2的表达缺陷则将红细胞的分化阻滞在早期阶段。研究结果表明,虽然作用窗口不同,但碳酸酐酶1和碳酸酐酶2在红系分化的过程中均发挥着重要的调控作用,这一发现对将来在体外红细胞生成具有指导意义。     

小鼠胎肝红系细胞在分化过程中形态学观察及时序性表达分析

小鼠胎肝是小鼠发育早期主要的造血器官,红系细胞在胎肝造血过程中形态特征和组成成分等方面发生了明显变化。根据红系细胞体积的变化,利用Countstar细胞计数仪对小鼠E12．5-E17．5胎肝中直径8-14岬细胞进行数量统计,再结合观测到的红系细胞的形态特征和血红蛋白表达量的不同,将E9．5．E17．5胎肝中的细胞分为10类。统计结果显示,随着胎肝造血系统的发育,哺乳类红系细胞在终末分化时出现细胞体积减小、细胞核固缩、排核和血红蛋白表达量增加等时序性变化。红系细胞表面特异标志Terll9和CD71在EryD中高表达而在成体骨髓细胞和外周血细胞中表达较低的结果表明胎肝中红系细胞具有较高的分化能力。这些数据为研究红系分化、克隆红系分化相关基因及探讨红白血病发生的机制提供了理论依据。     

Changes in the Expression of the αα Form of Enolase During Neuroblastoma Differentiation

Abstract: The relative amounts of the different enolase isozymes present in neuroblastoma cells change during differentiation. When differentiation is induced by low serum in the presence of DMSO (dimethyl sulfoxide), there is a 50% decrease in the concentration of enolase activity associated with the form αα, and an increase in the activity associated with the γ-containing isozymes (αγ plus γγ); in the absence of DMSO, there is no decrease in αα or in total enolase activity. In order to study the mechanism of the changes in αα, cells differentiated with low serum with and without DMSO were compared. Measurements of the concentration of the α antigen by microcomplement fixation and by immunotitration demonstrate that the decreased enolase activity in DMSO cells is due to a decreased concentration of the α antigen. Measurements of the relative rate of synthesis of the antigen show that the decreased concentration of the α antigen is due to a decreased rate of synthesis. Enolase in differentiated cells is sufficiently stable (t_1/2 > 100 h) that a comparison of the relative rates of degradation has not been possible. The decreased synthesis of the α subunit of enolase that occurs under these conditions appears to be a useful model system for studying the de-expression of the α gene that occurs in vivo during neuronal differentiation.     

An Immunocytochemical Technique for Analysis of Regulation of Genes Encoding Early Differentiation Marker Antigens in an Oocyte Translation System

Monoclonal antibodies are useful probes for analyzing cells at the molecular level at various developmental stages. Although identification of the genes encoding tissue- and stage-specific antigens could be informative for further molecular analysis, gene cloning is usually a time-consuming step, particularly when a monoclonal antibody is the only probe available. We describe here an immunocytochemical method for preliminary and immediate analysis of the regulation of antigen-coding genes. mRNAs purified from stage 27 and 38 Xenopus tadpoles were fractionated by size and injected into newt oocytes, from which frozen sections were prepared for immunostaining with tissue-specific monoclonal antibodies. Both of the antigens we tested, which are early markers for differentiating epidermal cells of Xenopus tadpoles, were detected in mRNA injected oocytes, but not in control oocytes. Immunostaining for each of the antigens showed that their relative levels in stage 27 and 38 tadpole tissue were reflected in those oocytes injected with mRNA purified from tadpoles of the respective stages. We suggest that this oocyte translation system combined with immuaostaining provides for rapid analysis of changes in levels of antigen coding mRNAs throughout development.     

