脉冲电场对人红细胞膜结构影响的冷冻断裂研究

对外加脉冲电场处理的人红血球冷冻断裂和蚀刻的复型观察中发现在强电场作用下,细胞周围有颗粒状和纤维状结构。结合ＳＤＳ电泳分析证明了它们是由于在电场作用下,红血球膜的带３蛋白和膜骨架蛋白（血影蛋白）脱出的结果。在强电场作用下,由于膜蛋白和膜骨架蛋白的脱出造成了对细胞膜的损伤,使细胞膜稳定性降低,细胞易变形和形成伪足。由于膜蛋白的脱出,多余的自由脂进入细胞质内而形成泡状结构。外电场改变了蛋白－蛋白以及蛋     

人早幼粒白血病细胞(HL-60)的表面超微结构及其在诱导分化过程中的变化

应用扫描电镜技术,结合超薄切片和组织化学方法,观察分析了人早幼粒白血病细胞(HL-60)的表面形态结构。扫描电镜下,HL-60细胞最明显的表面形态特征是绝大多数(约90%)具有丝状伪足。丝状伪足呈丝状、指状或圆棒状,其平均长度为4—6μ,直径约为0.25—0.3μ。丝状伪足具有明显特征性分布,常以成簇或成束状态分布在细胞的周围或一侧。超薄切片和组织化学反应结果显示,丝状伪足的内部结构主要是微丝和糖原颗粒。大多数HL-60细胞具皱褶而极少数细胞显示微绒??毛。用维生素A酸处理HL-60细胞6天后,有些细胞的丝状伪足消失,其细胞表面变得不规则,出现大型钝形伪足。由于丝状伪足与大型钝形伪足的成分基本相同,以及钝形伪足的出现伴随丝状伪足的减少,故推测钝形伪足可能来自丝状伪足。文中讨论了HL-60细胞形态结构与生理功能的关系。     

感染环形泰勒焦虫(Theileria annulata)的牛外周血白细胞的形态观察

用光镜及扫描电镜观察了体外高代培养的含牛焦虫颗粒的牛外周血白细胞的形态及在细胞周期中细胞表面的特征性变化。这种经多年传代的含虫的牛外周血白细胞恢复了分裂和繁殖的能力,目前已成为较稳定的细胞系。细胞表面具多种伪足突起,如叶状、丝状及绒毛状。细胞周期中备期细胞表面的主要特征是:S期:细胞平扁,边缘具薄的时状伪足及丝状伪足;G_2期:细胞中部隆起,表面具少量绒毛状伪足;G_1期:绒毛状结构少或无,而出现丝状及小的叶状伪足,细胞仍保持球形;M期:细胞球形,表面密被以绒毛。作者根据扫描电镜的观察认为光镜下所观察的两类细胞,实际上是反映了一种细胞处于不同发育阶段时的特征。     

逆向溶血斑法检测小鼠睾丸间质细胞睾酮的分泌

用逆向溶血斑法检测单个小鼠睾丸间质细胞睾酮的分泌,为进一步研究单个睾丸间质细胞的结构和功能提供一种有效的途径,结果表明,分泌睾酮的睾丸间质细胞周围形成空斑,空斑面积随睾酮分泌增加而增大,说明只要获得抗体,逆向溶血斑法就可以检测任何细胞的分泌物。     

黏着斑激酶与细胞迁移

细胞迁移过程始于细胞前端板状伪足的形成、外周黏附的建立、细胞体的收缩和尾部的解离.黏着斑激酶是一种非受体酪氨酸蛋白激酶,通过其激酶活性和"脚手架"的功能在细胞迁移的各个过程中发挥关键作用.现重点介绍黏着斑激酶介导的信号转导通路及其在调控细胞迁移方面的研究进展.     

肿瘤细胞与肿瘤微环境间的相互作用

在肿瘤组织中,除了肿瘤细胞外还有其他成分包括间质细胞和细胞外基质(ECM),这些共同构成肿瘤微环境。这些间质细胞包括:成纤维细胞、内皮细胞、炎症细胞、脂肪细胞等。间质细胞的分布、细胞外基质的组成和代谢产物在同一肿瘤中明显不同。肿瘤细胞周围的间质细胞状态对肿瘤的转移产生重要影响,甚至可以用来预测肿瘤复发。肿瘤的发生发展包括众多成分之间的相互作用,本文论述了这一复杂网络的各个组成部分。     

