粉纹夜蛾新细胞克隆株Tn5B-40的研究(英文)

从Tn5B1-4细胞系中克隆并筛选出了新克隆株Tn5B-40,测定分析结果表明,该克隆株对病毒(AcNPV)的敏感性和生长特性与原始细胞无显著差异,但在重组蛋白表达方面,无论对β-半乳糖苷酶还是碱性磷酸酶都明显地高于野生型细胞系,其中β-半乳糖苷酶在第6天的表达为原始细胞株的2倍,碱性磷酸酶的表达在第9天最高为原始细胞株的1.4倍。因此,该细胞是一株高产病毒和高表达重组蛋白的新克隆株。     

A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1–4 CELLS

Abstract  The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B1–4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins, however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B1–4 cells. The growth characteristics, productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B1–4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing ß -galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B1–4 cells.     

抗苏云金杆菌毒素Cry1Ac粉纹夜蛾细胞系的特性研究

为了从离体细胞水平探讨昆虫对苏云金芽孢杆菌杀虫晶体蛋白的部分抗性机制,本文采用活化的Cry1Ac 毒素对粉纹夜蛾BTI-TN-581-4细胞连续筛选86代,获得了高水平抗性细胞,研究了其某些特性。它对Cry1c 产生了低水平的交互抗性,对低渗溶液的耐受性显著增强,双向电泳图谱表明抗性细胞膜蛋白组分发生了明显的变化。膜蛋白组分的变化可能导致了筛选细胞的耐低渗透压和抗Cry1C。     

小鼠原生殖细胞建系过程及其分化特性的研究

以小鼠8.5dpc、10.5dpc、12.5dpc胚胎为材料,分离其中包含PGC的胚胎组织,使其生长于饲养层细胞上,在生长因子LIF、SCF和bFGF的共同作用下存活增殖,形成PGC克隆,经过几次分散转移至新的饲养层细胞,产生稳定增殖的EG干细胞克隆,共建成5株EG细胞系,AKP染色以及oct-4基因表达产物的免疫荧光检测均显示阳性。EG1、EG2、EG3、EG4、EG5,分别来自8.5、10.5dpc的胚胎,没有得到长期培养的12.5dpc的EG细胞系。EG细胞系在有饲养层细胞或添加LIF的环境中可稳定传代,保持不分化状态,至少15代内正常核型细胞所占比例80%以上。去除抑制分化因素的前提下,悬浮培养的EG细胞形成胚体,分化出类似胚胎内胚层和外胚层的细胞结构;贴壁生长的胚体能产生不同类型的分化细胞,包括上皮细胞、成纤维细胞、神经细胞等。EG细胞在裸鼠体内形成畸胎瘤。以上结果证实我们建立的EG细胞系具发育多能性,为研究早期胚胎和生殖细胞生长分化提供了模型。     

纤维素酶降解人参细胞胞壁产生的激发子诱导人参培养细胞的反应

用纤维素酶降解人参细胞壁制备的激发子刺激人参细胞后,电子显微镜的观察结果显示激发子处理诱导了细胞内淀粉粒的降解,出现了细胞内动员能量物质的应急反应;同时还出现了大量拟脂类圆球体。进一步的研究表明,涉及降解淀粉的淀粉酶活力是升高了,而降解脂类物质的酯酶活力是降低的;同时细胞中可溶性糖和总类脂含量是增加的。这些都说明了人参细胞经激发子处理后能量物质的动员以及脂类代谢途径的调整。另外,适当浓度激发子的处理会诱导人参细胞内人参皂苷含量和苯丙氨酸解氨酶活力的升高,而过氧化物酶和多酚氧化酶活力是下降的。     