KLFs对珠蛋白基因表达和红系分化的调控作用

Krüppel样因子(Krüppel-like factors, KLFs)是一组与真核基因转录调控密切相关的锌指蛋白.KLFs高度保守的羧基末端含3个串联的Cys2His2型锌指结构,用于结合GC盒和CACCC盒等DNA序列. 红细胞中特异表达的珠蛋白基因和许多红系调控因子中都含有CACCC盒.已有研究发现,多个KLFs通过结合CACCC盒参与调控珠蛋白基因表达和红系分化,例如,KLF1通过结合β-珠蛋白启动子和位点控制区(locus control region, LCR),促进β-珠蛋白的表达、γ-向β-珠蛋白基因的转换和红系分化;KLF2、KLF11和KLF13分别促进ε-和γ-珠蛋白基因的表达;KLF4促进α-和γ-珠蛋白基因的表达;KLF3和KLF8则抑制ε-和γ-珠蛋白基因的表达. 本文综述了KLFs调控珠蛋白基因表达和红系分化的研究进展.     

Avian developmental antigens: Expression on erythrocytes of chickens,Japanese quail,and quail-chicken hybrids

Monoclonal and polyclonal antibodies were used to examine the expression of three erythroid developmental antigen systems in the chicken, Japanese quail, and quail-chicken hybrid. Chicken fetal antigen (CFA), quail fetal antigen (QFA), and chicken adult antigen (CAA) each represent a series of cell-surface glycorproteins associated with the development of avian hematopoietic cells. Monoclonal anti-CFA antibodies from clones 190-4 and 288-1.1.1.2 supernatants were shown to react against epitopes associated with CFA determinants 8 and 2, respectively. Using complement-mediated microcytotoxicity, these reagents permitted the identification of different erythroid subpopulations in the neonatal chicken and hybrid; therefore, heterogeneity in cell surface CFA determinants among mature peripheral erythrocytes should serve as a useful tool for analyzing erythroid development. In the case of CAA, erythrocytes from adult hybrids were found to express the same complement of CAA determinants identified in the chicken, and CAA appeared much earlier in the hybrid than in either of the parental species. Similarly, two species-restricted fetal antigens associated with similar glycoproteins, CFA8 and QFA, had similar developmental profiles in their respective species, the chicken and quail. In contrast, these antigens were dominantly expressed but exhibited different developmental profiles on erythrocytes from the hybrids. While quail-chicken hybrids exhibited apparent genomic interactions in the expression of these developmental antigens, no evidence for the existence of hybrid-specific fetal antigens was obtained.     

Membrane transport during erythroid differentiation

Summary Transport, unidirectional flux, of a monosaccharide, a nucleoside and three amino acids, all of which enter cells by independent, discrete carriers, was compared at three stages of erythroid maturation, the normal (anucleate) mouse erythrocyte, and in differentiated and undifferentiated Friend erythroleukemia cells. We found specific transport alterations during this developmental program. Transport of 3-O-methylglucose increased with each successive developmental stage. Aminoisobutyrate transport was maintained during Friend cell differentiation, but fell slightly in erythrocytes. Leucine, lysine and uridine transport began to fall two days after dimethylsulfoxide exposure, and diminished further in red cells. These studies of transport are not directly comparable to uptake studies reported by others.Median cell volume and thus surface area decreased more during differentiation than amino acid transport declined, so flux, transport past a unit area of membrane, actually increased. Monosaccharide flux also increased. Only uridine transport fell in parallel to surface area. Perhaps sites for nutrient transport required for energy production are preferentially maintained.     

Ganglioside Expression During Differentiation of Chick Retinal Cells In Vitro

The neural retina has been widely used to study the developmental patterns of ganglioside metabolism. Recent findings about in vitro differentiating chick embryo retina cells showed that: a) GD3 and GD1a ganglioside patterns undergo the most dramatic changes; b) when the cells emit neurites, GD3 ganglioside and a group of complex gangliotetraosylgangliosides (GTOG) are transiently coexpressed; c) synchronized developmental phenomena are dissociated by anti-GM1 antibodies; d) GD3 remains as a major ganglioside in differentiated neurons, though it is almost not immunoexpressed; e) GTOG affect antibody binding to GD3; f) the content of gangliosides involved in neural differentiation modifies their immunostain localization on cell membrane; g) after exogenous GTOG uptake, immature neurons mimic GD3 immunoflourescent localization of mature cells; h) a subset of purified retinal ganglion cells express GTOG characteristic of mature neurons.     

