表皮生长因子对兔眼角膜修复作用的量效关系研究

用角膜表皮刮磨方法人工造成兔眼角膜创伤直径为10mm的圆形创面，分析小鼠表皮生长因子（EGF）对其修复的剂量关系。5个浓度EGF（100、50、20、10和0．5μm／ml）治疗的试验组痊愈时间均比对照组提前，有非常显著的差异（P＜0.01），但在这5个浓度组之间，痊愈时间无显著性差异。结果表明，小鼠EGF对兔眼角膜损伤有促修复作用，实验所用的5个浓度与修复作用无剂量依赖关系。

猕猴胚胎干细胞的诱导分化和凋亡

采用单层培养法研究维生素A酸(RA)、神经生长因子(NGF)、上皮生长因子(EGF)和碱性成纤维生长因子(bFGF)对猕猴胚胎干细胞系R366.4的诱导分化和凋亡的作用. 结果表明: ①不添加任何生长因子的条件下, 细胞分化不定向, 各种细胞所占的比例表现出明显的随机性; ②添加单一生长因子能促进细胞的分化进程, 并使某一类或某几类的分化细胞比例上升, RA 和 NGF 均能促进神经样细胞的形成, EGF 促进内皮样细胞的形成, bFGF 提高成纤维样细胞的比例; ③在分化的过程中伴有细胞早期和晚期凋亡的发生, RA 和 NGF 可增加细胞凋亡的数量. 这种由生长因子诱导的动物胚胎干细胞的分化可能存在种间差异。

人胚胎神经干细胞的分离培养及鉴定研究

目的:研究人胚胎神经干细胞的培养条件及体外分化情况。方法:从12周龄人胚胎脑皮质分离细胞,采用无血清培养技术,协同应用碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和重组人白血病抑制因子(rhLIF)进行培养;5-溴脱氧尿嘧啶核苷(BrdU)标记检测细胞的增殖能力,间接免疫荧光化学法检测细胞的分化情况。结果:培养得到的大量半悬浮生长的神经干细胞球能够传代培养;BrdU标记阳性,可诱导分化为神经元、星形胶质细胞和少突胶质细胞。结论:人胚胎脑皮质分离细胞培养得到的细胞群具有神经干细胞的基本特征,可进一步用于基础及临床研究。

表皮生长因子对恒河猴创伤眼角膜的促恢复作用

人工造成恒河猴眼角膜创伤, 并除去双眼角膜上皮细胞层, 仅边缘残留少量角膜细胞, 右眼作实验处理, 左眼对照, 分别每日滴3次表皮生长因子(EGF)溶液于右眼, 滴3次生理盐水于 左眼。每日用荧光素钠溶液滴眼。结果实验眼的创面在3-4d完全恢复, 对照眼在5-6d恢复。表明表皮生长因子对恒河猴创伤眼角膜的恢复具有明显的促进作用。图1参11