MUCOPOLYSACCHARIDES SYNTHESIZED BY CULTURED GLIAL CELLS DERIVED FROM A PATIENT WITH SANFILIPPO A SYNDROME

—Glial cells were cultured from brain tissue obtained at autopsy of a patient with Sanfilippo A syndrome. Mucopolysaccharides were labeled by culturing the cells in the presence of [³⁵S]sulfate. After proteolysis, intracellular and media-elaborated mucopolysaccharides were fractionated by Dowex 1 chromatography. One fraction, identified as heparan sulfate by chromatographic, electrophoretic, and enzyme susceptibility properties, accumulated in Sanfilippo glial cells in greater amounts than in controls. Heparan sulfate was also excreted into the culture media by both Sanfilippo and normal cultures, and it constituted a major fraction of the sulfated mucopolysaccharides synthesized by glial cells. Sanfilippo and normal fibroblasts were also included in these studies for comparative purposes. Sanfilippo fibroblasts accumulated significantly increased amounts of heparan sulfate as compared to normal fibroblasts. Heparan sulfate was excreted into the culture media by Sanfilippo and normal fibroblasts in equivalent amounts, but in contrast to glial cells, it was only a minor component of the sulfated mucopolysaccharides produced. Cultured glial cells should provide a useful system for investigating the role of heparan sulfate in glial cell function.     

长江江豚脐静脉细胞体外培养的初步研究

长江江豚(Neophocaena phocaenoides asiaeorientalis)属齿鲸亚目鼠海豚科, 是世界上唯一的江豚(Neophocaena phocaenoides)淡水亚种, 主要分布于我国长江中下游干流和洞庭湖、鄱阳湖及其部分支流1—3。       

CELL PROLIFERATION IN HAEMATOPOIETIC SPLEEN COLONIES OF MICE: DIFFERENCE BETWEEN COLONIES DERIVED FROM INJECTED ADULT BONE MARROW AND FOETAL LIVER CELLS

Cell proliferation in mouse spleen colonies, derived from injected foetal liver and young adult bone marrow, was studied by measuring incorporation of radio-iodine-labelled 5-iodo-2'-deoxyuridine (IUdR). Foetal liver-derived colonies incorporated significantly more IUdR than marrow-derived colonies on the 8th and 12th days after cell injection. The data are consistent with the view that foetal haematopoietic stem cells are capable, on average, of producing larger descendant populations than are stem cells from young adults.     

A NEW BANGIOPHYCIDEAN ALGA FROM ALABAMA1

A new species of red algae, Boldia angustata sp. nov., was discovered near Tuscaloosa, Alabama, during the spring of 1968. It differs significantly from the only previously described species, Boldia erythrosiphon Herndon, in length and width of young and mature thalli, cell size, shape of nuclei in first-order cells, shape of intercalary filaments, morphology of filaments derived from monospores, ontogenetic development of erect plants, morphology of the basal system, and regularity of first-order cells.     

B16及其克隆亚系细胞分泌乙型肝炎表面抗原的稳定性、动态和最佳条件

B16及其亚系细胞B16-7和B16-12在氨甲喋呤压力下连续传代7个月,以B16-7和B16-12分泌乙型肝炎表面抗原(HBsAg)最为稳定。停用氨甲喋呤,B16分泌HBsAg的量逐步下降。用单层细胞培养法对B16-7在不同条件下分泌HBsAg的动态研究证明:细胞常规传代长成单层后换液,血清量降至2％,孵育温度降至32℃,每48小时收获换液一次,连续收获4～5次,按Abbott试剂盒的曲线估计HBsAg的累积量可高达2.5mg/升左右,即1.3×10~(12)g/细胞/24小时。     

人红细胞膜带3蛋白重组人脂质体及其~(35)SO_4~(2-)运输活性

用Triton X-100选择性抽提法,部分提纯了人红细胞膜带3蛋白。在除去去污剂后,带3蛋白被插入由磷脂酰胆碱/胆固醇组成的脂质体中。研究表明,用0.05％Triton抽提膜能除去大部分唾液酸糖蛋白,不会损失太多的带3蛋白。再用0.5％Triton抽提,可增溶出大量带3蛋白和少量其它膜蛋白。把它重组入脂质体后,在最后超离心步骤中得到了小的单层囊泡和大的多层囊泡。本文研究了单层重组囊泡输入~(35)SO_4~(2-)的功能,并和那些由人红细胞“发芽”得到的天然囊泡输入~(35)SO_肋~(2-)的功能作了比较。     

Rapid initiation of suspension cultures of Trichoplusia ni insect cells (TN 5B-1-4) using heparin

The High Five or Trichoplusia ni 5B-1-4 cell line is more productive for heterologous proteins on a per cell basis than other commonly used insect cells lines. Adapting the TN 5B-1-4 cells to suspension culture and maintaining them as suspensions, which is usually difficult due to severe aggregation, but could be achieved by adding heparin at 100IU/mL. Unfortunately, the heparin inhibited the infection process even at very high values of multiplicity of infection (10 to 20 infectious particles per cell). This was reflected by the continued growth of the cells after virus addition. Since considerable cell growth was obtained after infection in the presence of 100IU/mL of heparin, final cell densities were higher and per cell production was lower most likely due to nutrient limitation.     

