Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD₆₀₀; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.     

Effect of combined administration of hydrocortisone and EDTA on the number of the antibody-producing cells in the spleen of mice immunized with sheep erythrocytes]

Experiments on mouse hybrids (CBA X C57BL)F1I indicated that injection of hydrocortisone in a dose of 1 mg/mouse 24 hours after the immunization with sheep red blood cells against the background of multiple EDTA injection resulted in a relative reduction of the plaque-forming cells in the spleen--more than 6-fold in comparison with control, and more than 3-fold in comparison with the effect of hydrocortisone or EDTA alone. This may possibly be the consequence of a more intensive hydrocortisone incorporation under conditions of prolonged hypocalciemic action of EDTA complexon.     

HSV-tk/GCV自杀基因系统与重组TNF联合杀伤肿瘤细胞的光学成像检测

目的:应用光学成像方法检测HSV-tk/GCV自杀基因系统与重组改造的人肿瘤坏死因子(recombinanthuman tumor necrosis factor,rmhTNF)的联合使用,对人涎腺腺样囊性癌细胞(adenoid cystic carcinoma,ACC-M)的杀伤效率。方法:实验分为四组,细胞模型对照组、单纯HSV-tk/GCV自杀基因系统治疗组、rmhTNF处理组、自杀基因联合rmhTNF治疗组。将稳定表达胸苷激酶-绿色荧光蛋白(thymidine kinase-green fluorescen protenin,tk-GFP)的ACC-M细胞(ACC-M-tk-GFP)按5 000个/孔接种到96孔板中,24 h后给药治疗;分别于加药前、给药后6 h、给药后24 h进行荧光成像。检测绿色荧光蛋白(green fluorescent protein,GFP)的荧光用于准确显示胸苷激酶(tk)基因表达的肿瘤细胞,检测碘化丙啶(propidium iodide,PI)的荧光用于显示死亡的肿瘤细胞。通过细胞的透射图及其PI和GFP荧光图像,分别计算tk 细胞和tk-细胞的死亡率。结果:HSV-tk/GCV自杀基因系统与rmhTNF联合治疗组的细胞在加药后6 h就已经出现细胞死亡迹象,而单纯HSV-tk/GCV自杀基因系统治疗组的细胞在加更昔洛韦(ganciclovir,GCV)后24 h才出现细胞死亡,单纯rmhTNF治疗组在加药后24 h还没有细胞死亡发生。对于给药后24 h的tk 细胞死亡率,联合治疗组(85.88%)明显高于GCV(38.13%)和rmhTNF单独治疗组(2.97%),并且联合治疗组给药24 h后会引发部分tk-细胞死亡(9.83%)。结论:HSV-tk/GCV自杀基因系统与rmhTNF联合治疗具有显著的协同抗肿瘤效应,能够明显提高对肿瘤细胞的杀伤率;结合光学成像方法,能为更直观方便地检测各组药物分别对tk 细胞和tk-细胞的杀伤率,可为深入研究"旁观者效应"提供参考。     

Basic characteristics of the process of Candida adhesion to human epitheliocytes

The adhesion of fungi belonging to the genus Candida to the epithelial cells of the mouth cavity reached its maximum at pH 6.2-7.0. The process of adhesion had similar dynamics at temperatures of 37 degrees, 28 degrees and 25 degrees C, but the adhesive activity decreased 2 times when temperature dropped from 37 degrees to 25 degrees and 4 times when temperature dropped to 4 degrees C. The introduction of the ions Ca2+ (1 and 10 mM) and Mg2+ (10 mM) led to the increase of adhesion by 80, 100 and 24% respectively. The heating of the fungal cells at 100 degrees C (for 1 hour) and at 63 degrees C (for 2 hours) decreased adhesion to 8 and 24% respectively, and treatment with formaldehyde (for 24 hours) decreased adhesion to 70% of that observed in experiments with live Candida cells.     

Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes

We developed a new in vitro model for a multi-parameter characterization of the time course interaction of Candida fungal cells with J774 murine macrophages and human neutrophils, based on the use of combined microscopy, fluorometry, flow cytometry and viability assays. Using fluorochromes specific to phagocytes and yeasts, we could accurately quantify various parameters simultaneously in a single infection experiment: at the individual cell level, we measured the association of phagocytes to fungal cells and phagocyte survival, and monitored in parallel the overall phagocytosis process by measuring the part of ingested fungal cells among the total fungal biomass that changed over time. Candida albicans, C. glabrata, and C. lusitaniae were used as a proof of concept: they exhibited species-specific differences in their association rate with phagocytes. The fungal biomass uptaken by the phagocytes differed significantly according to the Candida species. The measure of the survival of fungal and immune cells during the interaction showed that C. albicans was the more aggressive yeast in vitro, destroying the vast majority of the phagocytes within five hours. All three species of Candida were able to survive and to escape macrophage phagocytosis either by the intraphagocytic yeast-to-hyphae transition (C. albicans) and the fungal cell multiplication until phagocytes burst (C. glabrata, C. lusitaniae), or by the avoidance of phagocytosis (C. lusitaniae). We demonstrated that our model was sensitive enough to quantify small variations of the parameters of the interaction. The method has been conceived to be amenable to the high-throughput screening of mutants in order to unravel the molecular mechanisms involved in the interaction between yeasts and host phagocytes.     

地塞米松影响念珠菌对抗真菌药物敏感性的实验研究

目的 探讨地塞米松在体外试验中是否影响念珠菌对抗真菌药物的敏感性,以了解糖皮质激素与抗真菌药物直接作用于念珠菌时是否存在相互作用。方法 用微量液体培养基稀释法分别测定26株白念珠菌与地塞米松（0．2mg／ml）共同孵育前、孵育24～48h及7d时氟康唑、伊曲康唑、两性霉素B的最低抑菌浓度（MIC）值,并作对照。结果 白念珠菌与地塞米松孵育24～48h后、孵育后第7d氟康唑和伊曲康唑的MIC值升高,分别与孵育前的MIC值存在统计学差异,但孵育24～48h后的MIC与孵育后第7d的MIC无统计学差异;白念珠菌与地塞米松共同孵育24～48h后两性霉素B的MIC值也较孵育前升高,但第7d的MIC值与孵育前无差异。结论 地塞米松可增加三种抗真菌药物对于白念珠菌的MIC,但三种抗真菌药物间存在差异,表明地塞米松对于氟康唑和伊曲康唑体外抗白念珠菌的活性有拮抗作用,但没有时间依赖性,地塞米松对于两性霉素B的影响较氟康唑和伊曲康唑小,且影响时间较短。     

Presence of fungi in stool of children

A total of 258 children were tested for the presence of fungi in stool. One group consisted of 148 children with non-specific gastrointestinal tract disorders while the other was a group of 110 asthmatics. A quantitative method of enzymatic and mechanical homogenisation was used. The findings were divided into three ranges as follows: < 10(3), 10(3)-10(5), > 10(5) fungal cells in one gram of stool. The number of > 10(5) fungal cells in one gram of stool was considered as pathogenic and requiring treatment. Such a number of fungi in stool was found in 48.1% of children in the first group and in 35.9% in the second one. However, the percentage of fungal presence was higher in the group of asthmatics (83.6% vs. 70.3%). Candida albicans considerably outnumbered the remaining fungal species in the isolates. It was found out that other than C. albicans Candida species were more resistant to the antifungals.     

Sensitivity of fungi of the genus Candida to antifungal drugs

Seventy two Candida strains isolated from patients with candidiosis of the oral mucosa were studied with respect to their sensitivity to nystatin, levorin, decamine, ethonium, sanguiritrin and clotrimazole. At concentrations of 0.5 to 5 micrograms/ml all the Candida species i.e. C. albicans, C. tropicalis, C. krusei and C. quilliermondii were highly sensitive to clotrimazole. Fungistatic action of levorin, nystatin and sanguiritrin was observed in 91, 67 and 38 per cent of the strains respectively. The Candida strains were resistant to decamine and ethonium used in the above concentrations.     

