应用抑制性消减杂交分析全反式维甲酸和1,25-二羟维生素D_3诱导结肠癌细胞的相关基因

 为探讨结肠癌细胞诱导分化的机制 ,采用抑制性消减杂交 (suppression subtractivehybridization,SSH)研究联合使用全反式维甲酸和 1 ,2 5-二羟维生素 D3诱导分化结肠癌 Lo Vo细胞前、后差异表达的基因 .经比较消减 c DNA文库的序列与基因库的序列 ,发现 :有 1个基因的序列与正常鳞状上皮细胞中的 1个表达序列标签 (expressed sequence tag,EST)高度同源 ,同时发现6个新 EST (基因库登录号为 AW2 66492、AW2 66493、AW2 66494、AW58751 8、AW58751 9和AW58752 0 ) .说明诱导分化涉及到多个基因的表达 ,结肠癌的发生是多基因综合作用的结果 .进一步研究这些基因和 EST的功能对于结肠癌的防治将有重要意义 .     

印迹基因及其对胚胎发育的调控

某个基因位点呈单等位基因表达,且通过某种基因修饰作用来特异地抑制另一等位基因的表达,将这一基因称为印迹基因,它是等位基因排斥作用的一种特殊形式. 多数印迹基因与胚胎发育有关,可以调节胚胎的生长、发育及新生儿的生长,印迹功能的紊乱可以导致多种发育异常及死胎. 印迹基因的形成、特异识别及印迹性表达缺陷的机制还不清楚.     

Regulation of Neutrophil Apoptosis by Sodium Butyrate

Neutrophils have a very short half life because they constitutively undergo apoptosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay apoptosis, but this agent also primes functions such as the respiratory burst and receptor upregulation. Here, we show that sodium butyrate, which has been shown to increase gene expression and differentiation in a variety of cell types, is more effective than GM-CSF in delaying neutrophil apoptosis. Thus, sodium butyrate preserves cell morphology and function, and butyrate-treated cells express express high levels of CD16 after overnight culture. However, neither GM-CSF nor sodium butyrate appear to affect mRNA levels for CD16.     

Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a ‘sticky’ cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles.     

Use of cloned populations of mouse lymphocytes to analyze cellular differentiation

We describe a method for generation of homogeneous cell populations that each arise from clonal expansion of cells at a discrete stage of differentiation within a single lineage. We have used this to produce continuously propagatable lymphocyte clones. Each clone represents a cell at a progressive stage of thymus-dependent cellular differentiation. These cloned cells bear stable surface membrane glycoproteins characteristic of precursor cells and mature progeny; conditions allowing maximal cloning efficiencies for each cell type (10–85%) have been established. Mature lymphocyte clones continue to express specialized function and provide material for biochemical analysis of T lymphocyte functions; one fully differentiated clone from the “inducer” lymphocyte set synthesizes a molecule that activates other lymphocytes to secrete immunoglobulin. This activity is associated with a highly purified molecule having a molecular weight of 45,000 daltons and an isoelectric point of approximately 6.0. This molecule, together with clones of precursor and mature T lymphocytes, may provide a system to further study the mechanisms of gene activation during cellular differentiation.     

Chronic denervation of rat hemidiaphragm: maintenance of fiber heterogeneity with associated increasing uniformity of myosin isoforms

During several months of denervation, rat mixed muscles lose slow myosin, though with variability among animals. Immunocytochemical studies showed that all the denervated fibers of the hemidiaphragm reacted with anti-fast myosin, while many reacted with anti-slow myosin as well. This has left open the question as to whether multiple forms of myosin co-exist within individual fibers or a unique, possibly embryonic, myosin is present, which shares epitopes with fast and slow myosins. Furthermore, one can ask if the reappearance of embryonic myosin in chronically denervated muscle is related both to its re-expression in the pre-existing fibers and to cell regeneration. To answer these questions we studied the myosin heavy chains from individual fibers of the denervated hemidiaphragm by SDS PAGE and morphologically searched for regenerative events in the long term denervated muscle. 3 mo after denervation the severely atrophic fibers of the hemidiaphragm showed either fast or a mixture of fast and slow myosin heavy chains. Structural analysis of proteins sequentially extracted from muscle cryostat sections showed that slow myosin was still present 16 mo after denervation, in spite of the loss of the selective distribution of fast and slow features. Therefore muscle fibers can express adult fast myosin not only when denervated during their differentiation but also after the slow program has been expressed for a long time. Light and electron microscopy showed that the long-term denervated muscle maintained a steady-state atrophy for the rat's life span. Some of the morphological features indicate that aneural regeneration events continuously occur and significantly contribute to the increasing uniformity of the myosin gene expression in long-term denervated diaphragm.     

