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卵巢癌细胞系RMG-I Lewis(y)抗原含量变化对其卡铂耐药性的影响

生物化学与生物物理进展 2008年第10期35卷 1175-1182页

作者：赵越林蓓郝莹莹严丽梅刘娟娟朱连成张淑兰中国医科大学附属盛京医院妇产科沈阳110004 大连医科大学附属二院妇产科大连116021

利用Lewis(y)抗原稳定高表达细胞株RMG-I-H,研究细胞表面Lewis(y)抗原含量变化与细胞对卡铂耐药性的关系.利用四甲基偶氮唑盐(MTT)法测定不同浓度卡铂作用后细胞系RMG-I—H及其对照组RMG-I、RMG-I—C的细胞生长抑制率(IR);利用流式细胞... 详细信息

利用Lewis(y)抗原稳定高表达细胞株RMG-I-H,研究细胞表面Lewis(y)抗原含量变化与细胞对卡铂耐药性的关系.利用四甲基偶氮唑盐(MTT)法测定不同浓度卡铂作用后细胞系RMG-I—H及其对照组RMG-I、RMG-I—C的细胞生长抑制率(IR);利用流式细胞仪(FCM)分析细胞凋亡,同时检测药物作用后细胞的凋亡比率;利用荧光染色法和透射电镜进一步观察细胞凋亡状态.结果表明,RMG-I-H的半数抑制浓度(IC50)为(58.07±2.42),明显高于对照组RMG—I(28.83±3.57)和RMG-I-C(25.71±8.24)的IC50(P〈0.01),后两者间无统计学差异(P〉0.05),流式细胞仪分析证实3组细胞均存在凋亡,经30mg/L、60mg/L卡铂处理后细胞再进行流式细胞仪检测,RMG—I-H的相对凋亡率分别为(20.43±0.71m和(38.11±0.33)%,为对照组的49%-63%,明显低于对照组(P〈0.05),2个对照组间比较无统计学差异(P〉0.05),荧光染色和透射电镜直接观察了细胞形态变化,每个浓度组RMG-I-H的凋亡程度均低于RMG-I和RMG-I-C.说明细胞表面Lewis(y)抗原含量增加的同时,卵巢癌细胞对卡铂的耐药性增强.

关键词： Lewis血型系统半数抑制浓度卡铂流式细胞术/仪器和设备卵巢肿瘤/病理学细胞凋亡药物耐受性荧光染料女(雌)性

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微电流刺激表皮角朊细胞体外增殖的培养模型

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中国临床康复 2005年第34期9卷 74-76页

作者：岳海岭彭代智董征学林恒李芳周新刘敬第三军医大学西南医院全军烧伤研究所重庆市400038

目的:建立微电流作用于角朊细胞的体外模型,观察微电流对角朊细胞增殖等细胞行为的影响。方法:实验于2003-08/2004-08在第三军医大学烧伤研究所实验室完成。所用细胞来自西南医院泌尿外科3例行包皮环切术患者的手术切除包皮标本,患者均... 详细信息