脉冲电场对人红细胞膜结构影响的冷冻断裂研究

对外加脉冲电场处理的人红血球冷冻断裂和蚀刻的复型观察中发现在强电场(3KV/cm)作用下,细胞周围有颗粒状和纤维状结构。结合SDS电泳分析证明了它们是由于在电场作用下,红血球膜的带3蛋白和膜骨架蛋白(血影蛋白)脱出的结果。在强电场作用下,由于膜蛋白和膜骨架蛋白的脱出造成了对细胞膜的损伤,使细胞膜稳定性降低,细胞易变形和形成伪足。由于膜蛋白的脱出,多余的自由脂质进入细胞质内而形成泡状结构。外电场改变了蛋白-蛋白以及蛋白-脂分子间的作用可能是电穿孔的主要机理。本文还对当前公认的冷冻断裂中所观察到的膜中间颗粒的来源提出了疑问,并提出了它们还可能与冰晶有关。而冰晶的形成又与膜的亲水与疏水性有关。     

黄芩苷通过降低活性氧生成抑制血管平滑肌细胞伪足形成和迁移北大核心CSCD

已知黄芩苷(baicalin)通过削弱肌动蛋白相关蛋白(actin-related protein,Arp)2/3复合物的活性抑制血管平滑肌细胞(vascular smooth muscle cell,VSMC)伪足形成和迁移,然而,其抑制该信号途径的机制尚不明确。本研究证明,黄芩苷通过抑制VSMC活性氧(reactive oxygen species,ROS)生成降低Arp2/3活性,发挥阻止细胞伪足形成和迁移的功能。分别利用TRITC-鬼笔环肽和ROS荧光探针标记VSMCs,结果显示,黄芩苷能显著抑制血小板源性生长因子(platelet-derived growth factor,PDGF)-BB诱导的VSMC伪足形成和迁移,伴有ROS生成减少。用超氧物歧化酶(superoxide dismutase,SOD)清除胞内过氧化物后,PDGF-BB引发的VSMC伪足形成被逆转,且该过程与降低皮层肌动蛋白微丝(F-actin)成核蛋白Arp2/3活性有关。免疫沉淀分析结果进一步表明,黄芩苷降低p47phox磷酸化水平,与ROS生成减少相一致。体内的实验也表明,黄芩苷(70 mg/kg/d)能有效抑制球囊损伤诱导的大鼠颈总动脉ROS生成。以上结果表明,黄芩苷通过抑制NADPH氧化酶介导的ROS生成,降低细胞皮质区F-actin成核活性,阻止细胞伪足形成、迁移,进而发挥血管保护作用。     

BjAB细胞感染Epstein-Barr病毒后表面形态的变化

在扫描电镜下观察BjAB细胞在感染EB病毒以后表面形态的变化。未受感染的BjAB细胞以表面布满丝状伪足者最多见。感染1天后,部分细胞丝状伪足缩短变粗,另部分则丝状伪足几乎全部消失,细胞表面略现皱褶和分布着疏散的“手指样”结构。感染后3天时细胞突出的变化是表面皱褶更明显,更多见;而且开始看到表面泡状结构。这种泡状结构的细胞在感染5天时数量增多,而7天后大部分细胞已呈“泡样瘤”状。观察感染后5个月的细胞,这种表面结构仍保持不变。由于用灭活的EB病毒在相同条件下感染这种细胞,表面形态未见改变,所以这种表面形态的变化应与EB病毒感染,并设想同EB病毒基因组的存在和表达有关。     

监测活细胞中诱导激活的Rac、Cdc42信号转导通路的FRET单分子探针

Rho家族鸟苷三磷酸酶,包括Rac1和Cdc42等．参与调节细胞形态、细胞迁移、转录激活和基因表达等一系列细胞过程。根据FRET（Fluorescent resonance energy transfer）技术原理,构建了包含红色荧光蛋白dsRed1与青色荧光蛋白ECFP的全长cDNA．效应分子Pak1或N—WASP的GTP酶联结区域．信号分子Rac1或Cdc42的全长cDNA的几种单分子探针。转染NIH3T3或Hela细胞,以胰岛素、缓激肽为诱导剂,分别激活Rac1、Cdc42信号转导通路。离体荧光光谱检测表明．在两种转染动物细胞中均产生了FRET现象。不同信号转导通路的FRET效率,在诱导激活5min后,均达到最高值,但增加幅度有显著差异。随着诱导时间的延长,FRET效率下降,但下降速率在不同信号转导通路间差异显著。Rac1、Cdc42激活试验证实．诱导激活的转染细胞中,Rac1、Cdc42均处于激活状态（GTP—bound）,其在不同诱导时间的相对激活程度与FRET效率表现相同。诱导激活的Rac1、Cdc42信号转导通路,分别导致了转染活细胞中片状伪足、线状伪足的产生。这表明,采用这些单分子探针,可直接监测激活的信号转导通路,在活细胞中的3D时空分布变化图像及其产生的细胞学效应。采用这些单分子探针．分析、判断了一些调节蛋白对Rac1、Cdc42的GEF或GAP特性。从而提供一种可大大简化现有的鉴定待测蛋白分子的方法。     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

