BALB／c小鼠胸腺的发生

用ＨＥ和Ｇｉｅｍｓａ染色的鼠胚连续切片观察ＢＡＬＢ／ｃ小鼠胸腺的发生。１１ｄ鼠胚第３对咽囊内胚层与第３对鳃沟外胚层相互紧贴，组成胸腺原基，并可见淋巴细胞样细胞迁入胸腺原基。１２ｄ鼠胚鳃沟外胚层包绕咽囊内胚层。１３ｄ胚胸腺原基已形成一个实体的细胞团。原基内淋巴母细胞样细胞增多，达细胞总数的１０％左右。１５ｄ胚胸腺皮、髓质已可大致辨认，皮质内淋巴母细胞样细胞明显多于髓质。１７～１９ｄ胚胸腺的皮、髓质分     

人巨细胞病毒单克隆抗体的研制检定和应用

采用人巨细胞病毒（ＨＣＭＶ）ＡＤ１６９株作为免疫原，制备出１３株鼠－鼠杂交瘤细胞系。对其中的６株进行了检定．免疫印迹试验结果表明：单克隆抗体（ＭｃＡｂ）７Ｂ４、７Ｄ７、７Ｅ１１、８Ｅ８和８Ｄ６相对应的ＨＣＭＶ多肽分子量分别为４６、１５０、３８、５１７２和６５ｋＤ．ＨＣＭＶ感染人胚肺二倍体细胞（２ＢＳ）后不同时间制成抗原片，与ＭｃＡｂ作间接免疫荧光试验。结果表明：ＭｃＡｂ８Ｂ８相应的病毒多肽为即刻早期抗原，其它５株ＭｃＡｂ相应的病毒多肽均为晚期抗原，６株ＭｃＡｂ等量混合后，标上辣根过氧化物酶，用于ＩｇＭ抗体捕获法ＥＬＩＳＡ（ＭａｃＥＬＩＳＡ）中，并与间接ＥＬＩＳＡ（ＩＥＬＩＳＡ）同时检测ＨＣＭＶ－ＩｇＭ．在未经选择的１００份脐带血中，两法均为阳性的３份，两法均为阴性的９４份；ＭａｃＥＬＩＳＡ阳性而ＩＥＬＩＳＡ阴性的２份血清的特异性试验证明，ＨＣＭＶ－ＩｇＭ确为阳性．ＩＥＬＩＳＡ阳性而ＭａｃＥＬＩＳＡ阴性的１份血清的特异性试验证明，它是由ＲＦ引起的假阳性。     

应用重组质粒pAM－HBsAg在果蝇细胞中表达乙型肝炎病毒表面抗原及基因免疫的初步研究

应用果蝇（ＤＳ２）表达系统，构建了含有乙型肝炎病毒表面抗原（ＨＢｓＡｇ）、摄金蛋白启动子（ＭＴｎ－ｐｒｏｍｏｔｅｒ）的共表达质粒ｐＡＭ－ＨＢｓＡｇ，转染细胞，经克隆，存活细胞株的培养上清液经硫酸铵沉淀、氯化铯（ＣＳＣｌ）密度梯度离心沉淀，获得的抗原用酶联免疫吸附试验（ＥＬＩＳＡ）、放射免疫分析（ＲＩＡ）法检测抗体，免疫吸印法（Ｗｅｓｔｅｒｎｂｌｏｔ）和电泳凝胶银染显色证实分子量为２３０００和２７０００，免疫电镜观察显示表达产物为２２ｕｍ球型颗粒，通过重金属离子（ＣｕＳＯ４、ＺｎＳＯ４）的诱导可增加抗原的表达量。用共表达质粒ｐＡＭ－ＨＢｓＡｇ的ＤＮＡ，注射Ｂａｌｂ／Ｃ小鼠的股四头肌。经ＥＬＩＳＡ、ＲＩＡ检测抗体产生情况，结果免疫后的小鼠经硫酸锌喂养抗体高于普通喂养的小鼠。Ｓｏｕｔｈｅｒｎ杂交证实鼠肌肉细胞存在ＨＢｓＡｇ基因。小鼠免疫接种实验表明，ＤＳ２细胞表达的抗原与直接用ＤＮＡ含有ＨＢｓＡｇ的重组质粒）免疫小鼠均获抗ＨＢｓＡｇ的抗体。     

