Negative regulation of transforming growth factor-beta by the proteoglycan decorin

Decorin is a small chondroitin-dermatan sulphate proteoglycan consisting of a core protein and a single glycosaminoglycan chain. Eighty per cent of the core protein consists of 10 repeats of a leucin-rich sequence of 24 amino acids. Similar repeats have been found in two other proteoglycans, biglycan and fibromodulin, and in several other proteins including Drosophila morphogenetic proteins. Expression of high levels of decorin in Chinese hamster ovary cells has a dramatic effect on their morphology and growth properties. We now report that this effect is due at least in part to the ability of decorin to bind transforming growth factor-beta, an autocrine factor that stimulates the growth of Chinese hamster ovary cells. As transforming growth factor-beta induces synthesis of decorin in many cell types, our results suggest that decorin may be a component of a feedback system regulating cell growth.     

Natural inhibitor of transforming growth factor-beta protects against scarring in experimental kidney disease.

The central pathological feature of human kidney disease that leads to kidney failure is the accumulation of extracellular matrix in glomeruli. Overexpression of transforming growth factor-beta (TGF-beta) underlies the accumulation of pathological matrix in experimental glomerulonephritis. Administration of an antibody raised against TGF-beta to glomerulonephritic rats suppresses glomerular matrix production and prevents matrix accumulation in the injured glomeruli. One of the matrix components induced by TGF-beta, the proteoglycan decorin, can bind TGF-beta and neutralize its biological activity, so decorin may be a natural regulator of TGF-beta (refs 3, 4). We tested whether decorin could antagonize the action of TGF-beta in vivo using the experimental glomerulonephritis model. We report here that administration of decorin inhibits the increased production of extracellular matrix and attenuates manifestations of disease, confirming our hypothesis. On the basis of our results, decorin may eventually prove to be clinically useful in diseases associated with overproduction of TGF-beta.     

Retinoids specifically enhance the number of epidermal growth factor receptors

Retinoids elicit many biological and biochemical responses from cells in vitro. One widely used criterion for the responsiveness of cells to retinoids is inhibition of growth; retinoids reduce the saturation density and/or growth rate of many normal and tumorigenic cell lines. Propagation of eukaryotic cells has been demonstrated to be dependent on the presence of macromolecular growth factors such as epidermal growth factor (EGF), which can stimulate proliferation of epithelial and fibroblastic cell lines. We now describe the effect of retinoids on the binding of EGF to its receptor. Retinoic acid enhances binding of 125I-labelled EGF to various fibroblastic and epidermal cell lines. It has no marked effect on the affinity of this growth factor for its receptor, but increases the number of EGF receptor sites. Retinoic acid has little effect on the binding of concanavalin A (Con A) and insulin, indicating the specific nature of the action of retinoids on cell-surface glycoproteins. Treatment of cells with the phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) and retinoic acid shows poor antagonism between these compounds on EGF binding. It has been previously shown that retinoids induce or stimulate differentiation of embryonal carcinoma cells. EGF binding can be used as a marker to monitor differentiation of these cells.     

Regulatory networks define phenotypic classes of human stem cell lines

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal and adult sources have been called stem cells, even though they range from pluripotent cells-typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation-to adult stem cell lines, which can generate a far more limited repertoire of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine has highlighted the need for a general, reproducible method for classification of these cells. We report here the creation and analysis of a database of global gene expression profiles (which we call the 'stem cell matrix') that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent and differentiated cell types. Using an unsupervised clustering method to categorize a collection of approximately 150 cell samples, we discovered that pluripotent stem cell lines group together, whereas other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis we uncovered a protein-protein network (PluriNet) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas and induced pluripotent cells). Analysis of published data showed that the PluriNet seems to be a common characteristic of pluripotent cells, including mouse embryonic stem and induced pluripotent cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotency and self-renewal are under tight control by specific molecular networks.     