大鼠前体脂肪细胞分化过程中波形纤维形态结构的变化和波形纤维蛋白基因表达的研究

通过对不同分化阶段的大鼠前体脂肪细胞中波形纤维蛋白的结构形态和分布的间接免疫荧光观察,发现随着前体脂肪细胞的分化,波形纤维形态结构发生特异性改变,即从前期的围绕细胞核聚集且向细胞周边平行延伸到后期的围绕脂滴间隔形成致密笼状结构。此外,通过地高辛标记的寡核苷酸探针的原位杂交和免疫印迹研究了前体脂肪细胞的分化对波形纤维蛋白基因表达的影响。结果表明,分子量为57kD的波形纤维蛋白在mRNA水平和蛋白水平的表达贯穿于前体脂肪细胞分化的全过程,表达量呈递减趋势。这提示在前体脂肪细胞分化中,波形纤维与脂滴的特异性结合对于脂滴的前体脂肪细胞分化有着功能性的联系,特别是对脂肪细胞的脂滴形成极可能起到支撑的作用。     

Monoclonal Antibodies Reveal Complex Structure in the Membrane Skeleton of Tetrahymena

ABSTRACT. Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena . Five were directed against T. pyrifomis and seven were directed against T. thermophila . Some cross-reactivity between species was found. Each monoclonal antibody recognized one of the three major components of epiplasm, i.e. the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins. It was found, using these antibodies, that the epiplasmic proteins A, B, and C have overlapping but independent distributions within the cell.     

巨峰葡萄花芽分化过程中成花基因LEA FY的表达

目的:研究LEAFY基因在巨峰葡萄花芽分化期间的表达差异,为实际生产提供理论依据。方法:用半定量RT-PCR方法研究LEAFY基因在巨峰葡萄花芽分化期间的表达。结果:LEAFY基因的表达量在所研究的8个时期内有明显差异,在5叶期、6叶期、7叶期、8叶期的表达量相对较高。结论:根据成花基因LEAFY表达上的差异,可以人为地提前或延迟巨峰葡萄的花期。     

鸡髓样分化因子88的原核表达及单克隆抗体制备

目的:克隆、表达、纯化鸡髓样分化因子88(MyD88),制备其单克隆抗体。方法:从脾脏cDNA中扩增857bp的MyD88基因片段,插入pMAL-c5X表达载体,转化大肠杆菌BL21(DE3)获得表达菌株,IPTG诱导表达,用SDS-PAGE分析MBP(麦芽糖结合蛋白)-MyD88重组融合蛋白的表达,切胶纯化目的蛋白;免疫BALB/c小鼠,制备针对MyD88的单克隆抗体,Western印迹检测抗体特异性,制备腹水并进行抗体亚型鉴定和效价测定。结果:构建了鸡MyD88原核表达载体pMAL-MyD88,并在大肠杆菌中获得高表达,目的蛋白以可溶性和包涵体两种形式存在;建立了3株抗鸡MyD88单克隆抗体细胞株,制备了腹水,亚型分别为IgG1、IgG1和IgG2a,轻链均为κ,腹水抗体的效价均为1∶2×105。结论:在原核表达系统中表达、纯化了重组鸡MyD88,制备了针对鸡MyD88的单克隆抗体,为后续的MyD88定量和功能研究奠定了基础。     