心脏粘液瘤免疫组织化学观察和组织发生的探讨

对５２例心脏粘液瘤进行免疫组织化学观察。其中有２例伴有腺样结构。免疫标记显示：５２例粘液瘤中全部瘤细胞Ｖｉｍｅｎｔｉｎ为阳性。ＦＶⅢ，Ｄｅｓｍｉｎ和Ａｃｔｉｎ为阴性。５例瘤细胞Ｓ－１００蛋白为阳性。２例伴腺样结构者ＣＥＡ，ＥＭＡ和Ｃｙｔｏｋｅｒａｔｉｏｎ为阳性。组织化学染色显示：腺样结构ＰＡＳ－ＡＢ（ｐＨ１．０），ＰＡＳ－ＡＢ（ｐＨ２．５）和ＨＩＤ－ＡＢ均为阳性。结果提示：心脏粘液瘤可能来源于胚胎     

抗hER合成肽单克隆抗体的制备与鉴定

用合成的人雌激素受体（ｈＥＲ）抗原决定簇多肽（氨基酸序列从１５１～１６５）与ＫＬＨ的偶联物为抗原，免疫ＢＡＬＢ／Ｃ小鼠，经鼠－鼠杂交，ＥＬＩＳＡ和免疫细胞化学法筛选，有限稀释克隆化，得到一株分泌抗ｈＥＲ合成肽的杂交瘤细胞株（２Ｂ１２）。用ＥＬＩＳＡ法测定。该纯化抗体１０ｎｇ／ｍｌ仍与抗原反应，其免疫球蛋白为ＩｇＧ２ａ．ｋ。与ＭＣＦ－７乳腺癌细胞溶解蛋白的Ｗｅｓｔｅｒｎｂｌｏｔ分析，在分子量６７ｋＤ     

用一种新的免疫方法制备抗NPY单克隆抗体

本文报道了在８淋巴细胞杂交瘤技术中采用脾内微量免疫及腹腔植入联合免疫方法获得了两株分泌抗神经肽Ｙ（ＮＰＹ）单克隆抗体（ＭｃＡｂ）的杂交瘤细胞系，用ＥＬＩＳＡ法测定腹水ＭｃＡｂ的效价为１０￣（－３）～１０￣（－５）。ＥＬＩＳＡ交叉试验结果表明，该ＭｃＡｂ不与ＡＣＴＨ反应，两种ＭｃＡｂ的亲和常数（Ｋａ）各为１×１０￣９Ｍ￣（－１）和５×１０￣７ｍｏｌ／Ｌ￣（－１），抗原决定簇分析结果表明此两种ＭｃＡｂ针对不同抗原决定簇。     

蛋白激酶C抑制剂对去甲肾上腺素促进大鼠巨噬细胞I_a抗原表达的影响

我室以前的工作证明，在去甲肾上腺素（ＮＥ）促进大鼠巨噬细胞（ＭΦ）Ｉａ抗原表达的效应中，细胞内的第二信使物质三磷酸肌醇（ＩＰ－３）和Ｃａ￣２＋起着必不可少的作用。然而，另一第二信使物质甘油二酯（ＤＧ）的作用如何，尚待阐明。为此，我们用４α－佛　波醇－１２，１３－二癸酸盐（４α－ｐｈｏｒｂｏｌ－１２，１３－ｄｉｄｅｃａｎｏａｔｅ，４α－ＰＤＤ）抑制ＭΦ内的蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ，ＰＫＣ）以阻断ＤＧ的作用，结果使ＮＥ促进ＭΦＩａ抗原表达的效应显著减弱（Ｐ＜０．０１，Ｐ＜０．０１），说明在ＮＥ促进ＭΦＩａ抗原表达的效应中，ＩＰ－３、Ｃａ￣２＋和ＤＧ都起重要的作用。     

