SRB法和MTT法抗肿瘤药物筛选结果相关性研究

为了探索抗肿瘤药物体外筛选中磺酰罗丹明B（SRB）法和四甲基偶氮唑盐（MTT）比色法实验结果的相互验证作用,作者以PC-3、BGC-823、Bcap-37细胞系为研究对象,利用SRB法和MTT比色法对供试化合物行体外抑制活性考察,并用SPSS12．0对两种筛选方法得到的实验结果进行差异性分析。结果表明,SRB法和MTT比色法实验结果相关性好,可以相互验证实验结果的准确性和可靠性。     

Comparison of 5 microplate colorimetric assays forin vitro cytotoxicity testing and cell proliferation assays

This paper describes a critical comparative evaluation of 5 miniaturised colorimetric assays applicable to cytotoxicity testing of anti-tumour drugs (and other toxins)in vitro. Each assay shows a different linear range for optical density versus cell number, a different sensitivity to change in cell number and a different minimum detectable cell number; the values of these parameters vary with experimental conditions and with cell line used. All the methods gave good correlation with viable cell number (determined by colony forming efficiency) in toxicity assays after 3 or 4 days of treatment, but they underestimated cell death after 2 days. Toxicity levels for individual chemicals (in a standard 6-day assay) are similar for the different assays, irrespective of the mechanism of action of the chemical being tested. Two of the more recently developed assays (APNaOH and SRB) were found to be very sensitive under the conditions examined.     

Improving fluorescence-based assays for the in vitro analysis of cell adhesion and migration

Cell adhesion and cell migration are two primary cellular phenomena to be approached in vitro in order to allow for the effective dissection of the individual events and the unravelling of their underlying molecular mechanisms. The use of assays dedicated to the analysis of cell adhesion and migration in vitro also affords an efficient way of conducting larger basic and applied research screenings of the conditions affecting these processes and are potentially exploitable in the context of routine tests in the biological and medical fields. Therefore, there is a substantial interest in devicing more rationale such assays and major contributions in this direction have been provided by the advent of procedures based on fluorescent cell tagging. In this article we describe three fluorescence-based model assays for the qualitative and quantitative assessment of cell adhesion and cell locomotion in static and dynamic conditions. The assays are easily performed, accurate and reproducible, and can be automatized for high-throughput screenings of cell behavior in vitro. Performance of the assays involves the use of certain dedicated disposable accessories, which are commercially available, and a few instruments that, due to their versatility, can be regarded as constituents of a more generic laboratory setup.     

Differences in sperm function in vitro but not in vivo between inbred and random-bred mice

Genetic variation in spermatozoa was used to examine mechanisms important for fertilization in the mouse. A significantly greater proportion of cauda epididymal sperm from C57BL/6 (inbred) males were motile than from random-bred (CFW) males. Random-bred sperm, however, were able to fertilize a significantly greater percentage of eggs in vitro than were inbred sperm. When sperm of these two genotypes were used for insemination in vivo, and the penetrated eggs cultured through the first cleavage, the levels of cleavage were similar, suggesting that neither levels of sperm motility nor sperm penetration in vitro accurately reflect the ability of the same sperm populations to penetrate eggs in vivo.     

Differences between Dianthus caryophyllus L. cultivar in in vitro growth and morphogenesis are related to their ethylene production

The involvement of ethylene in the vitro development of shoots from nodal segments of two cultivars of carnation (Dianthus caryophyllus L.) was studied. Shoots of cv. Barbaret Antares showed low hyperhydricity in contrast with the high levels showed by cv. Barbaret Tanga when both were cultured in airtight culture vessels. Longer shoots were produced, in both cases, when the rate of gas exchange in the culture vessel was increased by using vented closures, which also prevented hyperhydricity and increased the multiplication coefficient in cultures of Barbaret Tanga.The two cultivars produced ethylene throughout the culture period but, a higher amount was produced during the first, second and fourth weeks in culture by the cultivar more sensitive to ventilation (Barbaret Tanga). Trapping ethylene did not produce any effect on cv. Barbaret Antares but improved the quality of cv. Barbaret Tanga explants, decreasing hyperhydricity and increasing the number of shoots, the length of the main shoot and the multiplication coefficient. These effects were more marked when ethylene was trapped during the first two weeks in culture.     