目的:建立微电流作用于角朊细胞的体外模型,观察微电流对角朊细胞增殖等细胞行为的影响。方法:实验于2003-08/2004-08在第三军医大学烧伤研究所实验室完成。所用细胞来自西南医院泌尿外科3例行包皮环切术患者的手术切除包皮标本,患者均知情同意,每个标本均独立进行实验,分为对照组和电刺激组。模型建立:在细胞培养皿中部组建3.0cm×1.0cm×1.0cm大小的培养小室,底部分别用镍铬合金丝或纯铂丝作为电极,同时自行制作输出电流范围为4～1800μA的微电流发生器。电刺激后观察两种电极周围是否发生影响细胞生长的副反应,选择未发生副反应的电极应用于模型中。把手术切除的包皮皮片用中性蛋白酶和胰蛋白酶消化以获取角朊细胞,精制无血清培养基培养。电刺激组给予一定强度频率和时间脉冲交流电刺激后,用碘化丙啶染色,选流式细胞术分析两组角朊细胞周期变化。结果:①镍铬电极的培养模型,制作比较复杂,电极周围易发生通电引起的副反应,电刺激1d后,电极附近生出淡绿色电解反应产物;纯铂电极的培养模型,制作过程相对简单,持续7d电刺激后,电极周围无化学反应,该模型选用了纯铂电极。②自制的微电流发生器输出功率稳定,电流强度可在4.0～1800μΑ之间调节。③角朊细胞DNA合成前期对照组比电刺激组所占百分率明显降低[(52.76±3.57)%,(46.63±0.38)%,t=2.953,P<0.05];合成期、合成后期和分裂期电刺激组所占百分率稍高于对照组[(29.54±16.27)%,(27.02±18.27)%;(23.84±16.48)%,(20.21±17.90)%],说明该组角朊细胞DNA合成活跃,细胞分裂数目增多。④在纯铂电极的培养模型中,电刺激4d后,角朊细胞呈多角形,核分裂相多见,细胞生长旺盛,在小室中的融合现象更加明显。结论:微电流刺激下,角朊细胞DNA的合成活跃,细胞分裂数目增多。自制纯铂电极培养模型输出功率稳定,电流强度可控,能满足细胞长期电刺激培养的需要,为进一步研究电流对细胞增殖的影响提供了技术平台。

关键词：流式细胞术/仪器和设备角朊细胞细胞培养

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双光源流式细胞术三参数细胞周期分析(英)

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Oncology and Translational Medicine 2005年第2期4卷 76-82页

作者：冯永东陶德定覃吉超武汉华中科技大学同济医学院附属同济医院肿瘤研究所、分子医学中心430030

目的:建立双光源流式细胞仪三参数细胞周期分析方法,同时检测两种细胞周期素和细胞DNA含量,分析非时相性的细胞周期素的表达。方法:运用免疫荧光标记技术对两种cyclin标记,一种用直接法(FITC标记),另一种用间接法(RPE-Cy5标记的二抗),... 详细信息

目的:建立双光源流式细胞仪三参数细胞周期分析方法,同时检测两种细胞周期素和细胞DNA含量,分析非时相性的细胞周期素的表达。方法:运用免疫荧光标记技术对两种cyclin标记,一种用直接法(FITC标记),另一种用间接法(RPE-Cy5标记的二抗),同时进行细胞核染色(荧光素DAPI),双光源二点激发,三波段定量检测,数据运用cellquest软件分析。mAMSA和mimosine诱导非时相性的细胞周期素表达。结果:两种cyclin可同时检测,互不干扰,而且不需要荧光补偿,与单标相比无明显差别,cyclin及DNA均可半定量分析。CyclinA/cyclin E/DNA分析可将对数生长期的MOLT-4细胞分为六个亚细胞群(G10、G1e、G1l、S、G2、M),mimosine处理的细胞可见G1期cyclin B1表达,mAMSA处理的细胞有G2期cyclin E的异位表达。结论:运用免疫荧光技术在流式细胞仪上三参数细胞周期分析可以快速定量检测两种cyclin和DNA含量,更精确地对细胞增殖进行分析。

关键词： DNA/分析 G2期流式细胞术/仪器和设备细胞培养的细胞分裂细胞增殖细胞周期细胞周期蛋白类荧光抗体技术

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A three-dimensional numerical fluid dynamic model of antigen-antibody surface adsorption on piezoelectric immunosensors

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ANNALS OF BIOMEDICAL ENGINEERING 2002年第10期30卷 1313-1322页

作者： Barak-Shinar, D Rosenfeld, M Zisman, E Abboud, S Tel Aviv Univ Fac Engn Dept Biomed Engn IL-69978 Tel Aviv Israel Tel Aviv Univ Fac Engn Dept Fluid Mech & Heat Transfer IL-69978 Tel Aviv Israel Savyon Diagnost Ltd Ashdod Israel