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

树鼩原代肝细胞培养体系和乙型肝炎病毒体外感染模型的建立

乙型肝炎病毒（Hepatitis Virus type B, HBV）感染是一种严重威胁人类健康的世界性问题。由于HBV感染具有宿主和组织特异性，长期以来一直缺乏有效的体外感染模型，严重制约了HBV感染机制和治疗药物的研究。树鼩（Tree Shrews, Tupaia Belangeri）与人和灵长类亲缘关系较为接近，作为小动物模型在乙型肝炎研究中备受关注。但由于各种原因的限制，HBV树鼩感染模型仍处于“国际公认但实际短缺，国内首创但实际空缺”的状况。因此，复建和优化HBV树鼩体内外感染模型成为我们长期研究方向所必备的前提基础和开端。而树鼩原代肝细胞的分离和培养则是建立HBV体外感染模型的关键的第一步。我们通过与机械分离法的直接比较，验证了两步灌注法在树鼩肝细胞分离中的优越性。进而发现，在分离后的体外培养过程中，二甲基亚砜不仅能够促进和维持原代肝细胞的分化，而且能够显著地抑制纤维状细胞群的出现。同时，肝细胞生长因子（HGF）和表皮生长因子（EGF）能够促进肝细胞在体外长期存活。在此优化的条件下，原代培养可持续4-5周，并且较多的细胞聚集形成类似肝窦结构的形态，从而成功地复制和改进了树鼩原代肝细胞培养体系，为建立HBV体外感染模型提供了基础。与此同时，我们采集了2种来源的HBV，即以乙肝病人血清和HepG2.2.15细胞培养上清为来源。我们采用蔗糖浓度梯度离心法以纯化病人血清病毒，能较好地将HBV颗粒和亚病毒颗粒区分开来。在收集HepG2.2.15细胞株来源的病毒时，我们详细研究了HepG2.2.15细胞的生长曲线和病毒产量曲线，找到病毒产量与细胞生长状态之间的联系，确定了最佳收集病毒时间，并通过放大培养条件，大量收集HepG2.2.15细胞的培养上清。我们采用浓缩和未浓缩的HepG2.2.15病毒、纯化的血清病毒等3种方式制备的病毒感染树鼩原代肝细胞，通过不同水平的指标来检测感染结果。首先，细胞内HBVx基因mRNA的检测结果表明，3种来源的病毒都可以感染树鼩原代肝细胞。同时，细胞上清中分泌的病毒颗粒及病毒蛋白的检测结果表明，HepG2.2.15病毒感染树鼩原代肝细胞的效果比血清病毒的感染效果好。免疫荧光的检测结果进一步证实了HepG2.2.15病毒能够感染树鼩原代肝细胞。因此，在我们的实验中，树鼩原代肝细胞对2种来源和不同制备方式的病毒均具有易感性，且以未浓缩的HepG2.2.15上清病毒感染力优于血清病毒。综上所述，我们现有的结果验证了树鼩原代肝细胞对HBV具有易感性。就感染效率而言，尽管感染效率依赖于病毒滴度及其感染力，我们在病毒细胞比例为0.02:1的条件下，仍能确认易感性，并获得了与国际同类研究相当的感染效率。就本研究的应用前景而言，我们所建立的HBV体外感染模型，能够作为实验室的通用平台，用以来研究HBV的感染机制和抗病毒药物的筛选与评价。

猕猴多潜能成体肝前体细胞及猕猴ES 定向分化为胆管上皮细胞的研究

为解决供体器官的不足，以细胞移植为基础的替代疗法已成为治疗不可逆肝 脏疾病新的希望。 肝前体（干）细胞（Hepatic progenitor cellS，HPCs）和 胚胎干细胞（embryoic stem cells, ES）由于其特殊的细胞特性已成为细胞替 代治疗理想的种源细胞。 然而一方面包括人在内的灵长类动物的正常成体肝来 源的HPCs 的分离依然是很困难的；另一方面，ES 细胞来源的肝细胞和胆管细胞 的生成效率依旧很低。因此有必要建立稳定的高效的灵长类动物HPCs 细胞分离 培养体系及ES 细胞的肝细胞或胆管细胞分化体系以满足供体细胞的不足；这种 体系的建立还有利于研究肝细胞生物学如分化机制、自我更新机制等方面的重要 基础问题。 本研究以猕猴为实验模型，研究了正常成体肝来源的猕猴HPCs 分离、纯化 的条件，系统地鉴定了猕猴HPCs 的细胞特性和体内、外分化潜能，并评价了体 内移植效果。 同时以rES 为材料，建立了rES 高效分化为限定性内胚层 （definitive endoderm cells, DE）和胆管上皮细胞的分化体系。主要实验结 果包括：1）： FBS、EGF、HGF 及rat tail collagen (鼠尾胶原)是分离培养正 常成体猕猴来源的肝上皮前体细胞(rhesus monkey liver epithelial progenitor cells, mLEPCs)所必需的，mLEPCs 在此培养体系中至少可以扩增20 代或5 个月以上，并仍然保持原有的细胞特性；mLEPCs 呈现典型的上皮细胞形 态，并表达HPCs 细胞特有的表达模式即同时表达肝细胞和胆管细胞相关基因 （ALB，APOH，CX43，IB4）或蛋白（CK7，CK8，CK18）；在适宜的分化体系下， mLEPCs 可分化为功能性的肝细胞，形成具有胆管上皮细胞的胆管样结构，并能 转分化形成肌肉样细胞、肌样成纤维细胞及少突样细胞；移植入肝损伤的免疫抑 制的小鼠体内后，mLEPCs 能参与受体肝组织的再生，并能分化成ALB 阳性的肝 细胞；体内定位发现mLEPCs 与胆管区的细胞有相似的免疫原性，提示mLEPCs 可能来源于胆管区。2）：rES 在高浓度的acitvin A（100ng/ml）和低浓度的血 清(1%)单层诱导体系下可定向分化得到高比率的限定性内胚层细胞（definitive endoderm cells，DE 细胞）(约80%)； 高比率的DE 细胞的得到还与rES 细胞的接种密度相关；BMP4 和FGF1 可诱导DE 细胞高效向胆管上皮细胞分化（约90%）， 但并不能得到肝细胞；而Notch 信号通路可维持DE 细胞的存活，并决定着DE 细胞向胆管细胞分化，在Notch 信号通路失活的情形下，即使存在BMP4 和FGF1 都不能促使DE 细胞向胆管细胞分化。 本实验首次成功建立了正常猕猴成体肝HPCs 分离培养体系，证实了分离得 到的猕猴肝上皮前体细胞不但具有正常HPCs 的增殖活力和参与受体肝组织的再 生能力，而且还具有三个胚层的分化潜能，这一结果将为以HPCs 为基础的细胞 替代治疗人类肝脏疾病的实现提供了可能，并首次证明了HPCs 也可以像某些少 数成体干细胞一样具有三个胚层得分化潜能。 此外，本研究建立了rES 高效定 向分化为DE 细胞和胆管细胞的分化体系，这一方法的建立将促进灵长类动物的 DE 细胞的发育机制研究，同时也可为高比率的内胚层功能细胞（如胰岛细胞、 肝细胞、肺细胞）的获得提供丰富的种源细胞和平台。