HMBA诱导人肝癌SMMC-7721 细胞分化过程中 Nucleophosmin 的表达与定位变化

通过选择性抽提经环六亚甲基双乙酰胺（hexamethylene bisacetamide,HMBA）诱导处理前后的人肝癌SMMC-7721细胞核基质,并运用亚细胞蛋白质组学等分析技术,研究nucleophosmin (NPM)在核基质上的表达和定位变化,及其与相关基因产物的共定位关系,观察研究了nucleophosmin 在诱导分化前后人肝癌SMMC-7721细胞核基质中的存在、分布及其与相关基因产物的共定位关系.双向凝胶电泳和质谱鉴定结果显示,nucleophosmin 存在于 SMMC-7721 细胞核基质蛋白组分中,在 HMBA 处理后细胞核基质中表达下调.蛋白质印迹杂交实验结果确证了 nucleophosmin 在核基质中的存在及其在诱导处理后细胞核基质中表达下调的变化.免疫荧光显微镜观察显示,nucleophosmin 定位在 SMMC-7721细胞核基质上,经 HMBA处理后出现分布位置与表达水平的变化.激光扫描共聚焦显微镜观察结果显示,SMMC-7721细胞中,nucleophosmin与 c-fos、c-myc、rb、p53 等基因产物具有共定位关系,但在诱导处理后细胞内的共定位区域发生了改变.研究结果证实,nucleophosmin 是一种核基质蛋白,定位于核基质纤维上,nucleophosmin 在人肝癌 SMMC-7721 细胞诱导分化过程中的表达分布,及其与相关癌基因、抑癌基因产物的关系对 SMMC-7721 细胞分化具有重要影响.     

Expression of Chicken Interleukin-2 in Insect Cells

Full-length chicken interleukin-2 (ChIL-2) protein was successfully expressed using the recombinant baculovirus/Sf9 insect cell system. The expressed protein was soluble and reached approximately 12 microg/ml. Similarly to native ChIL-2, baculovirus expressed ChIL-2 revealed two main bands corresponding to molecular masses of 22 and 20 kD as detected by SDS-PAGE and Western blot. Treatment of the expressed protein with N-endoglycosidase F for 2 h caused the complete disappearance of the 22 kD band, while the 20 kD band (which is close to the molecular weight predicted from the cloned cDNA sequence) remained unchanged. Together with results on native ChIL-2, it can be concluded that ChIL-2 is an N-glycosylated protein.     

Tissue-Specific Expression of Onco-Fetal Antigens During Embryogenesis

It has become evident from recent literature that especially in tumor virus systems, cell transformation leads to an arrest of differentation or to a retrodifferentiation. This may be reflected by the expression of embryonic antigens and it is therefore particularly important to characterize such antigens according to their specificity as well as to their Specificity during embryogenesis. We have demonstrated the expression of embryonic antigens which are cross-reactive in avian fibroblasts transformed either by Rous sarcoma virus or by methylcholanthrene. This paper is intended to demonstrate that these embryonic antigens are detected only at a certain period of embryogenesis and particularly in muscle cells. They are detected only occasionally or not at all in cells of other tissues such as brain, liver, lung, and the digestive organs. These antigens are absent from the target cells before transformation and are consequently induced by the transforming agent, either viral or chemical. Therefore, these results suggest that by transformation mechanism, cells become specifically reverted to an earlier stage of differentiation (retrodifferentiation).     

Changes in the DNA,RNA and Protein Contents During the Root-Tip Cell Differentiation of Triticum aestivum

One to two cm long root tips of Triticum aestivum L., after three days' germination at 25 ℃, each were cut into three regions--the meristem region, elongation region and mature region. The cells in different regions were stained with Hochest 33258, Pymnin G and FITC respectively. Nuclear DNA was measured by micmfluorometry with VIDAS digital image analysis system. The relative contents of RNA and protein in the cells were measured with a MPV-Ⅲ microspectrofluorometer. It was shown that the nuclear DNA content increased during root tip cell differentiation, being maximum in the mature region. The relative content of RNA was maximum in the meristem region, but decreased continuously during the growth of tissue. The relative content of protein was maximum in the elongation region, but minimum in the meristem region. The relationships among DNA, RNA, protein and cell differentiation were discussed.     