A piezoelectric crystal is a unit that changes its frequency in parallel with a change in its mass. This characteristic is exploited in designing flow cell-based immunosensors for detecting the concentration of antibodies in liquid samples. In the present study, computational fluid dynamic techniques are used to optimize the antigen-antibody binding process on an electrode surface placed on the base of a conical flow cell. The geometry optimization of the flow cell was determined to minimize the test time. This time is needed for the electrode to be saturated by the antibody, a process that requires the maximization of the adsorption rate and be accomplished by increasing the shear rate in the vicinity of the electrode. To validate the numerical model and to determine its parameters, experiments were carried out using an identical flow cell. In the experiments, the system did not reach saturation within an acceptable time frame, therefore, the model parameters were determined based on the unsaturated state. The experimental results confirmed the applicability of numerical simulations in predicting the effect of changing the inlet section area of the flow cells, proving the computational model to be very valuable in designing immunosensors based on flow cells. (C) 2002 Biomedical Engineering Society.

关键词：吸附抗体/分析抗体/化学抗体细菌/分析抗体细菌/免疫学抗体单克隆/分析抗体单克隆/免疫学抗原抗体复合物/分析抗原抗体复合物/化学生物传感技术/仪器和设备计算机模拟计算机辅助设计结晶电化学/仪器和设备电化学/方法电极设备设计/方法大肠杆菌/免疫学流式细胞术/仪器和设备流式细胞术/方法免疫测定/仪器和设备免疫测定/方法模型生物学模型化学

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A microfluidic device for measuring cellular membrane potential

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ANALYTICAL BIOCHEMISTRY 2001年第2期295卷 138-142页

作者： Farinas, J Chow, AW Wada, HG Caliper Technol Corp Cell Biol & Microfluid Grp Mt View CA 94043 USA

Recent developments in microfluidics have enabled the design of a lab-on-a-chip system capable of measuring cellular membrane potential. The chip accesses liquid samples sequentially by sipping from a microplate through a capillary, mixes the samples with cells flowing through a microchannel, contacts the cells with potential-sensitive dyes, and reads out cellular responses using fluorescence detection. The rate of cellular uptake of membrane-permeable, ionic fluorophores by THP-I cells was found to depend strongly on membrane potential. The ratio of the fluorescence of the anionic dye DiBAC(4)(3) and the cationic dye Syto 62 taken up by cells was found to double for every 33 mV change in membrane potential. The utility of this approach was demonstrated by assaying ion channel activity in human T lymphocytes. Because of the high sensitivity, low cellular and reagent consumption, and high data quality obtained with the microfluidic device, the lab-on-a-chip system should be widely applicable in high-throughput screening and functional genomics studies. (C) 2001 Academic Press.

关键词：巴比妥酸盐类/代谢细胞系细胞生理学计算机流式细胞术/仪器和设备流式细胞术/方法荧光染料异恶唑类/代谢膜电位机器人 T淋巴细胞/生理学

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Flow cytometry in biotechnology

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APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 2001年第3-4期56卷 350-360页

作者： Rieseberg, M Kasper, C Reardon, KF Scheper, T Inst Tech Chem D-30167 Hannover Germany Colorado State Univ Dept Chem & Bioresource Engn Ft Collins CO 80523 USA

Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook.

关键词：细菌/细胞学细菌/生长和发育细菌生理学生物技术/方法细胞培养技术/方法细胞死亡细胞分裂细胞生理学细胞存活流式细胞术/仪器和设备流式细胞术/方法酵母菌酿酒/细胞学酵母菌酿酒/生长和发育动物人类

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Using the microcyte flow cytometer to monitor cell number, viability, and apoptosis in mammalian cell culture

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BIOTECHNOLOGY PROGRESS 2000年第5期16卷 800-802页

作者： Harding, CL Lloyd, DR McFarlane, CM Al-Rubeai, M Univ Birmingham Sch Chem Engn Ctr Bioproc Engn Birmingham B15 2TT W Midlands England Aber Instruments Ltd Aberystwyth SY23 3AH Dyfed Wales

The Microcyte is a novel, portable flow cytometer based on diode laser technology,whose use has been established for yeast and bacterial analysis. We present data that demonstrate its suitability for routine mammalian cell counting and viability determination. To extend its range of applications in the field of animal cell culture biotechnology, a test to determine the number of apoptotic cells present has been developed for use with the instrument. Apoptosis was induced in hybridoma cell cultures by treatment with camptothecin. Apoptotic cells were labeled with biotinylated Annexin V and then visualized using a streptavidin-allophycocyanin conjugate. Their numbers were counted, and the cell size of the apoptotic cell population was determined using the Microcyte.