Histochemical localization of IGF-I and -II mRNA in the developing rat embryo

We describe the histological localization of embryonic and fetal tissues whose cells express the genes coding for insulin-like growth factors I and II (IGF-I and IGF-II) in the developing rat. Our studies span the period between early somite stages and full term. We have used oligodeoxyribonucleotide probes and obtained results which are both topographically precise and highly reproducible. The gene coding for IGF-II is predominant throughout development. It is strongly expressed in the liver and yolk sac. A variety of other tissues also expresses the IGF-II gene, especially many mesodermally derived structures in the process of differentiation. Many tissues do not express IGF genes. Thus no IGF mRNA was demonstrable in ectodermally derived structures, including the central and peripheral nervous systems as well as the skin and its derivatives.     

Effects of colonization processes on genetic diversity: differences between annual plants and tree species

Tree species are striking for their high within-population diversity and low among-population differentiation for nuclear genes. In contrast, annual plants show much more differentiation for nuclear genes but much less diversity than trees. The usual explanation for this difference is that pollen flow, and therefore gene flow, is much higher for trees. This explanation is problematic because it relies on equilibrium hypotheses. Because trees have very recently recolonized temperate areas, they have experienced many foundation events, which usually reduce within-population diversity and increase differentiation. Only extremely high levels of gene flow could counterbalance these successive founder effects. We develop a model to study the impact of life cycle of forest trees, in particular of the length of their juvenile phase, on genetic diversity and differentiation during the glacial period and the following colonization period. We show that both a reasonably high level of pollen flow and the life-cycle characteristics of trees are needed to explain the observed structure of genetic diversity. We also show that gene flow and life cycle both have an impact on maternally inherited cytoplasmic genes, which are characterized both in trees and annual species by much less diversity and much more differentiation than nuclear genes.     

植物根尖干细胞的表观遗传调控

干细胞是一类具有特化为不同细胞类型能力的多能性细胞,他为多细胞生物的器官发生、损伤修复和再生源源不断提供新细胞。干细胞的特化和维持需要复杂的基因调控网络来有序调控。此外,表观遗传调控在包括干细胞命运决定在内的许多生物学过程中发挥极其重要的作用。本文归纳了近年来对植物,主要是模式植物拟南芥（Arabidopsis thaliana（L.）Heynh.）根尖干细胞表观遗传调控方面的研究进展,重点论述了表观调控因子与控制干细胞的关键转录因子之间如何互作、调控植物根尖干细胞的自我更新和分化,并对今后研究的突破方向进行了展望。     

小鼠胚胎干细胞向脂肪细胞分化过程中PPARγ2基因的表达模式

将PPARγ2基因启动子和报告基因荧光素酶相连接克隆于特定载体构建成表达质粒,电穿孔转染小鼠ES细胞,筛选阳性克隆.诱导ES细胞向脂肪细胞分化,通过定量检测荧光素酶活性跟踪PPARγ2基因的表达情况,以此研究脂肪细胞分化过程中该基因的表达模式.结果显示PPARγ2基因在未分化的ES细胞和EB形成的前两天中不表达,从EB形成的第3天开始表达,直至脂肪细胞分化完成.该基因在已完成分化的脂肪细胞中的表达远强于在分化中的前脂肪细胞中的表达.首次报道了从小鼠ES细胞到脂肪细胞分化过程中PPARγ2基因的表达模式,支持了PPARγ2基因为脂肪组织特异性表达基因的已有报道,并为脂肪细胞分化机理研究提供了线索.     

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.     

Onset of odorant receptor gene expression during olfactory sensory neuron regeneration

Individual olfactory sensory neurons are thought to express only one odorant receptor gene from a repertoire of hundreds to thousands of genes. How do these sensory neurons choose just one specific odorant receptor to express during their differentiation? As an initial attempt toward understanding the process of odorant receptor gene regulation, we studied when odorant receptor expression is activated during sensory neuron regeneration. We find that receptor gene expression is activated in postmitotic neurons and can occur in the absence of the olfactory bulb. These results suggest that receptor expression is restricted to the terminal stages of olfactory neuron differentiation, and sensory neurons do not simply inherit the odorant receptor that is already expressed in mitotic precursor cells. Our results also support a model in which odorant receptor gene expression occurs independent of the olfactory bulb.     

p130/p107 expression distinguishes adipogenic potential in primary myoblasts based on age

Recent investigations have provided significant evidence that many mesodermally derived tissues contain stem cell-like precursors capable of being stimulated to undergo differentiation into a variety of cellular lineages. We have recently reported that primary myoblasts isolated from 23-month-old mice have an increased adipogenic potential when compared to their 8-month-old counterparts. To further characterize the degree of adipocyte differentiation in these myoblasts, we examined early and late markers of adipocyte differentiation. Within the first 24h of adipocyte differentiation, expression of p130 and p107, two members of the retinoblastoma tumor suppressor gene family, are regulated and this event is an important one early in adipogenesis. Consistent with the increased adipogenic potential of the older myoblasts and in contrast to the younger cells, the p130:p107 pattern of expression is very similar to that observed in adipogenesis where there is a transient increase in p107 expression accompanied by a decrease in p130 expression. Interestingly, while these older cells accumulated lipid and expressed genes associated with lipid metabolism, they failed to express adipsin and leptin, two well-established markers of terminal adipocyte differentiation. These results suggest that older myoblasts are capable of initiating and progressing through the adipogenic program to a point where they express genes associated with lipid metabolism, but do not reach a terminally differentiated state. This finding may have important metabolic implications in the aging population.     

