抗肿瘤药物筛选中MTT法和SRB法的比较

在抗肿瘤药物的体外筛选中 ,MTT法和 SRB法是常用的两种方法。我们用MTT法和 SRB法分别测定 3种已知植物抗癌药对 2 2株人肿瘤细胞的抗癌活性 ,对这两种方法进行了详细的比较。通过分析两种方法测出的细胞存活率 ( T/ C)的差异分布和相关系数以及 IC50 的二变量分布 ,比较了两种方法测定结果的异同 ;通过两种方法重复测定 3种药物对 7株人癌细胞的抗癌活性 ,比较了两种方法的重复性 ;通过分析两种方法测定结果 T/ C值随时间变化的程度 ,比较了两种方法测定结果的稳定性。实验结果表明 :MTT法和 SRB法的相关性较好 ,都可用于抗肿瘤药物的体外筛选 ,SRB法更适合于大规模筛选 ,3种抗癌药物的测定结果与临床资料基本一致。     

两种新型培养细胞数量检测技术的探索

将SRB比色法和结晶紫比色法与MTT比色法与MTT比色法在96孔板培养HeLa细胞的数量及活力测量精确度和操作方法上进行了对比。试验结果显示：SRB比色法和结晶紫比色法的最佳检测波长均为490nm。最佳染液浓度分别为0.4%和0.25%。SRB比色法的检测精确度略逊于MTT法，结晶紫比色法与MTT法无显著差异。另外，此两种检测方法的操作过程更为简便，所需时间也较短，可在一定范围内取代MTT比色法。     

MTT比色法抗肿瘤药物筛选实验条件和数据优化探索

MTT比色法是一种重要的体外抗肿瘤药物筛选方法.以PC-3细胞系为研究对象,对影响MTT比色法抗肿瘤药物筛选实验的主要因素一细胞密度、实验操作环节、OD值选取、以及数据的优化处理进行实验探讨.结果表明,当检测化合物6 h～72 h的抑制活性时,种细胞密度2 000个/孔为宜.同时,采用本文所提的实验条件和数据处理办法,可以实现实验结果的准确可靠,3次测试偏差不超过10%.     

两种体外细胞毒性检测方法的比较研究

目的比较两种常用的细胞毒性检测方法在医疗器械生物学评价中的相关性。方法分别采用MTT比色法和细胞增殖度法,在37℃条件下,将五种医疗器械／生物材料的浸提液分别与小鼠成纤维细胞（L-929）接触2天和2,4,7天,比较材料对细胞的毒性影响。结果5种不同的材料浸提液分别表现出不同程度的细胞毒性反应（0～2级）。将MTT比色法与细胞增殖度法（2天）的实验数据进行相关性分析,显示两者之间具有良好的相关性（R=0．977）。结论MTT比色法由于其检测所需的细胞量相对较少,试验步骤相对简便、检测周期短,因此具有一定的优越性,是个值得推荐的细胞毒性检测方法。     

热带太平洋深海微生物抗肿瘤活性的初步研究

以４个人体癌细胞株为模型、利用四甲基偶氮唑盐（Methyl Thiazolyl blue Tetrazolium bromide,MTT）比色法和磺酰罗丹明（SulfoRhodamine B,SRB）染色法对55株来自于热带太平洋深海微生物（细菌和霉菌）的培养液的乙酸乙酯抽提物以及菌体的甲醇提取物进行了细胞毒活性筛选,并主要采用分子生物学方法鉴定了该批菌株. 结果表明,55株微生物发酵样品共１１０个提取物中,９０％样品表现出细胞毒活性;其中13株微生物的活性较强（提取物有效抑制浓度≤16μg/ml）,具有较好的开发应用前景。同时还发现,菌体中检测到的活性菌株数大于发酵液中检测到的活性菌株数, 细菌的筛选得率高于霉菌的筛选得率.鉴定结果显示, 50株微生物分属于21属、29种,其中１３个较高活性菌株来源于８个属。本文为我国深海微生物资源的开发利用,提供了探索性研究信息。     