Differences between Brachiola (Nosema) algerae isolates of human and insect origin when tested using an in vitro spore germination assay and a cultured cell infection assay

Brachiola (Nosema) algerae is a microsporidian species generally believed to be an intracellular parasite of insects, especially mosquitoes. However, both mosquito and human isolates have been shown to infect mammalian cells. The present study was undertaken to determine if spores of two insect and two human isolates of B. algerae cultured at 30 degrees C and 37 degrees C differed in their ability to germinate and infect cultured green monkey kidney cells at these two temperatures. Spores from all four isolates exhibited an optimum pH of 9.5 for germination. Mercury (Hg2+) inhibited germination of all isolates equally. Germination of spores from all four isolates was significantly greater when the parasite was cultured at 30 degrees C than when cultured at 37 degrees C. However, spores from the insect isolates cultivated at 30 degrees C or 37 degrees C infected significantly fewer mammalian cells at 37 degrees C than did spores from the human isolates under the same conditions. Thus, there is no correlation between the effects of temperature on the germination and the infectivity of an isolate. In addition, while exposure of B. algerae to 37 degrees C has been reported to cause spore dysmorphism, we failed to observe any consistent ultrastructural changes that explained the greater infectivity of the human isolates at 37 degrees C.     

Cytomorphological lesions induced by chemotherapeutic agents in transitional cell carcinoma in vitro

Summary Transitional cell carcinoma from 20 patients and two human cell lines were maintained in short-term tissue culture. Each was studied ultrastructurally before and after incubaton with cisplatinum, adriamycin, or mitomycin C. Sequential ultrastructural changes were noted and were found to be specific for each agent tested. Ultrastructural changes in the nucleoli were produced by exposure to cisplatinum or mitomycin C; alterations in the heterochromatin of the nuclei were characteristic of treatment with adriamycin. The changes in the nucleoli seen with cisplatinum have not been described previously and support an alkylating property as a mechanism of action. Intravesical chemotherapeutic agents are now commonly used in clinical treatments. The morphological changes produced by these agents are specific and may be seen in the clinical setting. This research was supported by the Veteran's Administration Merit Review grant.     

Studies on in vitro colony formation by mouse bone-marrow cells using different sources of colony-stimulating factor

Summary Conditioned media of a primary mouse embryo and a mouse cell line were compared as sources of colony-stimulating factor. The incorporation of embryo cell conditioned medium into semisolid cultures of mouse bone-marrow cells induced the formation of a larger number of in vitro colonies than did the addition of equal volumes of LM cell conditioned medium. This finding did not appear to be the result of quantitative differences in the levels of C.S.F. between the sources since concentration of the LM cell conditioned preparation did not enhance effectively the number of colonies produced. Both the colonial morphology and the cellular components of the developing colonies were found to differ in accordance with the C.S.F. source employed for stimulation. Colonies that developed under the influence of embryo cell conditioned medium were typically larger and more disperse than were those produced in cultures stimulated with the LM cell conditioned source. The latter colonies were smaller, more compact and contained fewer cells. Differences also were noted in the relative proportion of granulocytes to mononuclear cells comprising the colonies. Those colonies stimulated with LM cell conditioned medium rapidly underwent a transition from primarily a granulocytic composition to one comprised principally of mononuclear cells. Cultures stimulated with embryo cell conditioned medium contained a greater number of granulocytic colonies which persisted for a protracted period during cultivation. The addition of 2-mercaptoethanol to cultures stimulated with embryo cell conditioned medium increased the number of colonies produced. Such synergy did not occur in cultures stimulated with the LM cell conditioned medium. Supported by United States Public Health Service Research Grant CA 13752.     

The effects of medium composition and culture conditions on in vitro rooting and ex vitro establishment of jackfruit (Artocarpus heterophyllus Lam.)