梅花鹿鹿茸软骨细胞和间充质干细胞永生化细胞株的构建及鉴定

分离梅花鹿鹿茸软骨细胞和间充质干细胞,使用SV40 LT抗原慢病毒载体建立永生化软骨细胞和间充质干细胞系并鉴定。将含有SV40 LT基因片段的慢病毒载体,转染人胚肾细胞293T获得包装后的病毒粒子,感染鹿茸软骨细胞及间充质干细胞,连续传代培养,通过形态观察、细胞增殖、real-time PCR、甲苯胺蓝染色法和流式细胞术等方法检测SV40 LT抗原表达以及细胞性质。实验所建立的永生化鹿茸软骨细胞系与间充质干细胞系能够稳定传代并具有较强的体外增值活性。RT-PCR检测到SV40T抗原的表达,通过甲苯胺蓝染色法和流式细胞术鉴定所得细胞系具有原代细胞的基本性质。成功获得永生化的软骨细胞与间充质干细胞系,为后续鹿茸生长及发育研究奠定了基础。     

MSH2 is required for cell proliferation, cell cycle control and cell invasiveness in colorectal cancer cells

We have investigated the role of MSH2,a mismatch repair gene in cell proliferation,cell cycle control and cell invasiveness in the SW480 human colorectal cancer cell line.RNAi-mediated inhibition of MSH2 expression was achieved using MSH2 shRNA lentiviral expression vectors.Effective knockdown of endogenous MSH2 expression was determined by real-time PCR analysis.The most efficient MSH2 knockdown vector was selected for subsequent studies using SW480 cells.Endogenous MSH2 mRNA levels decreased after lentiviral delivery of the MSH2-RNAi,indicating efficient silencing of MSH2 expression in SW480 cells.Cell proliferation,cell cycle progression and cell invasiveness were quantified by MTT assays,flow cytometry and transwell assays,respectively.RNAi-mediated inhibition of MSH2 expression in SW480 cells resulted in decreased cell proliferation,cell cycle arrest at the G0/G1 phase and decreased cell invasiveness.Taken together,these results provide evidence that MSH2 stimulates cell proliferation,promotes cell cycle progression and positively regulates cell invasiveness.     

c-myc oncogene protein synthesis is independent of the cell cycle in human and avian cells

Several lines of evidence suggest a role for the myc oncogene in cell proliferation. Most recently, mitogenic stimulation of quiescent lymphoid, fibroblast and epithelial cells has been demonstrated to lead to a sharp increase in c-myc RNA levels. To determine how c-myc expression is linked to the cell proliferative cycle, we have used centrifugal elutriation to enrich for populations of avian and human cells at different stages of the cell cycle. Centrifugal elutriation is a counterflow centrifugation method that separates cells on the basis of volume, a parameter correlating well with progression through the cell cycle. Using myc-specific anti-peptide antibodies, we show here that the synthesis, half-life and modification of c-myc proteins are constant throughout the cell cycle of normal and transformed cells.     

外加植物生长调节剂和钙调素对白芷悬浮培养细胞增殖效应的关系

利用３ＨＴｄＲ标记ＤＮＡ合成的方法研究了外加植物生长调节剂和钙调素对白芷悬浮培养细胞增殖的影响．ＭＳ培养基中有和无植物生长调节剂时,钙调素对细胞增殖的促进效应分别为４１％和４３％．结果表明外加钙调素对细胞增殖的效应与培养介质中的激素浓度可能无关     

蛋白聚糖NG2中硫酸软骨素糖胺聚糖链调节稳定转染NG2的U251细胞迁移能力

应用PCR反应使编码蛋白聚糖NG2的核苷酸在3064-3065位置的AG突变成为GC, 构建突变型NG2/S999A. 用表达野生型NG2和突变型NG2/S999A及空白表达载体分别稳定转染人成胶质细胞瘤U251. 应用Western Blot方法鉴定转染后的U251细胞表达野生型及突变型NG2的状态, 证实突变型NG2A999稳定转染的U251细胞株表达的NG2缺失硫酸软骨素葡糖胺聚糖链（C S-GAG）. 比较了3种U251细胞株的迁移, 表明蛋白聚糖NG2中CS-GAG链影响细胞的迁移能 力.     

长非编码RNA LINC00941在结直肠癌中的表达及对细胞增殖的影响研究

为了研究与细胞分化相关的长非编码RNA(long non-coding RNA, lncRNA) LINC00941在肿瘤发生发展中的作用,通过实时荧光定量PCR技术检测LINC00941在6种不同类型的人类癌细胞和正常胚胎肾细胞HEK-293细胞中的表达水平,结果表明,LINC00941在结直肠癌细胞HCT116和HCT116 p53-/-、肺癌细胞A549和NCI-H1299、黑素瘤细胞Stilling中均有较高的表达水平,在结直肠癌细胞中表达水平最高. 以结直肠癌患者肿瘤组织和癌旁正常组织为材料,实时荧光定量PCR检测LINC00941的表达水平发现,肿瘤组织中LINC00941 RNA的表达水平显著高于癌旁组织. 通过shRNA(short hairpin RNA)干扰技术降低HCT116细胞中的LINC00941 RNA水平,导致细胞增殖速度下降,说明LINC00941与结直肠癌的发生发展相关.     