Behavior and Ultrastructure of Primary Mesenchyme Cells at Sessile Site during Termination of Cell Migration in Early Gastrulae

The behavior and ultrastructure of primary mesenchyme cells at two ventrolateral sessile sites in early gastrulae were examined by time-lapse videomicroscopy, scanning electron microscopy, and immunotrans-mission electron microscopy using the sea urchin, Hemicentrotus pulcherrimus and the sand dollar. Clypeaster japonicus . At sessile sites in early gastrulae, PMCs terminated their migration after "touch-and-go" behavior, and even after the termination they retained a pulsatile movement. These behaviors indicate that the termination of PMC migration is not due to deprivation of cell motility nor the establishment of firm adhesion between PMCs and the site. PMCs used short cell processes during migration, and extended longer ones during the early period of migration termination. During the final period of migration at the sessile sites, PMCs extended characteristically thin and long cell processes to the basal lamina. These cell processes, as far as present results indicate, never attach to the blastocoel wall cells through the basal lamina. Thus it is indicated that the primary interaction site for PMCs to terminate their migration is the basal lamina.     

Changes in the morphology of the germinal dense bodies in primordial germ cells of the teleost,Oryzias latipes

Summary In many organisms, the germinal dense bodies (GDBs) are known to be organelles unique to the cells of germ-line. In the present study, GDBs in primordial germ cells (PGCs) of the teleost, Oryzias latipes, were examined by electron microscopy. An obvious change was noticed in the morphology of GDBs. In PGCs situated in the endoderm, GDBs consisted of a loosely woven strand-like structure, whereas, GDBs in PGCs in the gonadal anlage, which were amorphous bodies of various sizes and shapes, were composed of electron-dense fine fibrils. The changes in the morphology of GDBs proceeded gradually according to the progress of the stages in migration of the PGCs. GDBs of intermediate morphology were found. The change in the morphology of the GDBs began at the stage of movement of the PGCs from endoderm to mesoderm. It is suggested that the differentiation of PGCs proceeds during their migratory stages under the influence of surrounding somatic cells.     

蚕豆花粉母细胞染色体畸变同染色质胞间转移的关系

采用压碎、石蜡切片及植物染色体分带技术观察蚕豆花粉母细胞减数分裂过程发现:1.蚕豆花粉母细胞减数分裂的凝线期也出现染色质胞间转移现象;2.在8.08%的花粉母细胞内,染色体偏离正常的数目(n=6);3.染色体结构也有显著改变,其中包括染色体断裂、桥和长度的增减;4.出现双核和无核的花粉母细胞。大量证据表明,这些畸变现象同凝线期的染色质胞间转移有关。     

Developmental regulation of locomotive activity in Xenopus primordial germ cells

Primordial germ cells (PGCs) arise in the early embryo and migrate toward the future gonad through species‐specific pathways. They are assumed to change their migration properties dependent on their own genetic program and/or environmental cues, though information concerning the developmental change in PGC motility is limited. First, we re‐examined the distribution of PGCs in the endodermal region of Xenopus embryos at various stages by using an antibody against Xenopus Daz‐like protein, and found four stages of migration, namely clustering, dispersing, directionally migrating and re‐aggregating. Next, we isolated living PGCs at each stage and directly examined their morphology and locomotive activity in cell cultures. PGCs at the clustering stage were round in shape with small blebs and showed little motility. PGCs in both the dispersing and the directionally migrating stages alternated between the locomotive phase with an elongated morphology and the pausing phase with a rugged morphology. The locomotive activity of the elongated PGCs was accompanied by the persistent formation of a large bleb at the leading front. The duration of the locomotive phase was shortened gradually with the transition from the dispersing stage to the directionally migrating stage. At the re‐aggregating stage, PGCs became round in shape and showed no motility. Thus, we directly showed that the locomotive activity of PGCs changes dynamically depending upon the migrating stage. We also showed that the locomotion and blebbing of the PGCs required F‐actin, myosin II activity and RhoA/Rho‐associated protein kinase (ROCK) signaling.     

