MTT显色反应实验条件分析

目的 :检验分析MTT实验方法本底高、灵敏度低、稳定性差的影响因素 ,改进实验条件提高灵敏度和稳定性 ,降低本底。方法 :用MTT比色方法 ,细胞毒试验及细胞增殖实验 ,分析比较不同条件下MTT显色反应的性能特点。结果 :比较了 13种溶剂对Formazan的溶解性能。分析检验了琥珀酸脱氢酶在细胞上的分布特点、pH值对MTT反应体系的影响、酚红和牛血清对检验结果的影响 ,提出相应对策。检测了MTT反应时间曲线和细胞活力曲线。分析了改进后的MTT实验方法的灵敏度和稳定性。结论 :以MTT为底物的酶在细胞代谢中主要分布在细胞膜表面。MTT反应体系的pH值为 6 7,比较好的Formazan溶剂是正丙醇、异丙醇和乙二醇乙醚。去掉细胞培养上清 ,检测细胞上的酶 ,用溶剂使蛋白质变性 ,经沉淀去掉蛋白质影响 ,能使MTT实验方法的本底降低 ,灵敏度提高。通常情况下该方法检测K 5 62细胞和人淋巴细胞的灵敏度分别为 10 0 0和 10 0 0 0个细胞左右。     

Studies of effects of anticancer agents in combination with/without hyperthermia on metastasized human bladder cancer cells in chick embryos using the polymerase chain reaction technique

Cultivated T24 cells derived from a human bladder cancer were inoculated into the chorioallantoic membrane vein of chick embryos. Hyperthermic treatment was performed following injection of anticancer agents 3 days after the inoculation of the T24 cells. DNA samples were obtained from the livers of the chick embryos, and the polymerase chain reaction technique was used to amplify a DNA fragment specific to the human -globin gene. The Southern hybridization method was used to evaluate the inhibitory effects of anticancer agents in combination with/without hyperthermia on T24 cells metastasized to the liver. The hyperthermia exerted an inhibitory effect on the growth of the T24 cells in the livers of the chick embryos, and this was dependent on the thermal dose. The antitumor effects of hyperthermia performed at 42.5° C for 20 min and at 43.0° C for 10 min were evidenced by 69.2% an 82.0% inhibition of the growth of the metastasized T24 cells, respectively, as compared with the growth of untreated T24 cell. Hyperthermia performed at 42.5° C for 10 min alone produced 26.7% tumor growth inhibition, and these conditions for hyperthermia were subsequently used as a criterion for evaluating the effects of its combination with various anticancer agents. Adriamycin (20 g/egg) alone, mitomycin C (10 g/egg) alone, carboplatin (10 g/egg) alone, and cisplatin (10 g/egg) alone produced 13.5%, 58.9%, 27.3%, and 29.1% tumor growth inhibition, respectively. Adriamycin and mitomycin C applied in combination with hyperthermia showed additive inhibitory effects on the growth of the metastasized T24 cells in this chick embryo model.     

Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of αv integrins

We studied invasion-related adhesion events in vitro using three squamous carcinoma cell lines (HSC-3, poorly differentiated type; OSC-19, well-differentiated type; and KB cells, undifferentiated type). An in vitro invasion assay through matrigel in the transwell chamber revealed that HSC-3 cells were most invasive, OSC-19 cells moderately invasive and KB cells least invasive. Inhibition assay of invasion using synthetic peptides RGD, RGDV, RGDS, RGDT, IKVAV and YIGSR, showed that invasion of the three cell lines was significantly inhibited by RGDV. There were other peptides that inhibited invasion significantly including IKVAV for HSC-3, and RGDS and YIGSR for OSC-19. HSC-3 cells and OSC-19 cells adhered to fibronectin, laminin, vitronectin, and type IV collagen, and KB cells did not adhere to laminin but did to fibronectin, vitronectin and collagen type IV. Pretreatment of cells with RGDV peptide in the attachment assay reduced the ability of these cells to bind to vitronectin and fibronectin more efficiently than pretreatment with RGDS. Anti-v antibodies inhibited adhesion of HSC-3, OSC-19 and KB cells to vitronectin, but anti-1 antibodies did not inhibit adhesion. Immunofluorescent microscopic examinations showed that all cell lines were positive for anti-5 and anti-v antibodies, and only HSC-3 cells were positive for anti-3 antibody. 51 was not clearly demonstrated in any of the cell lines. RGDV was the most effective inhibitor of squamous cell carcinoma invasion among the synthetic oligopeptides used in this experiment, and it is suggested that it affects v3-and/or v5-mediated carcinoma cell invasion.Abbreviations BSA bovine serum albumin - MEM Eagle's minimal essential medium - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - PBS phosphatebuffered saline - FITC fluorescein isothiocyanate This work supported in part by a grant from the Osaka Cancer Research Foundation     

An investigation of the DNA-damaging ability of benzene and its metabolites in human lymphocytes,using the comet assay

