螅状独缩虫(Carchesium polypinum)类中间纤维的初步研究

利用间接免疫荧光和免疫印迹发现螅状独缩虫中存在两种分子质量为58ku和66ku的蛋白。这两种蛋白能分别与抗波形蛋白和抗核纤层蛋白B的抗体反应。分子质量66ku的蛋白位于大核边缘。另外,细胞经分级抽提后大核周围存在核纤层样结构。基于上述结果,可以认为螅状独缩虫中存在类中间纤维。     

用免疫荧光法检测鸡痘病毒感染的研究

对应用免疫荧光法检测鸡痘病毒感染鸡血清进行了研究。在鸡痘病毒感染细胞培养物抗原标本制备、免疫荧光染色条件以及反应特异性等研究探索的基础上，对来自于秦皇岛地区鸡痘高发区１５６０份鸡血清的免疫荧光法检测阳性反应率为１６．５４％，比常规的琼脂扩散法（阳性反应率６．９９％）高出９．５５个百分点     

眼虫中存在类中间纤维骨架体系

用分级抽提和DGD包埋去包埋技术处理小眼虫(Euglena gracilis),获得了完整的、清晰的细胞纤维网架结构。电镜结果显示纤维的直径在12～14nm,有时呈束状结构,通常以短纤维网络形式存在。免疫印迹证明类中间纤维蛋白的主要成分是一种66kD蛋白。免疫荧光显示类中间纤维蛋白主要分布在各类细胞器的表面,以及细胞表皮的下层。     

NgR在中菊头蝠(Rhinolophus affinis)脑中分布

Nogo和其受体相互作用可能在抑制神经再生中发挥着重要作用.运用免疫细胞化学方法,本研究观察了Nogo受体(NgR)在中菊头蝠脑中的分布.结果显示NgR在中菊头蝠脑皮层各层均有表达.在海马,NgR主要分布在CA1、CA3和DG区的神经细胞胞膜、胞质或/和突起上.杏仁核、丘脑、室旁核、视上核、视交叉上核等也有NgR阳性神经细胞着色.在脑的白质,轴突着色明显.小脑的分子层、Purkinje细胞层和颗粒细胞层均有NgR免疫阳性反应,其中阳性反应的颗粒细胞最多.这些提示NgR可能介导其配基对中菊头蝠脑多个区域的神经细胞起作用.     

核纤层蛋白及其基因的研究

核纤层是细胞核内极其重要的结构.近年来有关核纤层蛋白及其基因的研究取得了较大进展.文中就国际上有关该领域研究的新进展并结合作者所在实验室近年来的有关研究结果,从核纤层蛋白成分及功能、核纤层蛋白基因及其起源分化等方面进行了较系统的介绍和探讨,并提出了一些值得深入研究的新问题.     

用液晶(Liquid crystals)膜实现微波可见性的图形显示

<正> 胆甾相液晶(cholesteric liquid crystals)中的分子分层排列。在每层中的分子排列取向一致。但在层与层之间排列方法向扭转了一定角度,因此多层分子链的排列方向逐层扭转,而呈螺旋结构。当分子的排列方向旋转360°,又回到原来取向时为一个周期。在一个周期中,分子链排列完全相同的两层中间的距离称为螺距。当液晶受热而温度升高时,     

分子印迹球状环糊精聚合物的合成及粒度分布研究

研究了利用反相悬浮聚合技术,聚乙烯醇(PVA)包埋分子印迹环糊精聚合物合成球状分子印迹环糊精聚合物吸附剂的过程中,考察了催化剂用量、PVA溶液浓度、分散剂种类和用量、分散相及反应温度、搅拌速度等诸因素对合成球状环糊精聚合物粒度的影响.     

无蹼壁虎消化道组织学及组化观察初报

Ｃａｒｎｏｙ固定,常规石蜡包埋、切片、Ｈ．Ｅ染色、ＰＡＳ反应、ＲＮＡ反应,对无蹼壁虎消化道的组织学进行光镜观察．结果表明：其消化道由同心圆的粘膜层、粘膜下层、肌层和浆膜层组成．绒毛发达,绒毛的上皮细胞为单层柱状上皮．消化腺为分枝管状腺．ＰＡＳ反应显示柱状细胞纹状缘,杯状细胞和腺细胞呈强阳性,与分泌的粘多糖有关．ＲＮＡ反应显示腺细胞呈强阳性,与腺细胞分泌活动有关．     

利用CRISPR/Cas9系统构建LMNA基因突变 AC16人心肌细胞系

LMNA基因突变引发核纤层蛋白(LaminA/C)及其互作蛋白表达异常，引起机体一系列生理学反应，导致核纤层蛋白病发生，目前其具体致病机制尚不清楚。本文利用CRISPR/Cas9技术，选取符合sgRNA筛选原则的致病突变位点，成功构建了携带LMNA突变Q517X的细胞系，并对突变细胞系与LaminA/C相连或互作的LaminB1、PCNA、P53、PKCα等基因的RNA表达水平与蛋白表达水平及突变细胞核膜骨架形态进行检测，发现：Q517X突变细胞的LaminB1、P53、PCNA基因mRNA表达量显著降低约70%;LaminA/C蛋白几乎不表达，PKCα、LaminB1、P53等蛋白的表达量分别降低75%、60%、80%。结果表明，Q517X突变通过改变AC16人心肌细胞LMNA相关基因的表达进而影响细胞核正常骨架结构与功能。     