IGF-I and vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at low temperatures

In a previous study investigating the effects of low temperature on skeletal muscle differentiation, we demonstrated that C2C12 mouse myoblasts cultured at 30 °C do not express myogenin, a myogenic regulatory factor (MRF), or fuse into multinucleated myotubes. At this low temperature, the myoblasts continuously express Id3, a negative regulator of MRFs, and do not upregulate muscle-specific microRNAs. In this study, we examined if insulin-like growth factor-I (IGF-I) and a stable form of vitamin C (L-ascorbic acid phosphate) could alleviate the low temperature-induced inhibition of myogenic differentiation in C2C12 cells. Although the addition of either IGF-I or vitamin C alone could promote myogenin expression in C2C12 cells at 30 °C, elongated multinucleated myotubes were not formed unless both IGF-I and vitamin C were continuously administered. In human skeletal muscle cells, low temperature-induced blockage of myogenic differentiation was also ameliorated by exogenous IGF-I and vitamin C. In addition, we demonstrated that satellite cells of IGF-I overexpressing transgenic mice in single-fiber culture expressed myogenin at a higher level than those of wild-type mice at 30 °C. This study suggests that body temperature plays an important role in myogenic differentiation of endotherms, but the sensitivity to low temperature could be buffered by certain factors in vivo, such as IGF-I and vitamin C.     

An endoderm-specific GATA factor gene, dGATAe, is required for the terminal differentiation of the Drosophila endoderm

GATA factors play an essential role in endodermal specification in both protostomes and deuterostomes. In Drosophila, the GATA factor gene serpent (srp) is critical for differentiation of the endoderm. However, the expression of srp disappears around stage 11, which is much earlier than overt differentiation occurs in the midgut, an entirely endodermal organ. We have identified another endoderm-specific Drosophila GATA factor gene, dGATAe. Expression of dGATAe is first detected at stage 8 in the endoderm, and its expression continues in the endodermal midgut throughout the life cycle. srp is required for expression of dGATAe, and misexpression of srp resulted in ectopic dGATAe expression. Embryos that either lacked dGATAe or were injected with double-stranded RNA (dsRNA) corresponding to dGATAe failed to express marker genes that are characteristic of differentiated midgut. Conversely, overexpression of dGATAe induced ectopic expression of endodermal markers even in the absence of srp activity. Transfection of the dGATAe cDNA also induced endodermal markers in Drosophila S2 cells. These studies provide an outline of the genetic pathway that establishes the endoderm in Drosophila. This pathway is triggered by sequential signaling through the maternal torso gene, a terminal gap gene, huckebein (hkb), and finally, two GATA factor genes, srp and dGATAe.     

An Immortal Cell Culture Model of Hypothalamic Gonadotropin-Releasing Hormone Neurons

The neuroendocrine hypothalamus has been the object of intensive study in vivo and in tissue slices. However, using these models it is difficult to approach questions at the molecular and cellular level and to differentiate between direct effects and those mediated by other neurons. By using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene to target expression of the oncogene SV40 T antigen in transgenic mice, we have produced hypothalamic tumors which were cultured to produce clonal cell lines (GT1 cells). These cells express GnRH and many other neuronal markers, but do not express glial cell markers or other hypothalamic hormones. They have a distinctive neuronal phenotype, process the GnRH peptide accurately, and secrete GnRH in a pulsatile pattern. They respond to many neurotransmitters and neuromodulators including activin, norepinephrine, dopamine, nitric oxide, NMDA, and GABA, as well as GnRH itself. Thus, we have immortalized GnRH neurons by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene which is expressed late in the differentiation of that cell lineage. The GT1 cell lines serve as an excellent model for molecular, pharmacological, electrophysiological, and biochemical investigations into the regulation of GnRH and the characteristics of a pure CNS neuronal population. Moreover, their derivation demonstrates the success of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.     

Skin aging: a role for telomerase and telomere dynamics?

Skin is a complex tissue composed of two very different compartments -- the continuously renewing epidermis made up mostly by keratinocytes and the underlying matrix-rich dermis with the resting fibroblasts as its major cellular components. Both compartments are tightly interconnected and a paracrine mutual interaction is essential for epidermal growth, differentiation, and tissue homeostasis. Skin aging is commonly viewed as wrinkle formation, hair greying, and impaired wound healing. Nevertheless, the epidermis as the outermost shield needs to remain intact in order to guarantee an inside-out and outside-in barrier function throughout life time of a human being. Furthermore, the epidermis is one of the few regenerative tissues that express telomerase, the ribonucleoprotein complex that can counteract telomere erosion, one of the presently mostly favoured potential mechanisms causing cellular aging. This raises the question whether in the epidermis telomerase is able to counteract telomere erosion and thereby to prevents a telomere-dependent aging process and consequently which part of the skin is responsible for the most obvious changes associated with skin aging.