Effect of bone morphogenetic protein-2 on proliferation and apoptosis of gastric cancer cells

Objective: To investigate the effects of bone morphogenetic protein-2 (BMP-2) on the proliferation, differentiation and apoptosis of normal human gastric mucosal cells and gastric cancer cells.Methods: Poorly differentiated gastric cancer BGC823 cells, moderately differentiated gastric cancer cells and normal human gastric mucosal epithelial GES-1 cells were independently treated with recombinant human BMP-2 or its inhibitor Noggin. MTT assay was performed to detect the proliferation, flow cytometry done to measure the cell cycle and apoptosis and immunohistochemistry carried out to determine the expression of cyclin-dependent kinase 4 (CDK4).Results: BMP-2 exerted inhibitory effect on the growth of all types of cells and the inhibition become more evident with the increase of BMP-2 dose. After treatment with 200 ng/ml BMP-2, cancer cells arrested in G1 phase and those in S phase reduced. Gastric cancer cells had higher CDK4 expression than GES-1 cells. BMP-2 decreased CDK-4 expression in cancer cells but had no influence in GES-1 cells. Noggin conferred promotive effect on the growth of 3 types of cells. In 2 types of cancer cells, treatment with 2000 ng/ml Noggin significantly increased the proportion of cells in S phase but reduced that in G1 phase. However, Noggin did not affect the cell cycle of GES-1 cells. The CDK4 expression was markedly increased in 2 types of cancer cells but that of GES-1 remained unchanged after treatment with 2000 ng/ml Noggin.Conclusions: BMP-2 may inhibit the proliferation of both normal and malignant gastric epithelial cells, down-regulate CDK4 expression in gastric cancer cells and arrest gastric cancer cells in G1-phase in cell cycle. Through antagonizing BMP-2, Noggin, may accelerate the proliferation of gastric cancer cells. Thus, the abnormality of BMP signaling pathway may play an important role in the pathogenesis of gastric cancer.     

BMP-4 and Noggin signaling modulate dorsal fin and somite development in the axolotl trunk.

BMP-4, a member of the TGF-beta superfamily of growth factors, is involved in various developmental processes. We investigated the effects of BMP-4 and its antagonist Noggin on axolotl trunk development. Implantation of BMP-4-coated microbeads caused inhibition of muscle and dorsal fin formation in the vicinity of the microbeads. At some distance, myotomes developed with reduced height but increased width, which was accompanied by increased cell proliferation. These effects could be modulated by co-implanting Noggin-coated beads. Immunostaining of Pax7 further revealed that although the dermomyotome was absent in the vicinity of BMP-4-coated beads, at some distance from them, it was thicker than in controls, indicating that moderate amounts of BMP-4 stimulate this layer of undifferentiated cells. In contrast, Noggin generally inhibited the dermomyotome, possibly indicating premature differentiation of dermomyotome cells. We conclude that BMP-4 and Noggin are involved in the regulation of cell proliferation and differentiation during somite development.     

Multipotent and restricted precursors in the central nervous system

Acquisition of cell type‐specific properties in the nervous system is likely a process of sequential restriction in developmental potential. At least two classes of pluripotent stem cells, neuroepithelial (NEP) stem cells and EGF‐dependent neurosphere stem cells, have been identified in distinct spatial and temporal domains. Pluripotent stem cells likely generate central nervous system (CNS) and peripheral nervous system (PNS) derivatives via the generation of intermediate lineage‐restricted precursors that differ from each other and from multipotent stem cells. Neuronal precursors termed neuronal‐restricted precursors (NRPs), multiple classes of glial precursors termed glial‐restricted precursors (GRPs), oligodendrocyte‐type 2 astrocytes (O2As), astrocyte precursor cells (APCs), and PNS precursors termed neural crest stem cells (NCSCs) have been identified. Multipotent stem cells and restricted precursor cells can be isolated from embryonic stem (ES) cell cultures providing a non‐fetal source of such cells. Analysis in multiple species illustrates similarities between rat, mouse, and human cell differentiation raising the possibility that similar factors and markers may be used to isolate precursor cells from human tissue or ES cells. Anat Rec (New Anat): 257:137–143, 1999. © 1999 Wiley‐Liss, Inc.     