索拉菲尼联合阿霉素对人肝癌细胞株HepG2的抑制作用

目的：探讨索拉非尼（Sorafenib）和阿霉素（adriamycin）联合用药对肝癌细胞株nepG2的作用及可能的机制。方法：以不同浓度索拉非尼和不同浓度阿霉素分别组成单药组和索拉非尼＋阿霉素联合用药组作用于HepG2细胞,MTT法检测增殖抑制率、流式细胞仪分析细胞周期和凋亡率。结果：索拉非尼、阿霉素单药与联用均能抑制HepG2细胞增殖,呈剂量依赖效应,两药联用有协同效应（P〈0.01）。索拉非尼、阿霉素单药与联用均能诱导HepG2细胞凋亡,并以联合组更为明显,与对照组比较有显著的统计学意义（P〈0.01）。索拉非尼及阿霉素单药作用均可使细胞周期阻滞于G0-G1期,联合用药组G0/Gl期细胞比率低于索拉非尼及阿霉素单药组,S期细胞比率高于单药组;阿霉素能抑制HepG2细胞Survivin mRNA表达诱导细胞的凋亡。结论：索拉非尼与阿霉素联合作用于人肝癌HepG2细胞具有协同作用,其机制可能是通过多途径共同抑制HepG2细胞增殖及诱导细胞凋亡。     

Cytotoxicity of the dicarboximide fungicides, vinclozolin and iprodione, in rat hepatoma-derived Fa32 cells

Dicarboximide fungicides are widely used to control various fungal species. Their primary action is not known, due to a lack of knowledge concerning the mechanism of action of the dicarboximide group. The cytotoxicities of vinclozolin and iprodione in rat hepatoma-derived Fa32 cells were investigated. Cytotoxicity was measured by neutral red uptake inhibition after treatment for 24 hours. Iprodione was more toxic than vinclozolin. Vinclozolin was less toxic in glutathione-depleted cells than in control cells. This was also true for iprodione at lower concentrations, but iprodione became more toxic at higher concentrations. Both the fungicides increased the endogenous glutathione content by 20% after 1 hour. After 24 hours, the glutathione content was doubled by vinclozolin, but was not affected by iprodione. No effect on glutathione S-transferase activity or reactive oxygen species formation could be observed. Cytochrome P450-dependent ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities were moderately activated by iprodione and strongly activated by vinclozolin. A glutathione-related cytochrome P450-dependent metabolic attack of vinclozolin and iprodione could be responsible for their cytotoxicity in Fa32 cells. Further research is needed to fully elucidate these (or other) mechanisms.     

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) producing ROS affects growth and viability of filamentous fungi

CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) causes intracellular superoxide production and oxidative stress and enhances the susceptibility of Saccharomyces cerevisiae, Candida albicans, and C.?glabrata cells to cycloheximide, 5-fluorocytosine, and azole antimycotic drugs. Here, we demonstrate the antifungal activity of CTBT against 14 tested filamentous fungi. CTBT prevented spore germination and mycelial proliferation of Aspergillus niger and the pathogenic Aspergillus fumigatus. The action of CTBT is fungicidal. CTBT increased the formation of reactive oxygen species in fungal mycelium as detected by 2',7'-dichlorodihydrofluorescein diacetate and reduced the radial growth of colonies in a dose-dependent manner. Co-application of CTBT and itraconazole led to complete inhibition of fungal growth at dosages lower than the chemicals alone. Antifungal and chemosensitizing activities of CTBT in filamentous fungi may be useful in combination treatments of infections caused by drug-resistant fungal pathogens.     

Interaction of aminoglycosides and ionophores in the killing of Crithidia fasciculata

Crithidia fasciculata was utilized as a prescreen to determine the antiprotozoal action of aminoglycoside antibiotics alone and in combination with surface-altering agents. Paromomycin was tested with the carrier ionophores nigericin and valinomycin, the channel ionophore gramicidin and the polyene antibiotics amphotericin B and nystatin. After exposure to the drugs in suspension, organisms were plated out to determine the survival of C. fasciculata. Killing was time dependent for both the antibiotic and the ionophore. Paromomycin action was found to be potentiated by all the surface altering agents. The aminoglycosides kanamycin, gentamycin and streptomycin were studied alone and in combination with nigericin. Synergistic effects were demonstrated both with kanamycin and gentamycin in combination with nigericin. Streptomycin was ineffective both alone and with surface-altering agents.     