A sucrose level of 20 g/l in the culture medium and a 12 h photoperiod were required for optimal in vitro rooting of proliferated jackfruit shoots and these culture conditions also promoted subsequent establishment of plantlets in the glasshouse. High agar levels of 10 and 20 g/l reduced the humidity in culture but also reduced in vitro rooting and had no effect on the establishment of plantlets in the glasshouse. A 12 h photoperiod in ex vitro conditions gave greater growth and development of plantlets than 8 h or 16 h photoperiods.     

In vitro effects of ecdysterone on the spermatogonial cell cycle in Locusta

The effect of ecdysterone on specific phases of the cell cycle of Locusta migratoria migratorioides spermatogonia was assayed in vitro. An increase in labeling index was noted, indicating a decrease in duration of the G₁ phase. On addition of the hormone to adapted organs in vitro, spermatogonial mitotic index increases rapidly, then declines to a basal level within a time period which approximates that of the G₂ phase. Such a response indicates a removal of a G₂/M inhibition by the hormone.     

Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development.     

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.     

小鼠睾丸支持细胞体外培养特性

支持细胞是睾丸的重要组成部分,其主要功能是为生精细胞提供适宜的生长环境。从幼鼠睾丸中分离得到支持细胞,并通过苏木精-伊红和Fas-L免疫组化染色对分离得到的细胞进行了鉴定。通过对原代小鼠睾丸支持细胞的贴壁、生长和培养液中葡萄糖、谷氨酰胺、氨基酸等营养底物及其副产物乳酸、铵根离子等的代谢以及培养液渗透压和pH的研究,发现支持细胞的贴壁时间主要集中在接种后2～4h;当培养液中氨根离子浓度高于2.3mmol/L,渗透压高于326mosm/kg,pH≤6.8时支持细胞生长进入衰亡期;在氨基酸代谢方面,发现培养过程中丙氨酸和谷氨酸浓度迅速增加,缬氨酸、亮氨酸和异亮氨酸浓度略有降低,丝氨酸、精氨酸和甘氨酸浓度基本保持不变。因此培养液中铵根离子浓度的过量积累、渗透压和pH的异常和贴壁面积不足是限制支持细胞静态生长的主要因素。研究结果为支持细胞大规模培养及工艺优化奠定了基础。     

Use of skin cell cultures for in vitro assessment of corrosion and cutaneous irritancy

Skin cell culture is one of the most promising tools for in vitro evaluation of both cutaneous irritancy and corrosion. New culture methodologies, including three-dimensional reconstruction of skin, allow the evaluation of a wide range of compounds and complex formulations. A number of tests have already been developed for the evaluation of cytotoxicity and many end-points are now currently used, including cell viability, alteration of cell growth or cell function. In recent years parameters more closely related to in vivo irritancy effects such as synthesis of inflammatory mediators and/or their release by keratinocytes after exposure to potential skin irritants have been evaluated. This paper reviews technological aspects and results of validation using skin cell culture for in vitro assessment of corrosion and skin irritancy. Advantages and limits of skin cell cultures are also presented. Current questions about the validation process of cutaneous irritation and corrosion are also considered.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitution in vitro. The experimental results showed that lamin was involved in the nuclear assembly in vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear lamina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly.     

In vitro activity of immunoconjugates between cisplatin and an anti-CA125 monoclonal antibody on ovarian cancer cell lines

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10±0.42 and 2.65±1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immuno-staining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.     