Radial glial cell heterogeneity--the source of diverse progeny in the CNS

Here, we discuss the identity, heterogeneity and functions of radial glial cells mostly in the developing central nervous system (CNS). First, we define radial glial cells by morphological, cell biological and molecular criteria as true glial cells, akin to astroglia. We then describe the appearance of radial glial cells during neural development as a precursor intermediate between immature neuroepithelial cells and differentiating progeny. Then we review the diverse progeny arising in different lineages from radial glial cells as observed by clonal analyses and time-lapse imaging. This leads us to discuss the molecular mechanisms involved in the regulation of the lineage heterogeneity of radial glial cells - including their diversity in distinct regions of the CNS. We conclude by considering the possible mechanisms allowing neurogenic radial glial cells to persist into adulthood in various vertebrate classes ranging from fish to birds, while neurogenic glial cells become restricted to few small regions of the adult forebrain in mice and men.     

脊髓神经管神经上皮干细胞的分离培养和诱导分化

目的：从胚胎大鼠脊髓神经管中分离神经上皮干细胞并诱导其向多巴胺能神经元方向分化。方法：利用无血清悬浮培养、单细胞克隆技术分离神经上皮干细胞；采用5－溴－2－脱氧尿苷（BrdU）标记新生细胞，免疫细胞化学单标或双标染色技术，检测神经上皮干细胞蛋白（nestin）和分化后特异性神经细胞抗原的表达；用纹状体组织提取液，诱导神经上皮干细胞向多巴胺能神经元方向分化。结果：从胚胎大鼠脊髓神经管 中分离的细胞可以连续传代，表达nestin，它们分化后可以表达神经元、星形胶质细胞和少突胶质细胞的特异性抗原；与对照组3％相比，纹状体组织提取液可以诱导这些细胞中的12％分化成为多巴胺能神经元。结论：分离自脊髓神经管的细胞具有自我更新能力和多分化潜能（multipotent)，是增殖能力很强的神经干细胞；这些干细胞在一定的体外环境中能被诱导成为特定的神经元，提示其可以为神经移植提供材料。     

Sox10 overexpression induces neural crest-like cells from all dorsoventral levels of the neural tube but inhibits differentiation.

SoxE genes (Sox8, Sox9, and Sox10) are early response genes to neural crest induction. Although the early role of Sox9 has been examined in chick and frog, later roles in neural crest migration and differentiation remain largely unexplored. We first examined which SoxE genes were expressed in trunk neural crest cells and then investigated their function using in ovo electroporation. The results of this analysis reveal that Sox10 is present in migrating neural crest cells, whereas other SoxE genes are only expressed transiently after induction. Ectopic expression of Sox10 in the neural tube at trunk level induced expression of HNK-1 in neuroepithelial cells followed by extensive emigration from all levels of the dorsoventral neuraxis, including the floor plate. Sox10-expressing cells failed to express neuronal, Schwann, or melanocyte markers up to 6 days posttransfection (E8), suggesting these cells were maintained in an undifferentiated state. Overexpression of Sox8 or Sox9 had similar but not identical effects on neuroepithelial cells. These results show that high levels of Sox10, Sox9, or Sox8 expression in the neural tube are capable of inducing a migratory neural crest-like phenotype even in the absence of dorsal signals and can maintain these cells in an undifferentiated state.     

BMP signaling regulates the fate of chondro‐osteoprogenitor cells in facial mesenchyme in a stage‐specific manner

Background : Lineage tracing has shown that most of the facial skeleton is derived from cranial neural crest cells. However, the local signals that influence postmigratory, neural crest‐derived mesenchyme also play a major role in patterning the skeleton. Here, we study the role of BMP signaling in regulating the fate of chondro‐osteoprogenitor cells in the face. Results : A single Noggin‐soaked bead inserted into stage 15 chicken embryos induced an ectopic cartilage resembling the interorbital septum within the palate and other midline structures. In contrast, the same treatment in stage 20 embryos caused a loss of bones. The molecular basis for the stage‐specific response to Noggin lay in the simultaneous up‐regulation of SOX9 and downregulation of RUNX2 in the maxillary mesenchyme, increased cell adhesiveness as shown by N‐cadherin induction around the beads and increased RA pathway gene expression. None of these changes were observed in stage 20 embryos. Conclusions : These experiments demonstrate how slight changes in expression of growth factors such as BMPs could lead to gain or loss of cartilage in the upper jaw during vertebrate evolution. In addition, BMPs have at least two roles: one in patterning the skull and another in regulating the skeletogenic fates of neural crest‐derived mesenchyme. Developmental Dynamics 245:947–962, 2016. © 2016 Wiley Periodicals, Inc.     

