云南红豆杉培养细胞系的建立

紫杉醇(Taxol)最初是从红豆杉属植物短叶红豆杉(Taxus brevifolia)树皮中分离出的一种二萜类化合物^[1]．对卵巢癌,转移性乳腺癌和恶性黑色寮瘤等患者疗效显著^[2],全世界红豆杉属植物有近11种,都含紫杉醇成分．但含量很低,加之现存数量很少,生长极为缓慢．造成了紫杉醇原料供应的危机^[3]。紫杉醇化学合成已经成功^[4-6],但繁杂的反应过程及前体化合物来源的限制使得它们无法实现商业化生产。最近从短叶红豆杉中分离出一种生产紫杉醇的内寄生真菌Tgromyer andreanae^[7]．由于紫杉醇含量仅为24～50ng/L．没有实用价值。植物细胞和组织培养可能是解决天然抗肿瘤药物长期供应的有效方法之一^[8]。自1991年Christen等人申请利用红豆杉细胞培养物生产紫杉醇专利以来^[9]．有关红豆杉细胞培养的研究已有不少报^[10-12]。但云南红豆杉(T.yunnanensis)仅见愈伤组织诱导的报道^[13]。本文报道云南红豆杉愈伤组织诱导和细胞培养的初步结果,并分析了细胞培养物中紫杉醇含量。     

一株高水平表达重组蛋白昆虫细胞系的建立

报道了一株来自粉纹夜蛾Trichoplusiani脂肪体的传代细胞系 ,在辅以 5%胎牛血清的商品无血清培养基Excell 4 0 0中 ,细胞群体倍增时间为 2 2 9h ,最高密度可达 2 2× 10 6 mL ,该细胞对苜蓿丫纹夜蛾多粒包埋型多角体病毒 (AcMNPV)极为敏感 ,增殖AcMNPV多角体平均每个细胞达86个 ,表达由AcMNPV构建的重组蛋白的水平较高 ,β 半乳糖苷酶的表达水平为 ( 2 2 5 5± 13 4 )IU mL ;碱性磷酸酶的表达水平为 ( 4 7± 0 61)IU mL ,是一株高水平表达重组蛋白的传代细胞系 ,命名为HNU Tn FB1。     

红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展

紫杉醇是一种四环二萜酰胺类化合物,是从红豆杉科红豆杉属植物中提取分离出来的次生代谢物,是世界公认广谱、活性强的天然抗癌新药。但直接从植物中提取紫杉醇的传统生产方式,不仅产量低,且会对野生红豆杉资源造成严重破坏,同时紫杉醇的化学全合成也由于其结构复杂而不具备商业价值。与之相反,细胞培养技术具有受外界影响少、生产成本低、次生代谢产物多、细胞生长周期短的优势,是目前最具前景的紫杉醇生产方式。近年来随着科研水平的不断提升,紫杉醇无论在生理代谢调控、关键基因挖掘,还是新药物制剂与剂型及其类似物的开发和运用等方面,都取得了进展,但要建立紫杉醇商业化高产体系,还必须和前人的研究经验相结合。该文对红豆杉高产悬浮细胞系建立及其紫杉醇诱导的研究进展进行了综述,主要包括前人对红豆杉属植物组织与细胞培养相关的外植体、培养基、激素、培养条件、褐化等问题的研究,以及从代谢调节、培养方式、基因工程等多方面提高紫杉醇含量的最新进展,最后总结了当前研究的不足,并对今后通过多种组合方式来提高紫杉醇含量的生产途径进行了展望。以期促进红豆杉组织培养技术的进步,为药用资源保护和利用提供一定的理论基础与生产指导。     

两种昆虫细胞的微载体培养

斜纹夜蛾细胞系(SL-1)和家蚕细胞系(BmN)均能在国产微载体上正常生长增殖。以3mg/ml微载体培养细胞,两种昆虫细胞生长最高密度分别为8.2×10~5细胞/ml和7.6×10~5细胞/ml。扫描电镜观察显示一个微载体可贴附几十个,有的多达上百个细胞。两性昆虫细胞的微载体培养特征与常规静止培养无甚差异。     

昆虫细胞的大规模培养

昆虫细胞的大规模培养李文青,肖成祖（北京军事医学科学院生物工程研究所,）昆虫细胞培养的鼻祖是德国人ｆｏｒｈａｒｄＢｅｎｄｉｃｔ（１８７８—１９５８）［１］,他在１９１５年发表了有关昆虫细胞培养的第一篇文章［２］。早期,昆虫细胞的培养是为了进行昆虫的生...     