猕猴胚胎干细胞无饲养层、无血清培养及诱导分化成O2A 胶质前体细胞的研究

灵长类胚胎干细胞（ES 细胞）不仅能为研究生殖发育生物学基础理论提供良好的 模型，而且可为细胞替代治疗提供大量的供体细胞，因此具有重要的研究价值。当前 灵长类ES 细胞研究还有很多问题需要解决，如分离建立更多的胚胎干细胞系，优化培 养体系，提高ES 细胞定向分化为特定细胞的比例，研究ES 细胞自我更新和分化的机 制等。本文一方面概括了灵长类ES 细胞的研究进展，另一方面并对制备抗体，免疫外 科手术法分离灵长类胚胎内细胞团，建立猕猴ES 细胞的无饲养层、无血清培养体系和 诱导猕猴ES 细胞分化成高纯度的O2A 神经胶质前体细胞进行了研究。主要结论如下： 1）分别以猕猴脾脏淋巴细胞和人外周血单个核细胞作为免疫原，免疫日本大耳白兔， 得到免疫血清。在补体介导的细胞毒作用下，兔抗人和兔抗猕猴免疫血清可以裂解人 和猕猴囊胚滋养层细胞，从而分离出内细胞团，用于分离培养人和猕猴胚胎干细胞。2） 猕猴ES 细胞在以层粘连蛋白（laminin）为胞外基质，含转化生长因子beta1（TGFβ1） 的无血清培养基（SFM）中可以稳定的增殖至少22 代，保持不分化，并具有分化成三 个胚层细胞的能力。进一步的研究发现去除TGFβ1 后，猕猴ES 细胞出现分化，整合 素表达降低，推测TGFβ1 可能通过促进猕猴ES 细胞整合素的表达，加强其与胞外基 质的相互作用，从而维持ES 细胞的自我更新。然而猕猴ES 细胞不能在纤粘连蛋白 （fibronectin）和明胶上生长。3）无饲养层、无血清培养体系中长期培养的猕猴ES 细 胞，分化出拟胚体，14 天的拟胚体在血清中分化培养一周后，在含碱性成纤维生长因 子bFGF、表皮生长因子EGF 和胰岛素＋转铁蛋白＋亚硒酸钠ITS 的培养基中培养， 获得97％的O2A 胶质前体细胞，得到的O2A 细胞能够稳定增殖，并且可以自发分化 为II 型星型胶质细胞和少突胶质细胞。本实验的结果有助于猕猴ES 细胞分离建系和培 养系统的优化、推动猕猴ES 细胞自我更新和诱导为神经胶质细胞机制的研究，便于建 立ES 细胞替代治疗的猕猴模型，从而为人类ES 细胞的临床疾病治疗提供参考。

