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。     

超声激活血卟啉对S180细胞杀伤作用及形态学研究

利用频率为1.1 MHz及不同强度超声激活血卟啉, 对小鼠腹水型S180肿瘤细胞和诱发的在体肉瘤进行不同的实验处理. 通过光镜、电子显微镜技术、荧光标记、细胞化学等方法, 探讨超声激活血卟啉对S180细胞杀伤作用及形态学变化. 研究表明: 超声激活血卟啉对腹水型S180肿瘤细胞显微结构及表面超微结构破坏随声照强度增加而加剧; 声照后细胞显微结构、超微结构、酶活性的变化及DNA的降解丢失是癌细胞生长抑制、死亡的重要因素, 实验中发现的一定超声强度激活血卟啉可诱导癌细胞凋亡现象提示声动力疗法有促细胞死亡和诱导细胞凋亡两种模式. 通过细胞形态学变化, 以及辐照处理后细胞存在的由原发性损伤到继发性损伤直至死亡的动态变化过程, 探讨超声激活血卟啉对S180细胞杀伤作用, 以期为声动力疗法杀伤机制的研究积累资料.     

巨噬细胞激活在维甲酸和维胺酸抗肿瘤活性中的作用

巨噬细胞是体内执行免疫监视机能的重要效应细胞之一,可通过分泌和释放可溶性细胞毒因子对肿瘤细胞进行细胞外杀伤。本文报道维甲酸(RA)和维胺酸(R Ⅱ)可使小鼠腹腔巨噬细胞释放过氧化氢的能力增强。电镜观察表明,经药物激活的巨噬细胞对艾氏腹水癌细胞有明显的细胞外杀伤作用。预先给小鼠腹腔注射药物,然后腹腔接种10~4个艾氏腹水癌细胞,100%的动物不形成肿瘤。但增加瘤细胞接种数或改变给药途径,预防作用消失,这提示RA和R Ⅱ的抗肿瘤作用部分是通过激活的巨噬细胞完成的。     

光动力学结合声动力学治疗对小鼠鳞癌细胞超微结构的影响

目的:评价光动力疗法(PDT)联合声动力学疗法(SDT)对小鼠鳞癌超微结构的影响。方法:以镓卟呤衍生物ATX-70作为光敏剂和声敏剂,分别用光动力疗法、声动力疗法以及两者联合应用处理小鼠鳞癌,利用透射电镜观察不同时间段取材的细胞超微结构的变化。结果:激光或超声激活ATX-70对鳞癌细胞超微结构的破坏程度随取材时间的延长而加剧,联合应用对肿瘤细胞破坏程度更明显。损伤位点主要集中在胞膜、线粒体、内质网及细胞核上,同时还观察到一些肿瘤细胞表现出明显的凋亡特征。结论:光动力学结合声动力学疗法比单用肿瘤细胞破坏程度更明显,主要通过破坏细胞超微结构杀伤肿瘤细胞,部分通过诱导凋亡杀伤。     

条索状透明质酸在乳腺肿瘤细胞BT549化疗耐药中的作用

目的观察透明质酸的新存在形式——条索状透明质酸在乳腺肿瘤细胞BT549表面的表达情况,探索该结构在BT549细胞耐受紫杉醇杀伤中的作用。方法采用免疫细胞荧光技术观察BT549细胞表面条索状透明质酸的分布情况;分别用流式细胞术和RT—PCR检测透明质酸的条索状结构被破坏前后,紫杉醇诱导的肿瘤细胞凋亡、坏死率的改变以及凋亡相关基因c-junmRNA表达的改变。结果BT549细胞天然表达大量的条索状透明质酸,条索状结构被破坏后,紫杉醇诱导BT549细胞的凋亡和坏死率显著增高（P〈0．05）,凋亡基因c扣n的表达水平也显著增高（P〈0．05）。结论乳腺肿瘤细胞BT-549高表达条索状结构的透明质酸,这种透明质酸的新存在形式可能在BT549细胞耐受紫杉醇的杀伤过程中起到一定的保护作用。     