MTT法检测细菌细胞数的主要影响因素分析

MTT法是生物活细胞计数的灵敏、快速和便捷方法。报告对MTT法应用于细菌细胞数定量分析时的分析前培养时间、MTT反应时间、MTT剂量和检测波长等主要影响因素的分析结果,同时报告MTT反应产物溶解检测与直接检测,以及MTT法与平板菌落计数法定量检测的结果比较。结果如下：Formazan生成量与细菌活细胞数呈正相关,对数生长期变化最明显;MTT为0.25 mg/mL,反应2 h时吸光值与菌落数具有良好的线性关系（r=0.999 8）;Formazan的最大吸收波长为570～580 nm;MTT反应产物溶解检测和直接检测结果经统计学分析无显著性差异（P〉0.05）;细菌细胞数在107～109 cfu/mL范围内,MTT法与菌落计数法定量结果呈现良好的线性关系（r≥0.991 7）。     

东亚钳蝎毒素对Caco-2人结肠癌细胞的体外药理学研究

研究东亚钳蝎毒素对人结肠癌细胞Caco-2增殖的影响。以不同浓度的东亚钳蝎（Buthus martensii Karsch）毒素（10、20、40滋g/mL）干预体外培养的Caco-2细胞,分别于24 h、48 h后,用四甲基偶氮唑盐（MTT）比色法,观察毒素对Caco-2细胞的增殖抑制作用。运用淋巴细胞转化实验和乳酸脱氢酶（LDH）释放实验检测蝎毒素对Caco-2细胞的作用途径。结果表明：东亚钳蝎毒不仅能抑制Caco-2细胞的增殖而且能促进淋巴细胞转化,毒素对Caco-2细胞增殖的抑制作用与浓度和作用时间密切相关。     

蝎毒抗癌组分SVⅡ分离法的优化研究

目的比较三种柱径的分子筛G-50凝胶层析柱分离东亚钳蝎蝎毒的柱效;并对分离所得组分作MTT（酶反应比色法）抗肿瘤活性作用研究,为从中研制和开发出高效、低毒的新型抗癌特效药筛选出目标组分。方法（1）采用三种规格的分子筛层析柱分离蝎毒;（2）HPLC色谱分析比较各组分的指纹图谱;（3）MTT法观察不同浓度（1、10、100mg／L）的蝎毒及其组分对四种肿瘤细胞（HL-60、A549、K562／ADR、K562／S等）的毒性作用。结果经过分子筛柱层析,可从蝎毒（Scorpion venom,SV）获取三个组分SVⅠ、SVⅡ、SVⅢ;经HPLC色谱分析,各组分明显含有四种以上单体成分;MTT法研究表明,SVⅡ对四种肿瘤细胞的细胞毒性较原毒强,剂量-效应关系较好,而SVⅠ、SVⅢ对四种肿瘤细胞抑制作用不明显。结论（1）利用大柱径的层析柱分离蝎毒的柱效较高;（2）组分SVⅡ是蝎毒抗癌的目标组分,且其对耐药细胞株（K562／ADR）的抑制作用比阳性对照组强,有待进一步的分离纯化,筛选出色谱纯的抗癌活性成分（多肽单体）。     

细胞色素C在apoptin诱导宫颈癌Hela细胞凋亡中的作用

目的研究肿瘤特异性凋亡基因（apoptin）在诱导Hela细胞凋亡中的信号转导机制。方法用含有apoptin基因的真核表达载体瞬间转染体外培养的Hela细胞;采用MTT法检测Hela细胞的凋亡;以比色法检测caspase-8和caspase-3的相对活性;Western blotting检测凋亡细胞中细胞色素C的表达量。结果 MTT法证明ap-optin基因瞬间转染的Hela细胞凋亡率明显高于其他各组（P〈0.01）;caspase-3的活性升高,但caspase-8活性没有明显变化;细胞色素C释放量明显增多。结论 Apoptin基因可能通过促进线粒体释放细胞色素C激活caspase-3,进而诱导Hela细胞凋亡。     

