细胞生物学网络课程的构建与实施评价

在现代教育技术的推动下,网络课程已经成为现代教育的新趋势,它不仅克服了传统课堂教学的不足,而且增添了很多新的手段和内容。该文介绍了细胞生物学网络课程建设的体系,主要是多媒体课件及录像、在线课程学习、教学文件及相关资源等四大模块,并且通过网络课程的具体实践,实现了细胞生物学课程的网络教育和多媒体教学以及＂网络互动＂的教学模式。网络课程在一定程度上解决了高校普遍存在的学时少内容多、教师少学生多的矛盾,给学生的自主学习和师生交流提供了一个良好的平台。细胞生物学网络课程实施后,受到了省内近十所高校专家的高度评价,学生对本门课程教学的满意度也达到了93%以上,并且学生考试成绩优秀者所占比例也较网络课程实施前有显著提高,取得了明显的教学效果。     

微流控技术在细胞生物学中的应用

微流控技术是在尺度为几个或上百微米的通道中操纵纳升或纳升以下流体的技术,作为一种全新的领域,它给化学合成、生物分析、光学和信息学带来了重大的影响。本文将综述微流控技术在细胞学等领域的应用,并对其发展前景进行展望。     

免疫学技术在细胞生物学教学中的应用

免疫学技术在细胞生物学理论与实验课程中具有重要的应用价值,两者的有机结合能使细胞生物学课程教学更丰富形象,有利于教学质量的提高。目前,免疫学技术普遍只停留于细胞生物学理论课本知识教授中,在对学生开展的细胞实验操作课程中应用较少。如能将免疫荧光标记、流式细胞术等相关技术引入到实验教程中,不仅能使细胞实验结果更形象生动,更能拓宽学生知识领域与实验技能,有利于其综合能力的培养。     

原子力显微技术在细胞生物学中的应用

对近年来原子力显微技术(AFM)在细胞生物学中的应用大致归纳为几个方面进行了简单介绍,还指出了细胞表面结构难于识别、细胞内部结构难以原位观察等AFM应用于细胞生物学中的难题,并提出了“形状探针”的概念以及超薄切片的思路以解决这些难题。AFM在细胞生物学中的应用研究还远远不足,需要更多的科学工作者加入其中。     

CD138免疫磁珠细胞分选的FISH技术在多发性骨髓瘤诊断中的应用

为了分析CD138免疫磁珠细胞分选的染色体荧光原位杂交(FISH)技术在提高多发性骨髓瘤(MM)细胞遗传学异常检测敏感性的作用。本研究选取我院收治的30例确诊MM的患者为研究对象,分离骨髓单个核细胞,应用探针组合,同时采用2种方法进行细胞遗传学检测:实验组采用CD138免疫磁珠分选浆细胞后行荧光原位杂交技术(MACS-FISH)检测;对照组直接荧光原位杂交技术(D-FISH)检测。结果:30例MM患者,实验组采用CD138 MACS-FISH检出率为83.3%,对照组D-FISH法细胞遗传异常检出率为46.7%,两组差异具有统计学意义(p<0.05)。研究结果表明:分析不同类型的细胞遗传异常,MACS-FISH法1q21检出率为46.7%,RB1检出率为50.0%,Ig H检出率为70.0%,P53检出率为20.0%;D-FISH法检出率分别为23.3%,30.0%、36.7%、10.0%。通过细胞核型分析,30例MM患者中,发现5例患者为异常核型,仅为16.7%,其中1例患者为单一结构异常,复杂异常核型患者为4例。我们的研究结论表明:进行CD138免疫磁珠分选浆细胞的FISH技术在多发性骨髓瘤诊断应用中可显著提高细胞遗传学异常检测敏感性,具有临床推广应用的价值。     

荧光漂白恢复技术在细胞生物学中的应用

荧光漂白恢复(FPR)技术已发展成为定量测定细胞膜分子的流动性的方法之一。本文着重介绍了FPR技术应用于测定细胞膜中和细胞质内分子的运动,这些测定将有助于研究活细胞中膜分子的运动方式、功能及其相互关系。     