P-选择素CR结构域功能的模拟预测

<正>选择素(selectin)与其配体间相互作用介导的细胞粘附在炎症级联反应、肿瘤转移和淋巴细胞归巢等病理、生理过程中起重要作用。其结构组成从远离细胞表面的N-末端到胞内C-末端,依次为:钙型凝集素功能区(Calcium-type lectin domain,Lec),类上皮生长因子功能区(Epidermal Growth Factor-like module,EGF),具有粘附补体蛋白作用的多个重复序列(Consensus Repeats,CRs),跨膜区(Transmembrane,TM)和胞内区

来自易变山羊草抗禾谷孢囊线虫基因的克隆与序列分析

禾谷孢囊线虫（Heterodera avenae）是严重危害禾谷类作物的病原线虫之一,它广泛分布于澳大利亚、欧洲、北美、印度和中国等世界主要小麦产区,使作物严重减产,造成巨大的经济损失。目前最有效的防治措施之一是将外源抗性基因导入栽培小麦(Triticum aestivum L.)，培育抗禾谷孢囊线虫的新品种。但迄今为止抗禾谷孢囊线虫基因克隆研究的相关报道却很少。 本实验根据此前从抗禾谷孢囊线虫材料E-10扩增得到的与来自节节麦（Aegilops tauschii）的抗禾谷孢囊线虫基因Cre3高度同源的序列Rccn4,设计出三条嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法,从E-10基因组DNA中得到一个长为1264 bp的扩增产物(命名为Rccn-L),测序比对结果显示,这一序列将Rccn4的3’端延伸了1209 bp,与抗禾谷孢囊线虫Cre3基因核苷酸同源性为86﹪，核苷酸编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构,编码一个342个氨基酸残基的蛋白质。该蛋白质等电点为5.19，分子量为38112.6Da。从序列的第113位开始到第332位是NBS-LRR类抗病性基因LRR区,呈现XXLXXLXXL重复。LRR编码区内亮氨酸残基的含量达17﹪，与抗禾谷孢囊线虫Cre3基因LRR编码区的核苷酸和氨基酸同源性分别为89﹪和78﹪。本实验首次将SON-PCR成功地运用于植物基因克隆，为植物基因克隆提供了又一有效方法。 此外,还根据Cre3基因及其他的NBS-LRR类植物抗性基因的NBS和LRR区保守序列设计了两对特异性引物，从禾谷孢囊线虫抗性材料易变山羊草基因组DNA中扩增到两个相应的目标条带。测序分析结果表明，它们的长度分别为532bp和1175bp，构成了一个有32bp的共同序列的NBS-LRR编码区。其序列总长为1675bp（命名为RCCN），含有一个不完整的开放阅读框，没有起始密码子、终止密码子和内含子结构。其中编码序列为1673bp，可编码一个557个氨基酸的蛋白质，等电点（pI）为5.39，分子量为63537.5Da。与Cre3的核苷酸和氨基酸同源性分别为87.8﹪和77﹪。RCCN氨基酸序列中含有已知抗病基因NBS区域的几个保守模体：kinase2区的ILDD、kinase3的（ⅰ）ESKILVTTRSK，（ⅱ）KGSPLAARTVGG，（ⅲ）RRCFAYCS及EGF。RCCN NBS区与Cre3 NBS区的核苷酸和氨基酸的同源性分别为96.4﹪和94﹪。从氨基酸序列的274位到548位为LRR保守区，呈现不规则的aXXLXXLXXL（其中a代表I，V，L，F或M）重复，其中亮氨酸的含量为15.6﹪。该区域与Cre3的LRR区的核苷酸和氨基酸同源性分别为80.8﹪和74﹪。推测该序列可能为一个抗禾谷孢囊线虫的新基因。 本文对抗禾谷孢囊线虫基因的克隆研究，为进一步克隆基因全序列，探索其结构与功能，和研究该基因表达与调控提供了关键信息。同时也为通过基因工程途径将抗性基因向优良小麦品种高效、定向转移，最终培育出小麦抗禾谷孢囊线虫新品种奠定了基础。 Cereal cyst nematode (CCN) is a damaging pathogen of broad acre cereal crops in Australia, Europe, North America, India and China. It affects wheat, barley, oat and triticale and causes yield loss of up to 80%. At present, Transferring resistance genes against CCN into wheat cultivars and breeding varieties are considered one of the most effective methods for controlling the CCN. However, there are very limited reports concerning the cloning studies of resistance genes against the cereal cyst nematode. According to the sequence of Rccn4 which had high similarity to the nucleotide binding site (NBS) coding region of cereal cyst nematode resistance gene, Cre3， We designed three 3’ nested primers. Using single oligonucleotide nested PCR (SON-PCR) we successfully amplified one band, Rccn-L, of 1264bp from E-10 which is the wheat-Ae.variabilis translocation line containing the cereal cyst nematode resistance gene of Ae.variabilis. We found that this band of interesting is the 3’ flanking sequence of 1209bp in size of Rccn4. The coding region was 1026bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86﹪nucleotide sequence identity with Cre3. This peptide had a conserved LRR domain, containing the imperfect repeats,XXLXXLXXL, which contains 17﹪ leucine residues and shares, respectively, 89﹪ nucleotide sequence and 78﹪ amino acid sequence identity with the LRR sequence of Cre3 locus. This research firstly used SON-PCR in the research of plant genome successfully, which indicated that SON-PCR is another method of cloning plant gene. At the same time, According to the conversed motif of NBS and LRR region of cereal cyst nematode resistance gene Cre3 from wild wheat (Triticum tauschlii L.) and the known NBS-LRR group resistance genes, we designed two pairs of specific primers for NBS and LRR region respectively. One band of approximately 530bp was amplified using the specific primers for conversed NBS region and one band of approximately 1200bp was amplified with the specific primers for conversed LRR region. After sequencing, we found that these two sequences included 32bp common nucleotide sequence and have 1675 bp in total, which was registered as RCCN in the Genbank. RCCN contained a NBS-LRR domain and an incomplete open reading frame without initiation codon, terminator codon and inxon. Its exon encodes a peptide of 557 amino acid residues. The molecular weight of the protein from the amino acid was 63.537 KDa. The amino acid sequence of RCCN contained conserved motif: ILDD, ESKILVTTRSK, KGSPLAARTVGG, RRCFAYCS, EGF，LRR. RCCN shares 87.8﹪ nucleotide sequence and 77﹪ amino acid sequence identity with cereal cyst nematode gene Cre3. It might be a novel cereal cyst nematode resistance gene. These research results of cloning the resistance genes against cereal cyst nematode bring a great promise for transferring resistance genes into wheat cultivars and breeding new wheat varieties against cereal cyst nematode by gene engineering. And these results also lay the hard foundation for the expressing researches of these genes.