关键词：细胞凋亡细胞分离细胞存活流式细胞术/仪器和设备杂交瘤/细胞学动物小鼠

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Multiparameter flow cytometry (Reprinted from Immunochemistry, pg 849-866, 1994)

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JOURNAL OF IMMUNOASSAY 2000年第2-3期21卷 255-272页

作者： Stewart, CC [a] Roswell Park Cancer Institute Buffalo New York

Click to increase image sizeClick to decrease image size

关键词：流式细胞术/仪器和设备流式细胞术/方法荧光抗体技术/方法免疫表型分型/仪器和设备免疫表型分型/方法淋巴细胞亚群/分类动物人类

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DNA ploidy status and proliferative activity as markers of malignant potential in Barrett's esophagus:: Flow cytometric study using routinely paraffin-embedded tissue

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WORLD JOURNAL OF SURGERY 2000年第1期24卷 72-77页

作者： Giménez, A Minguela, A de Haro, LM Parrilla, P Bermejo, J Pérez, D García, AM Ortiz, MA Molina, J Alvarez, R Virgen Arrixaca Uni Hosp Dept Pathol Murcia 30120 Spain Virgen Arrixaca Uni Hosp Dept Immunol Murcia 30120 Spain Virgen Arrixaca Uni Hosp Dept Surg Murcia 30120 Spain Univ Murcia Fac Med Dept Biostat Murcia 30080 Spain Virgen Arrixaca Univ Hosp Dept Gastroenterol Murcia 30120 Spain

Regular endoscopic surveillance is recommended for patients with Barrett's esophagus to detect dysplasia and to diagnose carcinoma while it is in an early and possibly treatable stage. However, there are numerous unknown aspects regarding the natural history of dysplasia in this disease, and there is still a need for more accurate markers of risk of a malignant change. The aim of this study was to investigate the usefulness of DNA pow cytometry in Barrett's esophagus to define subgroups of patients showing similar histologic findings but with a different malignancy potential. Routinely formalin-fixed and paraffin-embedded tissues of 43 patients with Barrett's esophagus were processed for flow cytometric measurements (ploidy, proliferative index) and the results were compared with the histologic evolution observed in these patients. Only in the group of patients with "indefinite" dysplasia did me find statistically significant differences between the samples from patients with and without progression to more severe lesions (mean proliferative index of stable patients: 5.2% versus 8.3% in patients with progression,p = 0,001, Student's t-test), The presence in the how cytometric analysis of a DNA aneuploid cell line is closely related to the presence of severe histologic alterations (i.e,, high-grade dysplasia: p < 0.001, Fisher's exact test). Our results suggest that this procedure is at least capable of distinguishing between a real, although incipient, neoplastic process and morphologic changes of a reactive or reparative type. The increment in the tissue proliferative index could be an indicator of an early genomic instability which, with time, will develop into lesions with a more altered DNA content: aneuploidy.

关键词：腺癌/遗传学腺癌/病理学方差分析 Barrett食管/遗传学 Barrett食管/病理学 DNA 肿瘤/遗传学食管肿瘤/遗传学食管肿瘤/病理学食管/病理学流式细胞术/仪器和设备流式细胞术/方法流式细胞术/统计学和数值数据倍性癌前状态/遗传学癌前状态/病理学成年人老年人女(雌)性人类男(雄)性中年人

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Detection of Cryptosporidium oocysts in water:: techniques for generating precise recovery data

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JOURNAL OF APPLIED MICROBIOLOGY 1999年第6期87卷 804-813页

作者： Reynolds, DT Slade, RB Sykes, NJ Jonas, A Fricker, CR Thames Water Util Reading RG2 0JN Berks England

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was del eloped for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (s.w. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique Mras found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.

关键词：小隐孢子虫/分离和提纯流式细胞术/仪器和设备淡水/寄生虫学免疫磁化分离/仪器和设备显微镜检查共焦孢子动物

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