花椒挥发油抗肿瘤药理作用研究

目的 提取花椒挥发油,并对其进行抗人肺癌杀伤及诱导凋亡试验。观察花椒是否具有抗肿瘤活性,并确定其最小杀伤肿瘤细胞的剂量;同时观察诱导细胞凋亡的最小剂量以确定其抗肿瘤机制。方法 利用改进水蒸气法提取花椒挥发油,利用Tween-80作为助溶剂对花椒倍比稀释,对人A549肺癌细胞进行抗肿瘤试验,利用SRB方法和形态学确认细胞杀伤率,采用荧光染料Hochest33258染色检测细胞的凋亡水平。将试验同时分为时间-效应组（24、48、72h）和剂量-效应组（0．125～16mg／ml）。结果 ≥4mg／ml以上剂量的花椒挥发油提取物对A549具有较高的抑瘤率,达到87．9％;最低抑瘤浓度为1mg／ml,抑瘤率为40．2％。形态学观察结果表明,被杀伤的细胞体积萎缩,细胞膜皱缩,成活细胞数稀疏,数量明显低于空白对照组。不同给药时间的研究结果表明,药物最佳作用时间为48h及72h。利用低于杀伤浓度的剂量作用于A549进行研究,观察其对A549细胞凋亡的影响,利用Hochest33258观察对细胞核的影响。结果表明,0．51mg／ml剂量的花椒挥发油提取物作用于细胞72h对肿瘤细胞具有明显的诱导细胞凋亡作用。结论 高浓度（4～16mg／m1）的花椒挥发油对人肺癌A549细胞株有杀伤作用,低浓度（1mg／ml）的花椒挥发油对A549有诱导细胞凋亡的作用。     

高强度聚焦超声对人膀胱癌细胞的生长抑制作用及机理研究

研究高强度聚焦超声（high intensity focused ultrasound,HIFU）对人膀胱癌细胞的杀伤作用,并探索其作用机理.用不同强度的HIFU处理人膀胱癌细胞T24,用台盼蓝排斥法染色、细胞增殖试验（MTT法）研究HIFU对癌细胞的杀伤和生长抑制作用;流式细胞术检测其对癌细胞周期、凋亡及相关基因表达的影响.HIFU处理后,癌细胞死亡率升高,增殖活性降低,G0/G1期细胞数增加,S期细胞数减少,凋亡指数升高,与对照组比较,差异有显著性意义（P＜0.05）;P53、BCL-2和FAS表达阳性细胞数与对照组比较,无显著性意义（P＞0.05）;HIFU强度为600 W/cm^2时,HSP70表达阳性细胞数升高,与对照组比较差异有显著性意义（P＜0.05）.HIFU对人膀胱癌细胞T24具有明显的杀伤及抑制作用,其机理与抑制DNA合成有关;HIFU具有诱导癌细胞凋亡的作用,其诱导凋亡作用可能与P53、BCL-2和FAS无关.     

脂蟾毒配基诱导人肝癌细胞凋亡作用的研究

目的 通过体外脂蟾毒配基对人肝癌细胞（Bel7402）的凋亡诱导作用,为研究其抑制肿瘤细胞生长的作用机制提供依据。方法 通过应用流式细胞光度术检测细胞凋亡;采用细胞免疫细胞化学显色检测和Western blotting分析凋亡相关基因蛋白的表达来研究脂蟾毒配基对人肝癌细胞（Bel7402）凋亡的诱导作用。结果 表明脂蟾毒配基能够诱导Bel7402细胞发生凋亡,凋亡率大于50%;脂蟾毒配基提取液(浓度1.0 μm）作用于Bel7402细胞24小时后,bc1-2蛋白的表达下调,到48、72小时后下调更明显;而Bax蛋白的表达从24小时后开始上调,到48、72小时后表达上调明显。使用方差分析法与对照组相比较,P<0.05 ,统计学有显著意义。结论 提示诱导肿瘤细胞凋亡可能是脂蟾毒配基抑制、杀伤人肝癌细胞的机制之一。     

用~（31）P磁共振谱对艾氏腹水癌和肉瘤S－180细胞代谢的研究

艾氏腹水癌细胞和肉瘤Ｓ－１８０细胞是抗肿瘤药物筛选常用细胞株．实验采用取自小鼠腹腔的第７～８天的艾氏腹水癌和Ｓ－１８０细胞,用３１Ｐ磁共振谱测得了细胞内小分子含磷代谢成分;计算了细胞内ｐＨ值;还用３１Ｐ谱探讨了作用机制不同的三种抗代谢物：碘乙酸、２,４－二硝基苯酚及棉酚对艾氏腹水癌细胞代谢的影响     

一株新发现新城疫病毒体外高效杀伤肝癌细胞作用机制初探

目的:与NDV疫苗株LaSota对比研究一株NDV D817株对肝癌细胞高效特异性的杀伤效应和作用机制,进一步筛选NDV溶瘤毒株.方法:用MTT法对比病毒对三株传代肝癌细胞株SMMC-7721、Bel-7404和HepG-2及一株正常肝细胞株HL-7702的杀伤效应,并用TUNNL法及透射电镜观察病毒诱导肿瘤细胞发生凋亡作用.结果:NDV D817株对肝癌细胞株SMMC-7721、Bel-7404和HepG-2杀伤效应高达80%,显著高于疫苗LaSota株(P<0.01),而对人正常肝细胞HL-7702无明显影响;病毒在肝癌细胞中明显复制增殖,对细胞的杀伤活性与病毒作用剂量和病毒作用时间成正比;NDV D817株有效诱导肝癌细胞发生凋亡.结论:NDV D817株有效诱导肝癌细胞发生凋亡,对SMMC-7721、Bel-7404和HepG-2细胞具有高效杀伤性,而对正常肝细胞HL-7702未见明显影响.推测为溶瘤株.     