A Tissue-Engineered Microvascular System to Evaluate Vascular Progenitor Cells for Angiogenic Therapies

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.

Tyrosine phosphorylated SOCS-3 inhibits STAT activation but binds p120-RasGAP and activates Ras

Suppressors of cytokine signalling (SOCS, also known as CIS and SSI) are encoded by immediate early genes that act in a feedback loop to inhibit cytokine responses and activation of 'signal transducer and activator of transcription' (STAT). Here we show that SOCS-3 is strongly tyrosine-phosphorylated in response to many growth factors, including interleukin-2 (IL-2), erythropoietin (EPO), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). The principal phosphorylation sites on SOCS-3 are residues 204 and 221 at the carboxy terminus, and upon phosphorylation tyrosine 221 interacts with the Ras inhibitor p120 RasGAP. After IL-2 stimulation, phosphorylated SOCS-3 strongly inhibits STAT5 activation but, by binding to RasGAP, maintains activation of extracellular-signal-regulated kinase (ERK). A tyrosine mutant of SOCS-3 still blocks STAT phosphorylation, but also strongly inhibits IL-2-dependent activation of ERK and cell proliferation. Moreover, it also inhibits EPO- and PDGF-induced proliferation and ERK activation. Therefore, although SOCS proteins inhibit growth-factor responses, tyrosine phosphorylation of SOCS-3 can ensure cell survival and proliferation through the Ras pathway.