Sensitivity of Candida strains to polyenic antibiotics in the treatment of oral candidiasis

Sensitivity of 146 clinical strains of Candida albicans to nystatin, levorin and amphoglucamine was studied on solid media with the replica method. The strains were isolated from 79 patients with candidiasis of the oral mucosa. It was found that sensitivity of the fungi to the polyenic antibiotics was different which should be considered in treatment of candidiasis. On the basis of the mean MICs for the clinical strains and their distribution by the MICs it was shown that the activity level of levorin and amphoglucamine was higher than that of nystatin. During the treatment resistance of the Candida strains to the polyenic antibiotics increased and cross resistance developed which required application of other treatment means.     

Fluid shear stress synergizes with insulin-like growth factor-I (IGF-I) on osteoblast proliferation through integrin-dependent activation of IGF-I mitogenic signaling pathway

This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR.     

Recombinant human Erythropoietin enhanced the cytotoxic effects of tamoxifen toward the spheroid MCF-7 breast cancer cells

Erythropoietin (EPO) is widely used to treat anemia in patients undergoing chemotherapy for cancers. The main objective of this study was to investigate the effect of rHuEPO on the response of spheroid breast cancer, MCF-7, cells to tamoxifen treatment. The MCF-7 spheroids were treated with 10 mg/mL tamoxifen in combination with either 0, 10, 100 or 200 IU/mL rHuEPO for 24, 48 or 72 h. The viability of the MCF-7 cells was determined using the annexin-V, cell cycle, caspases activation and acridine orange/propidium iodide staining. rHuEPO-tamoxifen combination significantly (p greater than 0.05) increased the number of spheroid MCF-7 cells entering early apoptotic phase after 12 h and late apoptotic phase after 24 h of treatment; primarily the result of the antiproliferative effect tamoxifen. Tamoxifen alone significantly (p < 0.05) increased the caspase-3 and −9 activities in the spheroid MCF-7 cells by 200 to 550% of the control. Combination rHuEPO and tamoxifen produced much lesser effect on the caspase-8 activity. The rHuEPO in the combination treatment had concentration-dependently caused decrease in the caspase activities. rHuEPO-tamoxifen combination markedly increased MCF-7 cells entering the SubG0/G1 phase of the cell cycle by more than 500% of the control, while decreasing those entering the G2 + M and S phases by 50%. After 72 h, the combination treatment produced greater (p < 0.05) change in the SubG0/G1 phase than tamoxifen treatment alone. Morphologically, spheroid MCF-7 cells subjected to combination rHuEPO-tamoxifen treatment showed nuclear condensation and margination, cytoplasmic blebbing, necrosis, and early and late apoptosis. Thus, the study showed that rHuEPO-tamoxifen combination induced apoptosis in the spheroid MCF-7 cells. The apoptotic effect of the rHuEPO-tamoxifen combination treatment on the MCF-7 cells was greater than that produced by tamoxifen alone. The rHuEPO-tamoxifen treatment enhanced the caspase-independent apoptotic effects of tamoxifen on the spheroid MCF-7 cells.     

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K⁺ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).     

The polyene antimycotics nystatin and filipin disrupt the plasma membrane, whereas natamycin inhibits endocytosis in germinating conidia of Penicillium discolor

Aims:  To investigate the differences in membrane permeability and the effect on endocytosis of the polyene antimycotics nystatin, filipin and natamycin on germinating fungal conidia.
Methods and Results:  The model system was Penicillium discolor , a food spoilage fungus. Filipin resulted in permeabilization of germinating conidia for the fluorescent probes TOTO-1 and FM4-64, but not for ferricyanide ions. Nystatin caused influx of all these compounds while natamycin did not. Untreated germinating conidia internalize the endocytic marker FM4-64. Pretreatment of germinating conidia with natamycin showed a dose and time dependent inhibition of endocytosis as judged by the lack of formation of early endosomal compartments.
Conclusions:  The results obtained from this study indicated that, unlike nystatin and filipin, natamycin is unable to permeabilize germinating conidia, but interferes with endocytosis in a dose and time dependent manner.
Significance and Impact of the Study:  Natamycin acts via a different mode of action than other polyene antimycotics. These results offer useful information for new strategies to prevent fungal spoilage on food products and infection on agricultural crops. For laboratory use, natamycin can be used as a specific inhibitor of early endocytosis in fungal cells.     