转化生长因子－β1诱导前单核自血病细胞分化过程中细胞表型变化的研究

本文对重组人转化生长因子－β１诱导前单核白血病细胞系ＴＨＰ－１细胞向ＭΦ分化过程中细胞表面特异性抗原表达变化进行了研究。流式细胞仪分析表明，实验细胞ＴＨＰ－１主要表现单核细胞的表型，巨噬细胞标志Ｍａｃ－１α、Ｍａｃ－１β、Ｍａｃ－２为阴性。ｒｈＴＧＦ－β１作用后，分化细胞表面单核细胞标志４Ｆ２表达下降，巨噬细胞特异抗原Ｍａｏ－１α、Ｍａｃ－１β、Ｍａｃ－２表达上升，细胞表现出ＭΦ表型。结合前文分化细胞表现出的脱氢酶硝基蓝四唑还原、α－萘酚醋酸酯酶活性和吞噬功能，可以发现分化细胞在细胞表面特异性抗原表达及细胞表型的转变与其ＭΦ生物学功能的获得在时相上基本一致。     

实验性器官硬化细胞外基质及间质效应细胞的研究

我们应用免疫组化方法结合形态观察，对大鼠实验性肝，肺和肾小球的纤维化，硬化过程中的细胞外基质（ＥＣＭ－ＦＮ，ＣｏｌⅠ，Ⅲ，Ⅳ，Ⅴ）；以及肝脏的Ｉｔｏ细胞，肺泡壁原始间叶细胞（ＰＭＣ）和肾小球系膜细胞的骨架蛋白（ＤＭ，ＶＭ，α－ＳＭＡ）表型变化，进行了动态的对比研究，发现在纤维化硬化过程中，上述三种细胞大量增生并伴细胞骨架蛋白表型和ＥＣＭ分泌的系列改变。同时对此类细胞在修复过程中演变的共同规律和某些     

高,低分化的鼻咽癌细胞膜电位比较

用玻璃微电极细胞内记录法，对体外培养的人胚鼻咽上皮细胞、人低分化鼻咽癌（ＣＮＥ－２Ｚ）和高分化鼻咽癌（ＣＮＥ－１）细膜膜电位（ＭＰ）进行研究，结果表明：（１）与人胚细胞相比，癌细胞ＭＰ负值增大，各细胞ＭＰ差异大，分布弥散，尤以ＣＮＥ－２Ｚ细胞为甚；（２）这３种细胞，细胞外Ｋ＾＋对数浓度与ＭＰ值均呈高度负相关；（３）比较３种细胞ＭＰ值与细胞外液Ｋ＾＋对数浓度的回归系数，ＣＮＥ－２Ｚ的最大，人胚的最小     

Yolk sac endoderm is the major source of serum proteins and lipids and is involved in the regulation of vascular integrity in early chick development

An important function of the vascular system is nutrient delivery. In adult animals, this is mediated through a close contact of the mesoderm‐derived vasculature with the endoderm‐derived enterocytes and hepatocytes. During embryonic development, the yolk sac (YS) endoderm has been suggested to play a similar role. Physiological and molecular nature of the contact between the YS endoderm and the vasculature is not well‐understood. To understand roles of the YS endoderm in early development, we used the avian model and carried out a gene expression profiling analysis of isolated area vasculosa YS endoderm tissues from embryonic day 2–4 chick embryos, covering the first 48 hr of postcirculation development. Genes involved in lipid metabolism are highly enriched, indicating an active modification of lipid components during their transfer from the yolk to the circulatory system. We also uncovered genes encoding major serum proteins and key regulators of vascular integrity. In particular, PTGDS, an enzyme controlling the last step of prostaglandin D2 production, shows high expression in the YS endoderm. Experimental introduction of prostaglandin D2 into embryonic circulation led to intraembryonic vessel rupture. These data suggest that the YS endoderm is the major, if not exclusive, source of lipid and protein constituents of the early embryonic serum and plays an important role in the regulation of vascular integrity in developing embryo. Developmental Dynamics 240:2002–2010, 2011. © 2011 Wiley‐Liss, Inc.     