昆虫细胞的培养和尿激酶原表达的研究

目前,尿激酶原（Pro－UK）作为～种新型溶栓剂,越来越多的受到重视。北京大学承担国家“863”课题“利用昆虫一杆状病毒系统表达Pro一UK”获得成功［川。表达P］－UK酶活为8O0～1600IU／rnl,本研究将Sfg细胞用4L转瓶和SL生物反应器培养,并用重组Pro－UK的病毒感染,以进行尿激酶原的表达。1材料与方法工．且材料1．1．ISfg细胞和表达Pro－UK的重组病毒北京大学胡美浩教授提供。1．1．24L转瓶机美国BellcoGlass公司。1．1．3SL生物反应器美国NBS公司。1．1．4尿激酶检测试剂中国药品生物制品检定所。1．2方法1．2．1细胞在4…     

《生物医药研究的细胞系使用指南》介绍

细胞系的混淆、微生物污染、基因和表型的不稳定,都是细胞培养过程中存在的问题。由多加英国研究机构联合颁布的指南就细胞系的建立、获得、鉴定、冻存、实验室间转移、污染、不稳定性和混淆等方面的关键问题进行了详细的描述。     

A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae)

Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

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…     

A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment

Summary A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were separately minced into approximately 1-mm³ pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing 5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 μm) and the cells collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture. This work was supported by research grants from the National Institute of Environmental Health Sciences, Bethesda, MD (ES3101) and the United States Environmental Protection Agency, Washington, D. C. (R810146).     

DNA content analysis of insect cell lines by flow cytometry

The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cell lines were stable among the passages and during the length of time culture. This technique was demonstrated to be useful for the detection of mixed cell lines and nucleopolyhedrovirus cell infection, using Autographa californica MNPV. The flow cytometry gives interesting results on the cell cycle and the ploidy level; it appears as a good tool for insect cell lines characterization. This revised version was published online in July 2006 with corrections to the Cover Date.     

油桐尺蠖卵巢细胞系BS-484克隆株特性的研究

通过有限稀释法由Bs—484细胞系中成功地分离出四个克隆株(Bs—484B,E,F,G)。克隆细胞侏的生长特性不同于原细胞株Bs—484,各克隆株之间的形态特征、细胞倍增时间、以及在维持油桐尺蠖核型多角体病毒的复制能力等方面均有差异。用三种同工酶(乳酸脱氢酶、苹果酸脱氢酶和酯酶)比较了各克隆株与原细胞株之间的异同。     

葡萄糖的添加对昆虫细胞Sf21悬浮生长的影响

添加葡萄糖对昆虫细胞Sf21(Spodoptera frugiperda)悬浮生长的影响,发现补加糖量在lg／L时·能明显提高细胞生长的速度和最高密度,细胞最高密度由2．5×10⁴／ml增加到4.9×10⁶／ml; 朴加糖量在2g／L时,则有显著的抑制作用,即使增加接种密度．细胞生长的最高密度也只有2．1×10⁶／ml。当采取流加葡萄糖方法来培养细胞时,则其生长的最大密度可提高到5．2×10⁶／ml。     

Establishment and characterization of a new human renal cell carcinoma cell line (KRC/Y)

Summary A new renal cell carcinoma (RCC) cell line (KRC/Y) has been established from a surgical specimen of a 41-yr-old Japanese female patient with RCC composed of both clear cells and granular cells. This cell line has been maintained for more than 15 mo. through 45 passages with a stable growth, KRC/Y cells have clear or eosinophilic polygonal cytoplasm and round to oval nuclei with one or two nucleoli, and proliferate in a pavementlike cell arrangement with a lack of contanct inhibition. By electron microscopy, these cells contain abundant fat droplets and glycogen granules or well-developed organells or both, which were also observed in the original tumor. The doubling time of these cells at the 15th passage was 73 h. The chromosome number was from 37 to 45 with a hypodiploid modal number of 42. Tumorigenicity was identified by tumor formation after subcutaneous injections of KRC/Y cells in nude mice, which showed close resemblance to the original tumor by light and electron microscope observations. This study was supported in part by Sarah Cousin Fund, Boston, MA.