Survival of amphibian embryos after continuous ultrasound treatment

The influence of continuous ultrasound on the embryonic development of grass frog Rana temporaria has been investigated. Intact embryos at the blastula stage were treated by ultrasound of different frequency (0.88 and 2.64 MHz), intensity (0.05-1.0 W/cm2), and duration (1-15 min). The treatment with ultrasound of frequency 0.88 MHz and intensity 0.05 W/cm2 for 1-5 min tended to increase the proportion of normally developing embryos up to hatch (10-25% of control). Increasing the intensity of ultrasound (0.88 MHz) to 0.7-1.0 W/cm2 and the duration of its action to 5-15 min induced the death of almost all of treated embryos. No significant differences were found between the development of control embryos and embryos treated with ultrasound of middle intensity (0.2-0.7 W/cm2) for 1-5 min. The exposure of amphibian embryos to ultrasound of frequency 2.64 MHz and intensity 0.05-0.7 W/cm2 for 1-5 min did not change their survival. Increasing the intensity of ultrasound (2.64 MHz) to 1.0 W/cm2 and the duration of its action to 5 min decreased the number of normal developing embryos (by 35%).     

超声激活血卟啉对S180 肿瘤细胞表面EGFR 表达量的影响

目的：探讨超声激活血卟啉处理S180肿瘤细胞后膜表面EGFR表达量的变化。方法：将腹水瘤细胞随机分为四组,U组和UH组细胞分别于频率1．8MHz、2．0W／cm^2的超声装置中照射3min,并分别在1h3h5h后取材,应用免疫组化方法在光镜下观察EGFR的表达情况。结果：1h、3h取材,U组和UH组平均光密度值明显低于Cr组和H组,UH组最低。而5h取材时,UH组平均光密度值显著下降,其它组基本无变化。结论：提示在高频低强度处理下,随着时间的延长,超声激活HpD对EGFR的抑制作用增加,显示可能是基因调控使EGFR表达下调,从而使肿瘤细胞增殖减慢。     

Influence of variuos ultrasound regimens on oxide-modified actomyosin superprecipitation reaction from skeletal muscle of rabbit

A comparative study of rabbit skeletal muscles oxide-modified actomyosin superprecipitation reaction in dependence on continuous and impulsive (2 ms) ultrasound regimens was studied. From the analyses of kinetic curves the effect of the value of superprecipitation (A(m) - A(0)), time t1/2, required for achievement of half of its value was determined, and the normalized maximal rate of this reaction V was also calculated. It is shown that the use of continuous ultrasound to oxide-modified actomyosin was associated with a significant decrease of superprecipitation relative to controls. However, pulsed ultrasound caused a significant increase in superprecipitation value except for the values (A(m) - A(0)) in the application of the intensity of 0.2 W/cm2. The oxide-modified actomyosin superprecipitation value under the effect of continuous and impulsive ultrasound at intensity 1 W/cm2 in relative to control and all other applied intensities decrease to the most extent. It is caused perhaps by thermal influence of ultrasound. In general, the data obtained give reason to assume that the effects of continuous and pulsed ultrasound on the reaction of oxide-modified proteins complex superpretsipitatsii identical.     

1.5 MHz ultrasound irradiation of human lymphocytes.

Human lymphocyte cultures are exposed to 1.5 MHz continuous wave ultrasound, and it is demonstrated that cell death, as monitored by pyknosis, follows immediately on sonication at intensities within the usual therapeutic range (less than 1.7 W/cm2,spatial average). The number of cells affected is determined by the ultrasound intensity only, but the rate at which they proceed through their pyknosis cycle is modified by both the intensity and the duration of exposure. There is a clear indication of an intensity threshold for the effect approximately 1.1 W/cm2. Pulsed 1.5 MHz ultrasound (70 mus, 1 :1 pulses, 1.7 W/cm2 space-time average) results in a 15-20 hour delay in the measurable response to sonication. It is shown that the intracellular presence of a lysosomal-enzyme inhibitor strongly modifies the course of the ultrasound action. Evidence is presented to suggest that the basic interaction mechanism is via a cavitation process, but there are some difficulties with this interpretation, which are also discussed.     