香菇的细胞生物学

引言香菇是真菌界担子菌门担子菌纲伞菌目侧耳科香菇属,学名为Lentinus edodes(Berkeleg)Singer.由于它美味可口芳香,自宋朝以来,我国就开始人工栽培,迄今已有800多年的历史,但由于我国重栽培轻研究,致使我国当前的香菇研究水平及经济效益落后于日本几十年.随着科学的发展,人们了解到担孢子的形成过程及机理,观察列了香     

基于荧光激活细胞分选技术的超高通量酶活性筛选方法及其应用

基于荧光激活细胞分选(FACS)技术的超高通量酶活性筛选方法是新出现的一类高通量筛选技术.它利用流式细胞仪高灵敏度、高通量的特点,能以极高的速度(108/天)对大容量酶基因文库进行筛选.FACS筛选技术的出现突破了常规筛选方法低效、耗时、费力等瓶颈问题,极大地提升了人类对大容量基因文库的探索能力,因此在新酶基因筛选、酶活性检测、酶定向进化等领域有广泛的应用潜力.综述了FACS超高通量酶活性筛选方法的最新研究进展,着重介绍了其在酶定向进化中的应用.     

神经系统小胶质细胞流式检测及分选

小胶质细胞是中枢神经系统中主要的免疫细胞,数量上占大脑的5%～10%。作为中枢神经系统的巨噬细胞,它们会不断清除中枢神经系统中的斑块、受损或不必要的神经元和突触以及感染因子。从中枢神经系统组织中分离小胶质细胞为研究基础细胞生物学和检测体内治疗对小胶质细胞免疫功能的影响提供了强有力的工具。该文描述了利用小胶质细胞特异性表面标志物,通过流式细胞分选(FACS)纯化小胶质细胞的方法。     

一个用于转染细胞分选的双顺反子载体的构建及其应用

为简化转染细胞的分选过程,构建了一个含有细胞表面标志 CD34 基因的双顺反子载体 p3.1-IRES-CD34. 利用来源于脑心肌炎病毒 (EMCV) 的内部核糖体进入位点 (IRES) ,实现目的基因与 CD34 基因的共同表达 . 将绿色荧光蛋白 (EGFP) 作为目的基因插入载体的多克隆位点,然后转染 NIH-3T3 细胞,通过免疫磁珠分选 (MACS) 方法来分选细胞 . 结果表明：对于转染细胞,均可实现快速分选 ( 瞬时转染细胞约 48 h ,稳定转染 10~15 天 ) ,并且获得较高纯度 (95% 以上 ) 的表达目的基因细胞 .     

生物体微弱磁场检测技术在某部特殊作业人员健康评估中的应用研究

目的:研究某部特殊作业人员的健康状况,为有针对性地提出防护措施提供参考依据。方法:随机抽取某部不同工作岗位的特殊作业人员145名,进行生物体微弱磁场检测分析,以获取包括疲劳、免疫、睡眠、脑机能、血压、心脏、消化、肝胆、泌尿生殖、呼吸、运动、钙代谢、糖代谢、脂代谢、嘌呤代谢等15个系统在内的108项健康评估检测指标。对于每一项指标,仪器自带有其正常值范围,凡低于下限或高于上限的指标被视为异常。结果:所测特殊作业人员总体在钙代谢系统、消化系统、心脏系统、血压系统、呼吸系统、运动系统、免疫系统等七个系统存在不适症状的较为突出,其中钙代谢失衡的占比83.45%,脾胃不和的占比78.62%,心脏功能欠佳的占比72.41%,血压不稳的占比64.14%,咽喉不适的占比59.31%,骨关节不适的占比58.62%,免疫功能下降的占比51.72%。出现运动系统"颈椎疾患"症状的人数占比,实验组明显高于对照组(P0.05)。结论:特殊作业环境可能会影响作业人员的身体健康,应采取有效的安全防护措施,以减弱或消除有毒有害化学物质污染、强噪声、电磁辐射等对人体健康的影响。     