Cellular Expression and Proteolytic Processing of Presenilin Proteins Is Developmentally Regulated During Neuronal Differentiation

Abstract: We have determined the expression of the Alzheimer's disease-associated proteins presenilin-1 and presenilin-2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin-1 and presenilin-2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin-1 and presenilin-2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin-1 and presenilin-2 differs slightly, with more presenilin-2 expressed in axons compared with presenilin-1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full-length presenilin-1 or presenilin-2 could be detected within cell lysates. At early developmental stages the expected ～34-kDa N-terminal proteolytic fragment of presenilin-1 and the ～38-kDa fragment of presenilin-2 were detected. Later during differentiation we predominantly detected a ～38-kDa fragment for presenilin-1 and a ～42-kDa fragment for presenilin-2. By epitope mapping, we show that these slower migrating peptides represent N-terminal proteolytic fragments, cleaved C-terminal to the conventional site of processing. It is noteworthy that both presenilin-1 and presenilin-2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.     

C2C12成肌细胞诱导肌性分化过程中miR-101a的表达

以C2C12成肌细胞为模型,在分化培养基中诱导C2C12建立体外肌性细胞分化模型.以poly (A)3′-端加尾和实时定量PCR方法研究miR-101a在C2C12细胞分化过程中的表达情况.结果发现,在细胞转入分化培养基进行肌性分化的1-5 d中,miR-101a的表达量逐渐增加,提示miR-101a可能在肌肉发生中发挥调控作用.     

一种用作阴性对照的siRNA可诱导人K562细胞向红系分化

我们发现,一种在RNA干扰实验中用作阴性对照的商业化siRNA具有明显的诱 导人慢性髓性白血病K562细胞系向红系方向分化的作用.它表现为K562细胞瞬时转 染该siRNA后,红系分化的特异表面标志CD235及ε 、γ 和β 珠蛋白的表达升高,GATA-2的表达降低,细胞增殖速度减慢,软琼脂克隆形成率降低,并且此 过程不伴随细胞凋亡. 而生物信息学分析显示,该siRNA序列与目前所有已知人类 基因均无明显同源性.研究结果提示,该siRNA不适于用作红系分化实验中的阴性 对照. siRNA的作用远比人们目前所知的要复杂得多,siRNA的脱靶效应应当引起 研究者的足够重视,在RNA干扰实验中阴性对照siRNA的选择会极大地影响对实验 结果的判读.     

Changes in the Expression of β and Actins During Differentiation of PC 12 Cells

Cells of the rat pheochromocytoma line PC12 cease proliferation and develop neurites in response to nerve growth factor (NGF). Quantification of beta and gamma isoforms of nonmuscle actin in extracts of these differentiating cells showed that the beta:gamma ratio decreased from 1.30 +/- 0.05 to 0.99 +/- 0.05 after 6 days of NGF treatment. Cells treated with N6,O2-dibutyryl cyclic AMP (dbcAMP) also showed a shift in the ratio of beta:gamma isoforms, although few of these cells extended neurites. Administration of dbcAMP or both NGF and dbcAMP to cells accelerated the decrease in the beta:gamma actin isoform ratio relative to treatment with NGF alone. Those cells treated with both NGF and dbcAMP also showed an accelerated rate of neurite outgrowth. Suspension-grown PC12 cells treated with NGF showed neither an isoform ratio decrease nor neurite development. Our results suggest that either cyclic AMP may be a "second messenger" for NGF or it may effect the isoform ratio change by an independent mechanism. In addition, our data demonstrate an alteration in actin isoform expression, which accompanies the morphological differentiation of PC12 cells.