Noggin signaling from Xenopus animal blastomere lineages promotes a neural fate in neighboring vegetal blastomere lineages.

In Xenopus, localized factors begin to regionalize embryonic fates prior to the inductive interactions that occur during gastrulation. We previously reported that an animal-to-vegetal signal that occurs prior to gastrulation promotes primary spinal neuron fate in vegetal equatorial (C-tier) blastomere lineages. Herein we demonstrate that maternal mRNA encoding noggin is enriched in animal tiers and at low concentrations in the C-tier, suggesting that the neural fates of C-tier blastomeres may be responsive to early signaling from their neighboring cells. In support of this hypothesis, experimental alteration of the levels of Noggin from animal equatorial (B-tier) or BMP4 from vegetal (D-tier) blastomeres significantly affects the numbers of primary spinal neurons derived from their neighboring C-tier blastomeres. These effects are duplicated in blastomere explants isolated at cleavage stages and cultured in the absence of gastrulation interactions. Co-culture with animal blastomeres enhanced the expression of zygotic neural markers in C-tier blastomere explants, whereas co-culture with vegetal blastomeres repressed them. The expression of these markers in C-tier explants was promoted when Noggin was transiently added to the culture during cleavage/morula stages, and repressed with the transient addition of BMP4. Reduction of Noggin translation in B-tier blastomeres by antisense morpholino oligonucleotides significantly reduced the efficacy of neural marker induction in C-tier explants. These experiments indicate that early anti-BMP signaling from the animal hemisphere recruits vegetal equatorial cells into the neural precursor pool prior to interactions that occur during gastrulation.     

神经调节蛋白-1与碱性成纤维细胞生长因子对大鼠神经干细胞增殖和分化的影响

目的:探讨神经调节蛋白-1(NRG-1)和碱性成纤维细胞生长因子(bFGF)对神经干细胞(NSCs)增殖和分化的影响.方法:从新生大鼠大脑组织分离NSCs,进行体外培养,分别用bFGF、 NRG-1和NRG-1加bFGF处理,观察各组神经球的形成情况;应用免疫细胞化学法鉴定NSCs及检测分化后神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)、 2,3-环核苷酸磷酸二酯酶(CNP)的表达.结果:NRG+bFGF组和bFGF组形成的神经球数量和直径的差别不明显,但都明显多于和大于NRG组和对照组.免疫细胞化学显色结果显示,NRG+bFGF组、bFGF组中的神经球分化出的NSE、 GFAP、 CNP阳性细胞数量明显多于NRG组和对照组,尤其是NRG+bFGF组分化的NSE、CNP阳性细胞的突起较bFGF组更长和增多.结论:NRG-1与bFGF合用能促进NSCs的增殖和分化,促进分化的神经元、少突胶质细胞突起的生长.     

小鼠放射状胶质细胞和小脑皮质的发育

目的探讨小鼠小脑皮质发育过程中放射状胶质细胞的分化。方法应用免疫荧光及5-溴脱氧尿嘧啶核苷(BrdU)检测技术,标记小鼠胚胎8d至生后180d小脑(57例,分为19组,每组3只)的神经干细胞、放射状胶质细胞、普肯耶细胞及颗粒细胞。结果放射状胶质细胞于胚胎13d的神经上皮出现,尔后该细胞分化为各种神经元和贝格曼胶质细胞,并在小脑皮质层状结构的形成中起着重要作用。结论放射状胶质细胞来源于神经上皮细胞,是神经细胞和神经胶质细胞的前体细胞。在小脑皮质的发育过程中,放射状胶质细胞能分化为普肯耶细胞和颗粒细胞,并为神经细胞的迁移提供路径和支架。     