Quantitative and qualitative analysis of the antifungal activity of allicin alone and in combination with antifungal drugs

The antifungal activity of allicin and its synergistic effects with the antifungal agents flucytosine and amphotericin B (AmB) were investigated in Candida albicans (C. albicans). C. albicans was treated with different conditions of compounds alone and in combination (allicin, AmB, flucytosine, allicin + AmB, allicin + flucytosine, allicin + AmB + flucytosine). After a 24-hour treatment, cells were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) to measure morphological and biophysical properties associated with cell death. The clearing assay was conducted to confirm the effects of allicin. The viability of C. albicans treated by allicin alone or with one antifungal drug (AmB, flucytosine) in addition was more than 40% after a 24-hr treatment, but the viability of groups treated with combinations of more than two drugs was less than 32%. When the cells were treated with allicin alone or one type of drug, the morphology of the cells did not change noticeably, but when cells were treated with combinations of drugs, there were noticeable morphological changes. In particular, cells treated with allicin + AmB had significant membrane damage (burst or collapsed membranes). Classification of cells according to their cell death phase (CDP) allowed us to determine the relationship between cell viability and treatment conditions in detail. The adhesive force was decreased by the treatment in all groups compare to the control. Cells treated with AmB + allicin had a greater adhesive force than cells treated with AmB alone because of the secretion of molecules due to collapsed membranes. All cells treated with allicin or drugs were softer than the control cells. These results suggest that allicin can reduce MIC of AmB while keeping the same efficacy.     

Drug sensitivity of Candida in patients with kidney diseases receiving immunosuppressive therapy

It was shown that prednisolone and its combination with azathioprine++ increased contamination of the patients with yeast-like fungi and promoted development of candidiasis in them to a greater extent than cyclophosphamide. In the patients treated with the immunodepressants there was observed a carrier state in regard to various yeast-like fungi: 12 species belonging to 6 genera were isolated from the pathological materials. Determination of sensitivity to antifungal drugs in 200 Candida strains revealed that amphotericin B was the most active agent. Then followed mycoheptin, nystatin and nitroxolin. Levorin was the least active drug. The MICs of the drugs for the majority of the cultures were 0.5, 4-8, 8-16 and 32-64 micrograms/ml respectively. Candida resistant strains (mainly to levorin and mycoheptin) were isolated only from recipients of kidney transplants during the early postoperative period when the patients were subjected to intensive immunodepressive and prophylactic antifungal therapy. Among the fungi of the Candida genus C. guillermondii and C. parapsilosis proved to be the most resistant. Under the hospital conditions and in vitro studies it was found that cyclophosphamide and combinations of prednisolone with cytostatics increased resistance of Candida to the antifungal drugs. Rapid increasing of the fungi resistance to levorin and mycoheptin was observed. The increase in the resistance to amphotericin B was somewhat lower and that to nitroxolin and nystatin was extremely low. The study of the combined effect of the immunodepressants and antifungal drugs demonstrated that the immunodepressants increased the antifungal activity of amphotericin B, levorin and nitroxolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

声化学诱导艾氏腹水瘤细胞凋亡机制初探

本研究采用频率1.43MHz,声强3W/cm2的高频聚焦超声处理艾氏腹水肿瘤细胞,研究超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡的途径及其与癌细胞内的氧自由基之间的关系。通过细胞免疫组织化学方法检测与癌细胞凋亡相关的Bax,细胞色素c和caspase-3蛋白的动态表达,黄嘌呤氧化酶法检测超氧化物歧化酶活性变化,硫代巴比妥酸法检测膜脂质过氧化物的含量。结果发现超声加血卟啉处理1h,癌细胞胞浆中的三种促凋亡蛋白表达增多,3h时表现为高表达;处理1h的癌细胞,超氧化物歧化酶活性下降,膜脂质过氧化物增多。研究结果表明超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡可能通过线粒体途径,且与癌细胞受损后产生的氧自由基有关。