Benzene and five of its known metabolites—muconic acid, hydroquinone, catechol, p-benzoquinone, and benzentriol—were examined for DNA damage in human lymphocytes using the alkaline Comet assay, and conditions were optimised to determine responses. Metabolic activation (S-9 mix) was included in the assay for varying times to try to enhance effects. In addition, the effects of catalase were investigated as it is known to be present in S-9 mix reducing oxidative damage, and some benzene metabolites are known to react through oxygen radical mechanisms. Effects were also examined in cycling cells to determine whether they were more sensitive to damage then noncycling cells. Comets were measured either by eye or by image analysis. Data have been presented according to length of treatments. When Comets were measured by eye after treatment with hydrogen peroxide (H₂O₂), the positive control, and each compound for 0.5 hr, only H lymphocytes. When comets were measured by image analysis, a 0.5-hr treatment with H₂O₂ and benzenetriol and catechol confirmed results analysed by eye, with S-9 mix greatly reducing responses. When treatments were increased to 1 hr in the presence and absence of S-9 mix, benzene at a 5-fold increased dose produced a significant positive response but not at the lower dose. When treatment times were increased to 2 and 4 hr, doses were also increased, and muconic acid, hydroquinone, catechol, and benzoquinone in the presence of S-9 mix showed positive time and dose-related responses, and at the highest dose of benzoquinone the morphology of the nucleus was affected. Effects tended to become more pronounced at high doses and after longer exposures, although this was not always consistent from experiment to experiment. In conclusion, benzene and all metabolites investigated gave positive responses. Where altered responses were observed, they were significantly different from the corresponding controls. © 1995 Wiley-Liss, Inc.     

四甲基偶氮唑盐法用于非小细胞肺癌药敏试验条件的筛选

目的筛选非小细胞肺癌药敏试验的条件。方法取25例非小细胞肺癌患者的瘤细胞,用四甲基偶氮唑盐（MTT）法观察9种化疗药物,在不同浓度、不同时间对瘤细胞的敏感性。结果9种化疗药物对瘤细胞的敏感性,有随着药物浓度的增加、作用时间的延长而增高的趋势,但每个药物都有各自的特点。结论非小细胞肺癌细胞MTT法药敏试验的浓度应为临床用药量的计算值,药物作用时间应以48h为宜。     

Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

Human pharmacokinetics of an analogue of atrial natriuretic factor

Summary The pharmacokinetics of ANP-270, a 26 amino acid analogue of alpha human natriuretic factor (-hANF) with a prolonged effect on isolated arterial preparations has been studied in 40 healthy males, in a doubleblind placebo controlled investigation. Placebo or ANP-270 0.3, 1.5 or 3.0 g/kg were given by intravenous bolus injection, each to groups of 10 subjects. Blood samples were assayed for ANP-270 by a specific sandwich ELISA.The disappearance of ANP-270 from plasma followed a two-compartment decay, with mean distribution and elimination half-lives of 2.6 min (n = 30) and 10.6 min (n=20), respectively. These estimates were similar to those obtained by other investigators for -hANF. Their brevity explains the lack of a prolonged effect of ANP-270 in vivo compared to -hANF.     

Overexpression and purification of enzymatically active recombinant integrase protein of rous sarcoma virus

The carboxy-terminal domain of polymerase gene of Rous sarcoma virus was cloned into an expression vector under the control oflac regulatory elements, resulting in the plasmid pMF1413. Upon isopropyl--D-thiogalactopyranoside induction, viral integration (IN) protein was expressed in large quantity inEscherichia coli. The expressed recombinant protein was prepurified by successive washing of the bacterial pellet with 0.1 M NaCl and detergents. Further purification was performed in high yield by standard chromatography methods. The purified enzyme revealed selective DNA cleaving activity on supercoiled plasmid with the LTR-LTR junction fragment. The reaction was metal ion dependent, with a preference for Mn²⁺ over Mg²⁺, and showed substrate specificity at 1 mM MnCl₂.     

The effects of urinastatin on the plasma levels of granulocyte elastase during open heart surgery under simple deep hypothermia

Changes in granulocyte elastase (GLE) and -gluculonidase (-gl) were observed during open heart surgeries which were performed under deep hypothermia with surface cooling. In addition, the effect of urinary trypsin inhibitor, urinastatin, on the activities of these enzymes was studied. The patients were divided into three groups, namely group U-I with intravenous injection of 6000u·kg^–1 of urinastatin before cooling, group U-II administered with an additional 6000u·kg^–1 after warming to 30°C, and an untreated group (Group C). The plasma level of GLE increased significantly in the three groups compared with the level before cooling respectively. In the group U-II, the GLE level after the warming was lower than that in the control group. The serum level of -gl increased significantly in the three groups at the end of rewarming (36°C). The release of GLE from lysosomes in granulocytes was inhibited in the group U-II. The insufficient inhibition of GLE release in the group U-I is probably due to relatively short half-life of urinastatin. Therefore double administration of 6000u·kg^–1, before and after the cooling, may be required to achieve the therapeutic effect. Consequently, urinastatin appears to be useful in open heart surgery under deep hypothermia with surface cooling.(Kawamura T, Shimoda Y, Wakusawa R: The effects of urinastatin on the plasma levels of granulocyte elastase during open heart surgery under simple deep hypothermia. J Anesth 6: 269–276, 1992)