Notch1 but not Notch2 is essential for generating hematopoietic stem cells from endothelial cells

Hematopoietic stem cells (HSCs) are thought to arise in the aorta-gonad-mesonephros (AGM) region of embryo proper, although HSC activity can be detected in yolk sac (YS) and paraaortic splanchnopleura (P-Sp) when transplanted in newborn mice. We examined the role of Notch signaling in embryonic hematopoiesis. The activity of colony-forming cells in the YS from Notch1(-/-) embryos was comparable to that of wild-type embryos. However, in vitro and in vivo definitive hematopoietic activities from YS and P-Sp were severely impaired in Notch1(-/-) embryos. The population representing hemogenic endothelial cells, however, did not decrease. In contrast, Notch2(-/-) embryos showed no hematopoietic deficiency. These data indicate that Notch1, but not Notch2, is essential for generating hematopoietic stem cells from endothelial cells.     

In vivo localization and biological effect of anti-yolk sac monoclonal antibodies

Two monoclonal antibodies directed against rat yolk sac antigen 1 (mab 6D1) and yolk sac antigen 2 (mab 3C3) were injected i.v. into pregnant or tumor-bearing rats. Immunofluorescent examination of the different organs from animals killed, 2, 24, or 48 hours after inoculation showed the specific binding of mab 6D1 to the free surface of visceral endoderm cells in pregnant animals and on visceral cells of yolk sac carcinoma. The mab 3C3 reacted only with the endoderm of parietal yolk sac and with a distinctive parietal pattern of the tumor. The reaction was strong after 2 and 24 hours following injection and much weaker after 48 hours. The 3C3 mab had an embryotoxic effect, whereas the 6D1 mab did not influence the development of the fetus.     

Transport across endodermal cells of the chick yolk sac during early stages of development

The endoderm of the chick yolk sac mediates the transfer of materials from the yolk mass to the embryonic circulation. There is little evidence of endocytotic activity in the area pellucida, but the endodermal cells of the area vasculosa possess many microvilli and bristle-coated pits and vesicles, as well as a canalicular system and vacuoles in the apical cytoplasm. Three tracers, horseradish peroxidase, ferritin, and latex spheres, were injected beneath the endoderm of both cultured embryos and embryos in ovo to study the pathway of uptake of extracellular materials. All tracers were sequestered in bristle-coated pits (200–500 nm in diameter) of the endodermal cells of the area vasculosa, but not those of the area pellucida. Both horseradish peroxidase and latex spheres (and probably ferritin) were incorporated into pleomorphic intracellular yolk drops through bristle-coated pits and vesicles, and then into apical vacuoles, which fuse with the intracellular yolk drops. Horseradish peroxidase and ferritin were also found within apical canaliculi. The apical junctions between endodermal cells prevented the intercellular passage of the tracers. A ‘topping-up’ hypothesis is proposed whereby endodermal cells of the area vasculosa continually sequester extracellular yolk material, which is incorporated into a digesting intracellular yolk drop while, at the same time, digested yolk products are being transported across the vascular pole of the endoderm to the extraembryonic circulation and thence to the embryo.     

Retinoid binding proteins in mouse yolk sac and chorio-allantoic placentas

 In the adult, as well as in the embryo, a number of specific extra- and intracellular binding proteins such as the plasma retinol binding protein (RBP), the cellular retinol binding protein type I (CRBP I), and also the cellular receptors for RBP are thought to regulate transport and metabolism of retinol (vitamin A). Since the regulation of materno-fetal transport of vitamin A is not well understood, we examined the localization of these proteins during the development of the mouse chorio-allantoic and yolk sac placentas. The labyrinthine region of the chorio-allantoic placenta, where exchange of substances can occur between the maternal and fetal circulations, did not contain RBP (mRNA or protein) or antigen(s) similar to the bovine RBP-receptor p63, whereas the visceral endoderm of the yolk sac placenta, the second site for materno-fetal transport, did. Furthermore, only the endodermal cells of the visceral yolk sac appeared to strongly accumulate radiolabelled retinoids. The cellular retinol binding protein (CRBP I) was detected both in the trophoblast layer of the placental labyrinth closest to the fetal endothelium (layer III), and in the visceral endoderm of the yolk sac. Together, these findings suggest that the yolk sac placenta mediates retinol transfer to the embryo/fetus throughout the entire gestation. The chorio-allantoic placenta, on the other hand, does not appear to have this capacity, while the presence of CRBP I does suggest a retinol-metabolizing capability. Accepted: 5 February 1997     