碱性成纤维细胞生长因子和表皮生长因子联合诱导骨髓基质细胞分化形成神经球

目的观察碱性成纤维细胞生长因子(bFGF)和表皮生长因子(EGF)联合诱导骨髓基质细胞(MSCs)能否分化形成神经球。方法采用全骨髓法培养MSCs,bFGF和EGF联合诱导MSCs,倒置显微镜下观察分化细胞的形态,免疫荧光法和免疫印迹法鉴定分化细胞。结果 bFGF和EGF联合应用,可有效地诱导MSCs形成神经球。此神经球能传代并分化成神经元样细胞。免疫荧光和免疫印迹法均证实神经球表达nestin蛋白,神经球分化的细胞表达神经元、胶质细胞和少突胶质细胞标志性蛋白。结论 bFGF和EGF联合应用,可有效地诱导MSCs形成神经球。此神经球能自我更新并分化为神经元、胶质细胞和少突胶质细胞。     

Immunohistochemical characterization of the rat carotid body

We studied the histochemical phenotype of carotid body (CB) cells in the adult rat. In addition to tyrosine hydroxylase (TH), type I cells expressed numerous growth factors such as glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), as well as the receptors p75, Ret, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-alpha (PDGFR-alpha). Type II cells expressed the glial fibrillary acid protein (GFAP), vimentin, the trophic factor bFGF and receptors p75, EGFR and PDGFR-alpha. Both types I and II cells exhibited a positive immunoreaction to markers of neural progenitor cells such as the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) and nestin, respectively, suggesting that CB contain some immature cells even at the adult stage. The possibility that these cells can be expanded and differentiated into mature neurons should be explored.     

大鼠胚胎神经上皮细胞侧脑室移植后存活及分化的实验研究

目的：观察大鼠胚胎神经上皮细胞同种脑移植后的存活及生长分化状况。方法：孕12d大鼠剖腹取胚胎，剥离神经管，胰酶消化后获取神经管壁上的神经上皮细胞，然后植入其父体侧脑室中，分别于移植后10d,14d,21d,28d灌注取脑，肜HF染色及神经元特异烯醇化酶（NSE），胶质纤维酸性蛋白（GFAP）免疫组织化学方法检测胚胎神经上皮细胞移植后的存活及分化状况。结果：神经上皮细胞侧脑室移植后多贴附于脑室壁形成细胞团块，有的随脑脊液循环至第三脑室生长，随时间延长移植物增大，HE染色见脑室内有成团的移植细胞，免疫组织化学染色显示移植细胞团内既有NSE－免疫阳性细胞，也有GFAP－免疫阳性细胞，移植物周围多为GFAP－免疫阳性细胞。结论：胚胎神经上皮细胞侧脑室移植后能够贴附脑室壁存活，并能分化为神经元和神经胶质细胞。     

小鼠大脑皮质发育过程中神经细胞、胶质细胞及血管之间的相互关系

目的探讨小鼠大脑皮质发育过程中神经细胞、胶质细胞及血管发生之间的关系。方法应用免疫荧光、5-溴脱氧尿嘧啶核苷(Brd U)和墨汁血管灌注等技术,对胚胎受孕15d(E15)至生后90d(P90)小鼠大脑内神经干细胞、神经前体细胞、神经胶质细胞和血管进行形态学观察。结果 E15左右大脑皮质内开始出现血管网,随着年龄的增长血管分支逐渐增多,血管体密度呈现增长趋势;成年后(P60)血管密度基本稳定。大脑皮质部位小血管走行方向与放射状胶质细胞长突起延伸方向一致,呈平行分布;大量放射状胶质细胞伸出膨大的足板参与血脑屏障的构筑;增殖的神经干细胞主要沿着血管走行方向及放射状胶质细胞长突起延伸方向进行迁移。结论在大脑皮质发育过程中,神经干细胞不断增殖,并沿着血管走行方向及放射状胶质细胞长突起延伸方向进行迁移。神经细胞、神经胶质细胞参与血管壁的构成,三者之间相互诱导,共同维持血脑屏障的完整与功能调节。