Development and comparison of nonradioactive in vitro kinase assays for NIMA-related kinase 2

NIMA (never in mitosis arrest)-related kinase 2 (Nek2) is a serine/threonine kinase required for centrosome splitting and bipolar spindle formation during mitosis. Currently, two in vitro kinase assays are commercially available: (i) a radioactive assay from Upstate Biotechnology and (ii) a nonradioactive fluorescence resonance energy transfer (FRET) assay from Invitrogen. However, due to several limitations such as radioactive waste management and lower sensitivity, a need for more robust nonradioactive assays would be ideal. Accordingly, we have developed four quantitative and sensitive nonradioactive Nek2 in vitro kinase assays: (i) a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using peptides identified from a physiologically relevant protein substrate, (ii) DELFIA using Nek2 itself, (iii) a homogeneous time-resolved FRET assay termed LANCE, and (iv) A method of detecting phosphorylated products by HPLC. The DELFIA and LANCE assays are robust in that they generated more than 10-fold and 20-fold increases in signal-to-noise ratios, respectively, and are amenable to robotic high-throughput screening platforms. Validation of all four assays was confirmed by identifying a panel of small molecule ATP competitive inhibitors from an internal corporate library. The most potent compounds consistently demonstrated less than 100 nM activity regardless of the assay format and therefore were complementary. In summary, the Nek2 in vitro time-resolved FRET kinase assays reported are sensitive, quantitative, reproducible and amenable to high-throughput screening with improved waste management over radioactive assays.     

Blackleg potential of potato seed: determination of tuber contamination by Erwinia carotovora subsp. Atroseptica by immunofluorescence colony staining and stock and tuber sampling

Two methods to determine numbers of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, in tuber peel extract were compared; (1) growth and cavity formation on crystal violet pectate (CVP) medium (Pérombelon, Lumb & Hyman, 1987) and (2) immunofluorescent colony (IFC) staining (Van Vuurde & Roozen, 1990) using an antiserum against the bacterium conjugated with fluorescein isothiocyanate. Detection, identification and quantification of the bacterium based on the differential effect of temperature on growth in the CVP method were severely restricted and in some cases could not be done at low peel extract dilutions containing > 106 saprophytic bacteria ml“1 and > 103 cells ml-1 of E. carotovora subsp. carotovora. In contrast, although recovery was c. 50% relative to growth of E.c. atroseptica alone on nutrient agar, numbers of the bacteria could be determined by the IFC method regardless of numbers of saprophytic bacteria and E.c. carotovora present. Moreover, the tedium of counting colonies on a UV microscope could be avoided by automation using an imaging system on photograph film negatives of the microscope fields. Readily accessible tubers from the top layer of one tonne boxes in commercial stores were c. 10 times less contaminated than those from the middle of the boxes. For the two methods of peel extract preparation examined, the estimated sample size needed with an allowable error of log1010 E.c. atroseptica cells ml“1 extract with 95% confidence, was c. five tubers per box and 14 boxes for extract prepared from individual tubers and c. three lots of 10 tubers per box and 10 boxes for extract from 10 pooled tubers. A blackleg potential index for seed stocks was proposed based on the summation of the weighted number of individually tested tubers in different classes of contamination level.     

楸树无性系离体培养特性差异研究

对选育的5个楸树无性系(004-1、1-3、2-6、015-1、1-4)进行组织培养比较研究,以明确楸树无性系再生芽增殖和生根培养中遗传因素及外界条件的影响。结果表明:楸树不同无性系是影响瓶苗生长的主要因素,继代培养无性系间的方差分量在93.89%～98.16%,芽增殖系数、增殖芽数、芽长、叶数、茎段基部愈伤组织横向膨大、茎段基部愈伤组织纵向膨大在不同无性系间达到极显著水平;生根率、生根数和根长无性系间差异极显著,方差分量分别为92.97%、88.75%、96.25%。5个无性系生长性状以004-1表现最好,增殖系数为10.74,生根率为61.11%,移栽成活率为74.44%,无性系1-4最弱。     

组蛋白与hAMFR基因启动子的结合对体外转录活性的影响

采用从鸡血红细胞中分离纯化的组蛋白,从ＨｅＬａ细胞核中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性抽提物,以及从非洲爪蟾卵细胞核中撮以的萃取物热处理物上清用于核小体构建,以含有人自泌移动因子受体基因启动子序列的ＤＮＡ片段为模板进行体外转录实验,结果表明,组蛋白和转录因子在ｈＡＭＦＲ基因启动子序列上的竞争性结合对转录活性具有重要的影响作用,在启动子区域预先构建的核小体能够抑制转录活性;