大鼠原生殖细胞培养和分化的研究

研究大鼠胚胎原生殖细胞（primordial germ cells,PGCs)的培养及分化,取受精后11-12.5天大鼠PGCs进行原代培养,光、电镜观察PGCs及其分化细胞的微细结构,碱性磷酸酶染色检测细胞的分化程度,结果显然显示大鼠PGCs大而圆,散在分布,或多个聚集成团,胞质中含有椭圆形的线粒体和丰富的核糖体,在鼠胚成纤维细胞饲养层存在的情况下,PGCs保持未分化状态,碱性磷酸酶反应呈强阳性,在缺乏饲养层的条件下PGCs很快分化,形态不规则,有伪足,碱性磷酸酶反应减弱,进一步分化可形成具有细长突起的神经元样细胞,胞质中含有细丝束的表皮细胞,可见节律性跳动的心肌细胞,具有分泌颗粒的分泌细胞及似血管,心脏形状的管腔结构等,由PGCs分化来的细胞碱性磷酸酶反应均呈阴性,结果表明大鼠PGCs能够分化形成三个胚层的衍生物,生殖嵴来源的PGCsp是一种具有发育全能性的胚胎多能干细胞,本研究同时证明鼠胚饲养层能抑制大鼠PGCs的分化。     

Polymorphous granular cells in the lung of the primitive fish,the bichir Polypterus senegalus

This study describes the distribution and the coexpression of specific neurochemical markers in both neuroendocrine‐like cells (NEC‐like) and polymorphous granular cells (PGCs) that populate the mucociliated epithelium of the lung in the air‐breathing fish Polypterus senegalus, using confocal immunohistochemistry. Using confocal immunohistochemistry, we determined the coexpression of specific neurochemical markers. Colocalization studies showed that 5HT is coexpressed with calbindin and nNOS and choline acetyltransferase (ChAT) is coexpressed with nNOS in the PGCs. This study also shows for the first time the simultaneous occurrence of piscidin 1 and 5HT in the PGCs. The function of these cells being equivalent to ones found in fish gill subepithelial parenchyma is still not known. Due to the importance of piscidin 1 in local immune defence, more research is to be useful to understand a possible interaction of PGCs with immune response in the bichir lung. In fact, the capacity of PGCs to produce NO and other neuroactive substances found in immune cells of fish may represent a primitive form of immunoregulation of innate immunity and specifically antimicrobial function as NO induction and respiratory burst activity are correlated with immune response.     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

Altered primordial germ cell migration in the absence of transforming growth factor beta signaling via ALK5

Transforming growth factor beta (TGFbeta) inhibits proliferation and promotes the migration of primordial germ cells (PGCs) towards explants of gonadal ridges in vitro. However, its effects in vivo are still unclear. Here, we analyzed the behavior of PGCs in embryos lacking TGFbeta signaling via the type I receptor ALK5. TGFbeta in vivo was neither a chemoattractant for PGCs, nor did it affect their proliferation during migration towards the gonadal ridges up to embryonic day (E)10. Unexpectedly, the absence of TGFbeta signaling in fact resulted in significant facilitation of PGC migration out of the hindgut, due to the reduced deposition of collagen type I surrounding the gut of Alk5-deficient mutant embryos. Migratory PGCs adhere strongly to collagen; therefore, reduced collagen type I along the gut may result in reduced adhesion, facilitating migration into the dorsal mesenterium and gonadal ridges. Our results provide new evidence for the role of TGFbeta signaling in migration of PGCs in vivo distinct from that described previously.     

Interactions between Germ Cells and Extracellular Matrix Glycoproteins during Migration and Gonad Assembly in the Mouse Embryo

Cells are known to bind to individual extracellular matrix glycoproteins in a complex and poorly understood way. Overall strength of adhesion is thought to be mediated by a combinatorial mechanism, involving adhesion of a cell to a variety of binding sites on the target glycoproteins. During migration in embryos, cells must alter their overall adhesiveness to the substrate to allow locomotion. The mechanism by which this is accomplished is not well understood. During early development, the cells destined to form the gametes, the primordial germ cells (PGCs), migrate from the developing hind gut to the site where the gonad will form. We have used whole-mount immunocytochemistry to study the changing distribution of three extracellular matrix glycoproteins, collagen IV, fibronectin, and laminin, during PGC migration and correlated this with quantitative assays of adhesiveness of PGCs to each of these. We show that PGCs change their strength of adhesion to each glycoprotein differentially during these stages. Furthermore, we show that PGCs interact with a discrete tract of laminin at the end of migration. Closer analysis of the adhesion of PGCs to laminin revealed that PGCs adhere particularly strongly to the E3 domain of laminin, and blocking experiments in vitro suggest that they adhere to this domain using a cell surface proteoglycan.