Assessment of fibroblast cells submitted to ultrasonic irradiation

Physiotherapists consider ultrasound an indispensable tool, which is commonly employed in clinical practice as a treatment aid for musculoskeletal dysfunctions. The aim of our study has been to analyze fibroblast cell structures following low-intensity pulsed ultrasonic irradiation. Fibroblast cell cultures irradiated with ultrasound were analyzed through electron microscopy to determine an ideal irradiation beam that preserved cell morphology and integrity. Analysis by fluorescence microscopy and transmission electron microscopy was used to follow morphological changes of the nucleus and cytoskeleton following different ultrasound irradiation intensities. According to the parameters used in the pulsed irradiation of fibroblast cultures, control over the intensity employed is fundamental to the optimal use of therapeutic ultrasound. Cell cultures submitted to low-intensity pulsed ultrasonic irradiation (0.2-0.6 W/cm2) at 10% (1:9 duty cycle) and 20% (2:8 duty cycle) maintained shape and cellular integrity, with little damage. In the group irradiated with an intensity of 0.8 W/cm2, a loss of adhesion was observed along with an alteration in the morphology of some cells at an intensity of 1.0 W/cm2, which resulted in the presence of cellular fragments and a decrease of adhering cells. In cells irradiated at 2.0 W/cm2, there was a complete loss of adhesion and aggregation of cellular fragments. The present study confirms that biophysical properties of pulsed ultrasound may accelerate proliferation processes in different biological tissues.     

Production of thymine base damage in ultrasound-exposed EMT6 mouse mammary sarcoma cells

Mouse mammary sarcoma cells, line EMT6/Ro, were exposed for 1 min to 1 MHz continuous wave ultrasound over a range of intensities from 0.5 to 30 W/cm2 (spatial peak). The presence of thymine base damage (TBD) products of the 5,6-dihydroxydihydrothymine type was determined by an alkali degradation assay. Production of damage was found to be greatest (approximately 2.7 X 10(-3%) t'/T) at an intensity of 10 W/cm2 and fell off rapidly above and below this intensity. The amount of base damage produced at 10 W/cm2 ultrasound was approximately equivalent to the damage produced by a gamma-ray absorbed dose of 12 krad. Assay of cells immediately after sonication at 10 W/cm2 showed that approximately 14% of the cells had been lysed. Tests showed that it was the DNA of the intact cells, however, which sustained all of the TBD. Survival data demonstrated that of the remaining unlysed cell population approximately 5% were viable, whereas cells exposed to 12 krad showed no survival. Additionally, cells were exposed for up to 5 min at 5 W/cm2. An increase in TBD was demonstrated with increasing time of exposure such that the rate of production at 5 min was approximately three times greater than that of a 1-min exposure. TBD was found to be completely suppressed when cells were sonicated at 10 W/cm2 for 2 min under 4 bar of hydrostatic pressure. Addition of the radical scavengers beta-MEA and cystamine eliminated TBD but had minimal effect on survival. The pressure and scavenger experiments demonstrate that TBD results from cavitation-induced free radicals. Based on the values for both the half-life and diffusion distance of such radicals, our results indicate that at least part of the bubble collapse occurs intracellularly.     

Effect of ultrasound on a bilayer lipid membrane.

The effects of continuous wave ultrasound at a frequency of 1 MHz in the intensity range of 0-1.4 W/cm2 on an oxidized cholesterol bilayer lipid membrane (BLM) were observed. Ultrasound at 1.5 W/cm2 broke the membrane; in the range from 0.5 to 1.4 W/cm2, it accelerated the draining of the bulk lipid solution from the annulus to the Teflon support. At all intensities it has no effect on the conductance, the capacitance, or the dependence of each on the voltage applied across the membrane. Electrical parameters were measured in the presence of aqueous solutions of NaCl, KCl, and distilled water. The motivation and results of this project are explained in relation to an overall objective of determining the specific effects of ultrasound on biological membranes.     

Bioeffect of ultrasound on endothelial cells in vitro

The effects of low-intensity ultrasound (US) on biological systems have been investigated extensively; however, the effects of ultrasound stimulation on endothelial cells were rarely studied. In this study, 1 MHz, pulsed 1:4, and four different spatial-average temporal-peak intensities (0.5, 1.0, 1.6, and 2W/cm2) of ultrasound were used to stimulate endothelial cells for 10 min per day. The results showed that ultrasound (intensity 1.6-2.0W/cm2) treatment after 6 days enhanced the nitric oxide (NO) and Ca2+ release from the endothelial cells but did not promote cell growth. In addition, ultrasound stimulation changed the cellular morphology and orientation, and increased extracellular matrix secretion from endothelial cells.