MTT法用于药物对L2细胞毒性的实验研究

目的：评价噻唑蓝（MTT）法检测药物对细胞的毒性作用的可靠性。方法：大鼠肺泡上皮L2细胞以叔丁基对苯二酚（TBHQ）10．100μM,BsO以1-10mM分别处理,用MTT法检测细胞活性、JC-1（5,5’,6,6’-四氯．1,1’,3,3’-四乙基苯并咪唑羰花青碘化物）荧光染料法检测细胞线粒体电位改变、台盼蓝排斥实验检测细胞死亡率,分析各指标的情况。结果：在处理剂量范围,MTT法检测到的光密度（OD）值未能达到一般判断的半数抑制浓度（ic50）水平,最高抑制率仅达到30％左右;台盼蓝排斥试验检测数据表明TBHQ的LC50值为50μM,丁硫氨酸亚砜胺（BSO）为5mM;利用JC-1荧光染料判断的半数凋亡剂量分别为50μM和7mM。结论：MTT法作为最常采用的细胞生长抑制检测手段,但在某些特定实验中可能不能客观地反映细胞的活性,建议多种方法结合进行评价。     

MTT比色法测定促肝细胞生长物质对肝细胞生长的刺激活性

本实验建立了用简便的ＭＴＴ比色法对促肝细胞生长物质的促肝细胞增殖作用的测定方法,确定了实验的最适条件。与传统的３Ｈ ＴｄＲ掺入法进行比较的结果显示,ＭＴＴ比色法与３Ｈ ＴｄＲ掺入法测定结果基本相符,灵敏度相近,但消除了同位素的污染,是一个测定促肝细胞生长物质刺激肝细胞增殖活性的简便方法。     

A study of MTT-hybrid assay using human bone and soft tissue tumor cells

We investigated a new chemosensitivity test, MTT-hybrid assay, which was a hybrid of MTT colorimetric assay and double-layered soft agar colony assay, using human bone and soft tissue tumor cells. MTT formazan crystals produced by viable cells in the soft agar medium were solubilized by SDS at 60 degrees C. The absorbance (560 nm) is directly proportional to the cell number over a wide range. The absorbance increased in proportion to colonial growth of osteosarcoma cells, while it decreased in a human diploid cell strain in a few days. Drug sensitivity of tumor cells is supposed to be assessed without contaminating normal cells by MTT-hybrid assay in primary tumor samples. Good correlation of IC50 was observed between MTT-hybrid assay and colony assay. The MTT-hybrid assay shows potential value as a rapid predictive test for chemotherapeutic agents in an individual patient.     

一种新的以影像为基础的细胞增殖和细胞毒性检测方法

3-(4,5-二甲基-2-噻唑)-2,5-二苯基溴化四唑盐)（MTT）比色法是传统上检测细胞增殖和细胞毒性的常用方法.
CloneSelectTM成像系统是一种以影像为基础的用于分析细胞生长的可视检测系统.本研究采用人结直肠癌HCT116细胞系,运用CloneSelect成像系统和MTT方法分别检测药物阿的平的细胞毒性,并采用Bland Altman作图法比较两种实验方法获得的pEC50值,分析两种研究方法获得的结果的一致性. 结果表明,CloneSelectTM成像系统和MTT法获得的pEC50值具有较好的一致性.与MTT方法相比,基于影像的CloneSelectTM成像分析技术检测快速、无损伤且结果更准确,获取资料不损伤细胞,允许后续其它时间点或动力学检测. 研究提示,这种新的以影像为基础的检测技术可以替代MTT方法,用于分析不同药物的抗细胞增殖活性.     