Inhibition of suppressor T-cell development following deoxyguanosine administration

The expression of immunodeficiency in patients with specific purine enzyme defects indicates a crucial role of the purine salvage pathway in the acquisition and expression of normal immune function. One current hypothesis links the failure of normal lymphocyte development in these diseases to the accumulation of deoxynucleotide triphosphates. In our studies of human in vitro IgM responses, we observed that antigen-induced T-suppressor cell activity was abrogated in the presence of micromolar concentrations of deoxyguanosine (dGuo). In contrast, more than 1,000-fold higher resistance to dGuo was found for both noin-proliferative T-helper cell activity and the differentiation and proliferation of the precursor B lymphocytes for direct haemolytic plaque forming cells (PFC). To determine whether these observations could have in vivo relevance, we monitored the generation of murine T-suppressor cells, capable of abrogating a primary IgM response. It was found that dGuo (but not guanosine) selectively inhibited the in vivo development of T-suppressor cells.     

A novel human lymphokine that inhibits haematopoietic progenitor cell proliferation

T lymphocytes in culture synthesize and secrete a variety of factors that activate and guide the differentiation, replication and maturation of haematopoietic cells in vitro. Malignant T-cell lines as well as T-cell hybridomas producing several of these factors have been established. We report here a factor produced by a human cell line that exerts a potent inhibitory effect on the growth of bone marrow progenitor cells. The properties of this factor, which we have termed colony-inhibiting lymphokine ( CIL ), differ from other inhibitors of haematopoietic progenitor cell proliferation, but resemble those of a T-cell-derived factor causally linked with some cases of severe aplastic anaemia in humans. Sensitivity of cells to this factor appears to correlate positively with expression of HLA-DR surface antigens.     

聚ADP核糖基聚合酶的抑制剂苯甲酰胺在肺癌细胞PG凋亡中的作用

研究了聚ADP核糖基聚合酶（PARP）抑制剂苯甲酰胺（BA）在DNA损伤所造成的肺癌细胞（PG）凋亡过程中的作用,分别用MNNG（N－甲基－N－亚硝基氮亚硝基胍）和BA,以及同时用MNNG和BA处理PG细胞,然后用非放射性细胞增殖测定试剂盒和流式细胞仪测定PG细胞的增殖活性和细胞凋亡的变化,并用免疫组化法检测细胞中Bcl-2蛋白表达的变化,结果表明,在MNNG的作用下细胞的增殖活性受到明显抑制,细胞凋亡显著,Bcl-2蛋白低表达,单独用BA处理PG细胞时,细胞的增殖活性,细胞凋亡数及Bcl-2的表达与空白对照组相似,用BA和MNNG共同处理PG细胞时,细胞的增殖活性,细胞凋亡数及Bcl-2的表达,与对照细胞和BA单独处理的细胞相比均无明显差异,这说明PARP对诱导细胞凋亡起重要作用,而且这种作用可被PARP抑制剂BA所抑制。     

Sequence and domain structure of talin

Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, we have cloned and sequenced murine talin. We describe a model for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane.     

Mitogens trigger a calcium-independent signal for proliferation in phorbol-ester-treated lymphocytes

The activation of T lymphocytes by mitogens requires at least two signals; the first, delivered to T cells by a mitogen in conjunction with accessory cells (monocytes/macrophages), leads to the generation of the second signal, interleukin-2 (IL-2). The first signal also induces the expression of IL-2 receptors on the surface of a subpopulation of T cells; binding of IL-2 to its receptor then initiates a cascade of events culminating in DNA synthesis by these cells. Certain compounds act synergistically with mitogens in promoting T-cell proliferation by substituting for the activities of interacting cells or their products. For example, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been shown to restore the ability of macrophage-depleted T-cell populations to respond to mitogenic lectins. Transmembrane fluxes of calcium, leading to increased free cytosolic calcium concentrations ([Ca2+]), have been demonstrated following mitogen binding to lymphocytes and have been implicated in the initiation of cell proliferation. We show here that the effect of TPA on lymphocyte proliferation occurs in the absence of extracellular Ca2+ or detectable changes in [Ca2+]i, but only in the presence of mitogens. This suggests that in cells which have been incubated with the phorbol ester, mitogens can induce proliferation by a calcium-independent signal.     

Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via specific muscarinic receptor subtypes

Little is known about the factors which regulate the growth and development of the mammalian brain. Although proliferation of neuronal cells ceases relatively early in development, certain types of glial cells proliferate and differentiate mainly perinatally. In the perinatal period, the ability of acetylcholine to stimulate phosphoinositide (PI) hydrolysis in brain reaches peak levels, and indeed the stable acetylcholine analogue carbachol can stimulate PI hydrolysis of primary neonatal astroglial cells. As PI hydrolysis is thought to be important in the regulation of cell proliferation, we investigated whether cellular DNA synthesis can be induced by carbachol. Our results show that carbachol stimulates DNA synthesis via muscarinic acetylcholine receptors (mAChRs), in primary astrocytes derived from perinatal rat brain, in an age-dependent fashion. Carbachol is also mitogenic in certain brain-derived astrocytoma and neuroblastoma cell lines, as well as in chinese hamster ovary (CHO) cells expressing recombinant muscarinic receptors. DNA synthesis is strongly activated by carbachol in those brain-derived cell lines and transfected CHO cells that express mAChR subtypes which activate PI hydrolysis efficiently, and poorly activated in cells expressing mAChR subtypes which only weakly activate PI hydrolysis. These results strongly support a role for acetylcholine in regulating astroglial cell growth in the developing brain, and indicate that the specificity of acetylcholine-induced cell proliferation may be determined by the expression of those mAChR subtypes which activate PI hydrolysis.     

Activated T lymphocytes produce a matrix-degrading heparan sulphate endoglycosidase

We have previously found that lines of activated T lymphocytes specifically autosensitized to the basic protein of myelin (BP), on intravenous inoculation into syngeneic rats, were able to penetrate blood vessels, accumulate in the nervous system and cause experimental autoimmune encephalomyelitis (EAE). An important question is how effector T cells reach such targets outside the walls of blood vessels. To investigate this we have studied in vitro the interaction of anti-BP effector T lymphocytes with the basement membrane-like extracellular matrix produced by vascular endothelial cells. We now report that activated but not resting T lymphocytes produce an endoglycosidase capable of degrading heparan sulphate side chains of the proteoglycan scaffold of the extracellular matrix. Moreover, the anti-BP T lymphocytes respond to BP presented by extracellular matrix by markedly enhanced elaboration of the endoglycosidase. These results suggest that tissue-specific antigens on blood vessel walls could direct lymphocyte homing by activating enzymes that facilitate penetration of the subendothelial basal lamina. They also suggest that effector T lymphocytes can recognize antigen which is not associated with a major histocompatibility complex signal.     

Extensive proliferation of mature connective-tissue type mast cells in vitro

There are two phenotypically distinct subpopulations of mast cells in rodents: connective tissue-type mast cells (CTMC) and mucosal mast cells (MMC). These populations differ in their location, cell size, staining characteristics, ultrastructure, mediator content and T-cell dependency. Several investigators recently reported a further subclass of mast cells which arise when normal mouse haematopoietic cells are cultured with interleukin-3 (IL-3); IL-3 is an activity similar or identical to mast-cell growth factor, histamine-producing factor, or P-cell stimulating factor. These cultured mast cells are in many ways similar to MMC; they stain with Alcian blue but not safranin, contain chondroitin sulphate E proteoglycan rather than heparin proteoglycan and have relatively low histamine content, as do MMC. Although proliferation of MMC is known to be T-cell dependent in vivo and thought to be IL-3-dependent in vitro, the factors on which CTMC proliferation depends remain elusive. Here we show that mature CTMC purified from mouse peritoneal cells can proliferate in vitro in methylcellulose culture and maintain the appearance and function of CTMC. We also present evidence that mature CTMC cannot proliferate in the presence of pure IL-3 alone.     

ERK信号转导通路在PDGF诱导的人动脉平滑肌细胞增殖中的作用

了解ERK信号转导通路在PDGF诱导的人动脉平滑肌细胞增殖中的作用.原代培养人脐动脉平滑肌细胞（hUASMC）,取生长正常的细胞分4组：对照组,PDGF（platelet derived growth factor）组,ERK阻断剂组和PDGF＋ERK阻断剂组.继续培养24h后,用免疫细胞化学技术测细胞核内核增殖抗原（PCNA）的表达;用MTT法测细胞的增殖活性.结果显示：1）与对照组相比,PDGF组细胞内PCNA 的表达明显增强,MTT法测得A值也升高（P〈0.01）.2）ERK阻断剂可完全抑制PDGF诱导的PCNA 的表达增多和A值的升高.结论：ERK信号转导通路参与了PDGF诱导的人动脉平滑肌细胞的增殖.