A NEW CLASS OF BIHETEROCYCLIC THIOGLYCOSIDES FROM PYRIDINE-2-(1H)-THIONES

ABSTRACT

A reported method for preparation of a new class of biheterocyclic thioglycosides via reaction of pyridinethiones with 2,3,4,6-tetra- O -acetyl-α-D-gluco- and galactopyranosyl bromides has been studied.     

ESTABLISHMENT AND SOME CHARACTERISTICS OF A CONTINUOUS CELL LINE DERIVED FROM FAT BODYS OF THE CABBAGE ARMYWORM (LEPIDOPTERA, NOCTUIDAE)

Fat bodies from fully grown larvae of the cabbage armyworm, Mamestra brassicae , were cultivated in a synthetic medium, MGM–431. Small free cells migrated out and multiplied rapidly. The first cell passage was done after culture for 26 days, and 100 passages were performed in the following 9 months. The established cell line was designated as NIAS–MaBr–85. The cell population is morphologically heterogeneous, but most of the cells are hexaploid with 180 microchromosomes. The cells could be stored for 3 months at 5°C, or for longer when frozen at -80°C in medium containing 10% glycerol. Qualitatively the cell line requires few amino acids: only cystine, histidine, isoleucine, methionine and threonine are essential as determined by omission of individual amino acids. The cells are susceptible to Chilo iridescent virus and Autographa californica nuclear polyhedrosis virus. This cell line can grow in serum-free M-M medium.     

印楝素A与印楝素B对粉纹夜蛾BTI-Tn-5B1-4的细胞毒性

为了明确印楝素A和B活性差异的机理,本研究比较了印楝素A和印楝素B对粉纹夜蛾Trichoplusia ni离体培养胚胎细胞系BTI-Tn-5B1-4的毒性。结果表明：印楝素A与印楝素B对BTI-Tn-5B1-4细胞具有良好的增殖抑制活性,处理后3 d,其IC₅₀值分别为2.9 μg/mL和9.85 μg/mL,印楝素A的细胞毒力显著高于印楝素B。倒置显微镜观察发现,印楝素A和印楝素B处理可导致细胞变形,贴壁能力下降,并出现明显空泡,印楝素A的影响明显高于印楝素B。流式细胞仪检测结果表明,印楝素可导致BTI-Tn-5B1-4细胞体积显著膨大,印楝素A处理细胞体积增大程度显著高于印楝素B;印楝素可以明显影响BTI-Tn-5B1-4细胞膜电位,1.25 μg/mL印楝素A和印楝素B处理后3 d,细胞DiBAC4（3）荧光强度分别增加88.12%和55.37%,印楝素A的影响显著高于印楝素B。荧光显微镜观察发现,印楝素对BTI-Tn-5B1-4细胞核具有明显影响,印楝素B的影响明显高于印楝素A,印楝素B处理后,细胞核受损细胞数更多,受损程度更严重。结果显示印楝素A和印楝素B的细胞作用机理存在差异,本研究从细胞学水平解释了印楝素的生长发育抑制作用机理。     

无血清培养昆虫细胞(BTI-Tn-5B1-4)的适应过程

昆虫细胞培养是近年来迅速发展起来的动物细胞培养工程中的一个新领域。人们可以利用杆状病毒在昆虫细胞内的感染、复制,来大量生产昆虫病毒作为生物杀虫剂[1]。而昆虫细胞杆状病毒表达载体系统的建立,则可通过昆虫细胞的体外培养大量表达病毒携带的外源基因。实践证明,这…     

分子佐剂C3d上调Raji细胞协同刺激分子B7-1和B7-2的表达(简报)

我们在以往研究中,引入选择性增强体液免疫效应的新型分子佐剂C3d,成功构建了重组避孕疫苗hCGB-C3d3,通过免疫Th2型优势的 BALB/c小鼠和,Th1型优势的C57BL/6小鼠,显示分子佐剂C3d在不同品系小鼠均使免疫效应从Th1型细胞免疫向Th2型体液免疫偏倚。