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 µg and 100 µg/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.     

A microplate spectrofluorometric assay for bacterial biofilms

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r ²=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.     

Calibration of MTT assay in proton beams using radiochromic films

PurposeThis study provides methodology of calibrating as well as controlling the output for an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay irradiated in a low energy proton beam using EBT3-model GAFCHROMIC^TM film, without correcting for quenching effect.MethodsA calibrated Markus ionization chamber was used to measure the depth dose and beam output for 26.5 MeV protons produced by a CS30 cyclotron. A time-controlled aluminum cylinder was added in front of the horizontal beam-exit serving as a radiation shutter. Following the TRS-398 reference dosimetry protocol for proton beams, the output was calibrated in water at a reference depth of 3 mm. EBT3 film was calibrated for doses up to 8 Gy at the same depth. To verify the dose distribution for each 96-well MTT assay plate, EBT3 film was placed at the reference depth during irradiation and cell doses were scaled by measured percent depth dose (PDD) data.ResultsThe radiochromic film dosimetry system in this study provides dose measurements with an uncertainty better than 3.3% for doses higher than 1 Gy. From a single exposure and utilizing the Gaussian shape of the beam, multiple dose points can be obtained within different wells of the same plate ranging from 6.9 Gy (sigma ∼4%) in the central well, and 2 Gy (sigma ∼8%) for wells positioned closer to the periphery.ConclusionsWe described a methodology for radiochromic film-based dose monitoring system, using low-energy protons, which can be used for the MTT assay in any proton beam, except within Bragg peak region.     

A rapid colorimetric assay for carbamyl phosphate synthetase I

A rapid, reproducible, and sensitive colorimetric assay for carbamyl phosphate synthetase I was presented. A four-fold increase in sensitivity and reduced assay time were afforded by this procedure. The method utilized the chemical conversion of carbamyl phosphate to hydroxyurea by the action of hydroxylamine instead of employing a coupling enzyme. The hydroxyurea was quantitated in 15 min by an improved colorimetric assay for ureido compounds by measuring the absorption of the resulting chromophore at 458 nm. Optimum conditions for both the formation and quantitation of hydroxyurea were established. Activity measurements of carbamyl phosphate synthetase I obtained by this uncoupled method were identical with those obtained by the ornithine transcarbamylase coupld assay.     

3种方法检测体外神经细胞存活的技术探讨

为了准确、客观、快速、高效地反映体外培养细胞存活的情况,将PC12细胞以不同密度接种于96孔板中,培养48h后,然后采用结晶紫比色法,中性红比色法,MTT比色法来检测细胞的存活情况,再将所得结果进行比较,比较3种方法的优缺点,寻求最佳检测细胞存活方案．结果发现,不同方法检测细胞存活的范围各不相同．其中结晶紫比色法细胞存活数与比色光吸收值（absorbance value,A值）呈正相关程度较其它两种好,而且检测细胞存活范围最为宽广．     

多孔板-MTT比色法评价植物和微生物代谢产物的抗真菌活性

多孔板-MTF比色法测定植物和微生物代谢产物对真菌抑制活性的步骤为:在多孔板的每孔中依次加入浓度为105孢子/mL的供试真菌孢子悬液90μL,不同浓度的药液10 μL.25℃暗培养48 h,然后每孔中加入8mg/mL的MTT溶液10μL,继续培养10 h后,离心去上清,加入DMS0 150 μL,振荡30 min,离心后上清液在510nm测定吸光值.采用上述条件测定了白屈菜红碱对稻瘟病菌和西瓜枯萎病菌的MIC值分别为80和1.5μg/mL,IC50值分别为21.99和0.78 μg/mL;Diepoxinζ对稻瘟病菌的MIC和IC50值分别为200和96.21 μg/mL.多孔板-MTT比色法为快速有效地筛选和评价植物和微生物抗真菌活性成分创造了条件.