免疫磁珠法分离鼻咽癌基质的T淋巴细胞

肿瘤内环境与肿瘤的发生密切相关.肿瘤细胞周围的组织在癌变发生时不会是一个沉默的旁观者,可能在肿瘤的发生和发展中扮演十分重要的角色.本研究分别采用不同的磁珠分选技术分离T淋巴细胞.采用CK LMP1,CD105和成纤维细胞表面蛋白,结合全血总T细胞试剂盒,间接法分离鼻咽癌基质的T淋巴细胞;采用CD3直接磁分选法分离鼻咽癌基质的T淋巴细胞,然后用免疫组化法鉴定分选的效果和细胞的质量.结果表明,免疫组化显示间接磁分选法分离出来的T淋巴细胞不能完全去除肿瘤细胞,RNA的质量不佳;而直接磁分选分离出来的T淋巴细胞为纯净的T淋巴细胞,而且RNA的质量良好.提示直接磁分选技术是分离鼻咽癌基质T淋巴细胞的首选方法.     

Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

We describe a novel technique, low surface energy Gas Expansion Molding （GEM）, to fabricate microbubble arrays in polydimethylsiloxane （PDMS） which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell cuTture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating （VAC） process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.     

Cell Sorting in Hydra vulgaris Arises from Differing Capacities for Epithelialization between Cell Types

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Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.     

Mitochondrial Staining Allows Robust Elimination of Apoptotic and Damaged Cells during Cell Sorting

High-speed fluorescence-activated cell sorting is relevant for a plethora of applications, such as PCR-based techniques, microarrays, cloning, and propagation of selected cell populations. We suggest a simple cell-sorting technique to eliminate early and late apoptotic and necrotic cells, with good signal-to-noise ratio and a high-purity yield. The mitochondrial potential dye, TMRE (tetramethylrhodamine ethyl ester perchlorate), was used to separate viable and non-apoptotic cells from the cell sorting samples. TMRE staining is reversible and does not affect cell proliferation and viability. Sorted TMRE⁺ cells contained a negligible percentage of apoptotic and damaged cells and had a higher proliferative potential as compared with their counterpart cells, sorted on the basis of staining with DNA viability dye. This novel sorting technique using TMRE does not interfere with subsequent functional assays and is a method of choice for the enrichment of functionally active, unbiased cell populations.     

Improved Phycocatalysis of Carotene Production by Flow Cytometry and Cell Sorting

Using flow cytometry, strains of Dunaliella salina that produce large amounts of beta-carotene were sorted from mixed populations. When grown in large outdoor bioreactors, one carotene-producing strain thrived and was phenotypically stable throughout the three year trial. The use of this high-yield strain in industrial-scale processes doubled beta-carotene productivity. Flow cytometry provided a rapid and precise method to identify and isolate algal strains of potential commercial value.     

Improved Phycocatalysis of Carotene Production by Flow Cytometry and Cell Sorting

Using flow cytometry, strains of Dunaliella salina that produce large amounts of beta-carotene were sorted from mixed populations. When grown in large outdoor bioreactors, one carotene-producing strain thrived and was phenotypically stable throughout the three year trial. The use of this high-yield strain in industrial-scale processes doubled beta-carotene productivity. Flow cytometry provided a rapid and precise method to identify and isolate algal strains of potential commercial value.     

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations. FACS is a technique of choice to purify cell populations of known phenotype. Other bulk methods of purification include panning, complement depletion and magnetic bead separation. However, FACS has several advantages over other available methods. FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density. In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker. FACS allows the purification of individual cells based on size, granularity and fluorescence. In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1). Negative selection of unstained cells is also possible. FACS purification requires a flow cytometer with sorting capacity and the appropriate software. For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser. The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells. The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2). The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb. Sorting parameters can be adjusted depending on the requirement of purity and yield. Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.