Ectopic somatic endoderm in secondary human yolk sac.

Three secondary human yolk sacs (SHYSs) showing heterotopic endodermal tubular structures of the gut-forming type were analyzed from a series of 180 SHYSs. These structures were similar to the early somatic endoderm involved in the formation of gut and lung. They may have arisen either as sequestrations in growth-disorganized embryos or as a phenomenon of differentiation from extraembryonal endoderm, which would indicate that extraembryonal tissues such as the SHYSs retain the capacity to differentiate somatic endoderm in developmentally altered embryos. It is possible that these structures may be the precursors of placental hepatic tissue and teratomas. Their morphologic resemblance to similar structures found in glandular, polyvesicular, and intestinal human yolk sac tumors provides yet another example of the similarity between SHYSs and yolk sac tumors.     

Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis

Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking. Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic. To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene. Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11). These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0. Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis. Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype. The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.     

In vitro endothelial differentiation of long-term cultured murine embryonic yolk sac cells induced by matrigel

The yolk sac of an early mammalian embryo contains progenitors of hematopoietic cells and vascular endothelial cells. We established a cell line, YS4, from murine embryonic yolk sac 10 years ago. The line has been successfully cultured since then. To determine whether these long-term cultured yolk sac cells still have the potential to differentiate into endothelial cells, an in vitro model of yolk sac cell differentiation into tubeforming endothelial cells was established in the present study by culturing the yolk sac cells on basement membrane proteins (Matrigel). The results indicate that upon plating onto Matrigel, YS4 cells attach quickly, align in tandem, and form a complete network of capillary structures within 12 h. By using antibodies against the known components of Matrigel in a tube formation inhibition assay, we found that extracellular matrix proteins such as laminin, collagen IV, vitronectin, and fibronectin are the most important components in the Matrigel which induce the yolk sac cells to undergo endothelial differentiation. New basement membrane proteins are also required for the endothelial differentiation process, as indicated by the fact that base membrane protein synthesis inhibitor, D609, can block the differentiation process. Furthermore, our experiments revealed the involvement of several signal transduction pathways, such as protein kinase A, C and protein tyrosine kinase in this differentiation process.     

Development, differentiation, and maturation of fetal mouse yolk sac macrophages in cultures

The development and differentiation of macrophages in the fetal mouse yolk sac were studied morphologically in four different culture experiments. In the culture of mouse embryos with yolk sac, the development of fetal macrophages was demonstrated to precede that of promonocytes and monocytes in the yolk sac. In vitro differentiation of the fetal macrophages was consistent with the results of our previous in vivo observation indicating that fetal macrophages were differentiated from primitive macrophages, but not from the monocytic cell series. Differentiation of primitive macrophages into fetal macrophages, before the development of promonocytes and monocytes, was reproduced in the culture of cell suspensions from the fetal mouse yolk sacs, with a mouse bone marrow stromal cell clone (ST2) particularly with those at 8 days of gestation. In the soft agar or liquid culture of yolk sac cells with LP3-conditioned medium, monocyte-macrophage colonies were effectively induced, but not fetal macrophage colonies. The results provide evidence for the existence, in yolk sac hematopoiesis, of two distinct macrophage populations: a fetal macrophage population and a monocyte-derived macrophage population. The data indicate an obvious difference in development and differentiation between the two populations and the temporal precedence of fetal macrophages appearing before monocyte-macrophages.