鳖卵细胞的组化分析观察

卵巢用Ｃａｒｎｏｙ固定,石蜡包埋、切片、用ＨＥ染色,ＰＡＳ反应和ＲＮＡ反应,光镜观察显示：中华鳖卵细胞可分为卵原细胞期,初级卵泡期,生长卵泡期和成熟卵泡期．ＰＡＳ反应显示卵透明带、卵黄颗粒呈强阳性,ＲＮＡ反应显示颗粒层、核仁呈强阳性．     

Prokaryotic expression, localization and function of the infectious laryngotracheitis virus glycoprotein G

The infectious laryngotracheitis virus （ILTV） glycoprotein G （gG） gene of E3 and Zhonghai strains was cloned, sequenced and compared with the gG gene of other Type Ⅰ animal herpesviruses. To find the localization and the function of the gG in the infected cells, the 35 kD fusion protein （His-GG） was expressed by inserting the coding region of gG except for the signal peptide into pET30a （＋）. After purification of the His-GG fusion protein, the rats＇ antibody to the His-GG was prepared and purified by using the protein G Sepbarose. Results of laser scanning confocal microscopy （LSCM） detection showed that the ILTV gG was in the perinuclear region and membrane of chicken embryo liver （CEL） and kidney （CEK） cells, and that the gG accumulated more in the coalescent part than in the other parts of the adjacent CEL or CEK cells. The plaque size and the one-step growth curve tests suggested that the ILTV gG was required for viral growth by cell-to-cell direct infection in tissue-cultured CEL cells.     

Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor

A pathological hallmark of Alzheimer's disease is the deposition of amyloid fibrils in the brain. The principal component of the amyloid fibril is beta/A4 protein, which is derived from a large membrane-bound glycoprotein, Alzheimer amyloid protein precursor (APP). Although the deposition of amyloid is thought to result from the aberrant processing of APP, the detailed molecular mechanisms of amyloidogenesis remain unclear. A C-terminal fragment of APP which spans the beta/A4 and cytoplasmic domains has a tendency to self-aggregate. In an attempt to establish a cultured-cell model for amyloid fibril formation, we have transfected COS-1 cells with complementary DNA encoding the C-terminal 100 residues of APP. In the perinuclear regions of a small population of DNA-transfected cells, we observed inclusion-like deposits which showed a strong immunohistochemical reaction towards an anti-C-terminal APP antibody or an anti-beta/A4 amyloid core-specific antibody. Electron microscope observations of the inclusion-carrying cells revealed an accumulation of amyloid-like fibrils of 8-22 nm diameter near and on the nuclear membrane. The fibrils showed a beaded or helical structure, and reacted positively with the anti-C-terminus antibody by immunoelectron microscopy. These results suggest that the formation of amyloid fibrils is an inherent characteristic of the C-terminal peptide of APP. The present system provides a suitable model for the molecular dissection of the process of brain amyloidogenesis.     

Spatio-temporal images of growth-factor-induced activation of Ras and Rap1.

G proteins of the Ras family function as molecular switches in many signalling cascades; however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses and how the signals for differentiation and survival are distinguished by neuronal cells.     

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241kD) of vesicular stomatitis virus (VSV) is the most important snbnnit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96—120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108—120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.     

Targeted transfection by femtosecond laser

The challenge for successful delivery of foreign DNA into cells in vitro, a key technique in cell and molecular biology with important biomedical implications, is to improve transfection efficiency while leaving the cell's architecture intact. Here we show that a variety of mammalian cells can be directly transfected with DNA without perturbing their structure by first creating a tiny, localized perforation in the membrane using ultrashort (femtosecond), high-intensity, near-infrared laser pulses. Not only does this superior optical technique give high transfection efficiency and cell survival, but it also allows simultaneous evaluation of the integration and expression of the introduced gene.     

凹叶厚朴种子的结构及细胞化学研究

用组织切片法及细胞化学染色研究了凹叶厚朴种子的结构和内含物质分布的情况.结果表明：凹叶厚朴种子有种皮、胚和胚乳三部分组成;胚小,胚乳丰富.种皮由外种皮、中种皮和内种皮组成.外种皮细胞内无淀粉粒,含少量的贮藏蛋白质和脂类物质;中种皮细胞内有较多的淀粉粒和脂类物质以及少量的贮藏蛋白质;胚细胞中无淀粉粒,也无贮藏蛋白质和脂类物质;胚乳细胞中含有较多的淀粉粒,也含有大量的贮藏蛋白质和脂类物质.凹叶厚朴种子的结构及细胞化学特征是自然条件下凹叶厚朴种子繁殖率低下的原因之一.     

使用碳纳米管AFM针尖的蛋白质高分辨率成像

原子力显微镜(AFM)是分析生物分子结构的有效手段,而目前使用的探针针尖的性质限制了高分辨率图像的获得。该文将碳纳米管安装到原子力显微镜的传统针尖上,制作出碳纳米管针尖以解决这个问题。运用碳纳米管针尖在大气常温条件下获得了由3个单元组成的小鼠抗体IgG蛋白质的Y形结构,并且分子的尺寸与X射线晶体衍射的结果非常接近,这种效果用传统针尖是无法获得的。获得的蛋白质分子超微结构的高分辨率图像为研究蛋白质分子功能提供了有价值的信息。     

肝细胞的三维受控组装

为解决肝病救治中肝供体缺乏的问题,采用基于快速成形技术的细胞组装机,将肝细胞和海藻酸钠与明胶复合材料的共混物作为成形对象,通过细胞材料直接三维受控组装技术,实现了堆积成形具有一定三维结构和预定义孔隙的肝组织前体。常规体外培养条件下,肝细胞存活并保持生物学活性达12d之久。这一技术有望成为肝脏及其他人体组织器官人工构造的新途径。