【作者】 谭红明；

【导师】 甘平；

【作者基本信息】 重庆医科大学 ， 生物医学信息技术， 2008， 硕士

【摘要】 本文利用现代生物医学信息学的技术和方法,分两大部份采用频率相同(30Hz)而强度不同(0.5mT、1.0mT和1.5mT)的磁场分别作用于离体的K562人红白血病细胞(第一部分)和在体的兔VX2肝肿瘤细胞(第二部分)进行生物学效应的相关研究。第一部分不同强度磁场对K562细胞生长和增殖的研究目的:通过研究强度不同而频率一致的磁场对K562细胞生长、增殖和分化的影响,探讨磁场的强度因素对离体K562人红白血病细胞增长和分化的影响。方法:经过传代培养的K562在频率一致(30Hz)而强度不同(0.5mT、1.0mT和1.5mT)的磁场中进行辐照后,利用四甲基偶唑盐法(MTT)研究不同强度磁场对K562细胞增殖及其分化的影响;采用流式细胞术(FCM)研究不同强度的磁场与细胞周期的调控关系;通过光镜、电镜观察和图像分析法研究经磁场辐照后K562的形态学变化。结果:(1)仅有一组(1.0mT)的细胞数量与辐照前比较具有明显的统计学意义(P<0.05)。离体的K562对特定的磁场强度(1.0mT,30Hz)具有“易感性”;(2) 1.0mT磁场组的K562在6h后即可见到细胞毒性现象的显现,细胞体积开始变大;在24h时可以见到细胞核固缩,胞浆中大量空泡,胞浆量减少,核膜不完整等现象。1.5mT磁场组的细胞在72h后可见核固缩、胞浆量减小。(3) FCM检测结果表明,随着磁场剂量的增加,G0/G1期细胞也增加,其中1.0mT组的增加最明显,凋亡率最高。第二部分磁场辐照结合HIFU治疗兔VX2肝肿瘤模型目的:在第一部分实验的基础上,研究磁场对在体VX2肝肿瘤细胞增殖的影响;同时结合高强度聚焦超声(HIFU)作为试验手段,观察磁场结合HIFU治疗在体肿瘤的效果。方法:将VX2肝癌模型兔随机分成治疗组和对照组。治疗组中磁场辐照的相关参数根据第一部分的实验结果,选取强度为1.0mT,频率为30Hz的磁场作为辐照源。在辐照完成之后,治疗组和对照组采用相同的HIFU进行治疗。治疗靶区的声强为15800W/cm2 ,扫描方式为直线线扫,速度2mm/s.层面间隔为5mm。HIFU治疗后48h取材,进行病理学检查和电镜检查,比较治疗组与对照组的形态、结构等差异。结果:(1)治疗组(1.0mT)的肿瘤细胞在10d和20d后与对照组相比,其细胞平均直径和体积较小,但是没有表现出统计学差异;(2)光镜下治疗组胞浆嗜伊红染色浅,细胞肿胀,胞浆疏松,胞浆内大量空泡,少量细胞核固缩、染色质边集,对照组中可见胞浆内大量大小不等空泡,染色质边集和细胞核固缩;电镜下治疗组胞浆内大量线粒体肿胀和内质网扩张,细胞核固缩和染色质边集,两组肿瘤组织均可见到细胞胞浆内大小不等空泡。(3)治疗组灰度级达到或超过32所用的时间平均为12s,而对照组为32s;并且对照组中,有8只兔子的皮肤出现了明显的红肿或溃烂;治疗组中仅有1只兔子的皮肤出现红肿现象。全文结论(1)1.0mT,30Hz的磁场能显著地抑制离体K562的生长、增殖;并且该抑制效应与辐照时间呈正相关关系;(2)1.0mT,30Hz的磁场能显著地阻止离体K562进入细胞分裂周期,主要抑制G2到M分裂期,并诱导细胞凋亡机制的启动;(3)1.0mT的低强度磁场对在体肝VX2肿瘤细胞的生长和增殖没表现出抑制效应,镜检中未发现凋亡小体的形成;(4)与对照组相比,磁场结合HIFU能够明显地缩短HIFU治疗VX2肝肿瘤细胞的时间,而且对组织皮肤的伤害更小。更多还原

【Abstract】 The Bio-effect of impulse MF (Magnetic field) on tumor cell both in vitro and in vivo has been becoming one of the research focuses since many years. Although the fact is that there are many papers published on this field of MF bio-effect, the most frequent method that is used to do this research is the combination of statistics and epidemiology investigation, especially to the study of MF. On this paper, MF will be employed to act on the tumor cell, in vitro and vivo with the same frequency and different intensity (0.5mT、1.0mT and 1.5mT),Moreover, modern biotechnology and method will be used to clarify the effect of different intensity MF to the cell’s growth、proliferation and differentiation.Part One The study of MF induced K562 cell’growth、proliferation and differentiation and cell cycleObjection: To clarify the mechanism and principle of LMF acted on tumor cell through the research of LMF influence on cell’s growth、proliferation and differentiation; Hopefully, it could provide some theory base and references on the clinic therapy of human tumor.Method: The cultured K562 cells which have been treated with 3 groups of different intensity MF, take use of the colorimetric 5-diphenyl tetrazolium bromide (MTT) to investigate the impact on the proliferation and differentiation of tumor cells. Besides, the corresponding relation between the LMF and the tumor cell cycle will be determined with Flow cytometry; the morphological changes on K562 cells induced by LMF can be observed in the presence of light、electron microscopy and photo analysis,Results:1. The results show that the proliferation amount of K562 cells, compared to pre-treated only one group (1.0mT) indicated that it is significant on statistics. The longer the cells of this group exposure on the MF; the stronger the inhabitation of this group cells become, There is no any significance on the other two groups. It is easy to conclude that MF is able to restrain the growth of tumor cells only under special intensive range.2.The poison phenomenon will be emerging after 6 hours on this group, 1.0mT, the cells became bigger and bigger. the volume、diameter and ratio of nucleus/cytoplasm of K562 cells are decreased, while the heterochromatin are condensed in nucleus after 24 hours. It differentiates toward mature direction. It is very normal on the group of 1.5mT, even after 72 hours.3. The cell amount of from G0 to G1 will go up while it will drop G2/M if the usage dose of MF increased. It is quite obvious on this group of 1.0mT. The occurrence ratio of apoptosis will sum up. Meanwhile, the results showed that different intensity LMF primarily affect the procedure from G2 to M in the process of cell cycle.Part Two Research of combined MF and HIFU tumor therapy. Objection: Explore the effect that LMF was applied to destroy VX2 tumor cell lines of New Zealand rabbit, and the impact of combined LMF and HIFU with the treatment of tumor.Methods: 50 New Zealand Rabbits were divided by 2 groups, one is the control, and another is the treatment group. The option of all MF parameters is based on the results of Part One, namely 1.0mT and Frequency 30Hz were selected as the treatment parameters.VX2 cell line were transplanted into the liver of rabbits in terms of activation and culture cells, and build up the tumor model.Control and Treatment will continue to be dealt by HIFU after they got MF done. The parameters of HIFU as follows: Frequency of treatment head is 1.6MHz, the intensity of ultrasound will reach to 2000W/cm2.All groups will get treatment under the scan of B Mode.Results1.The average diameter and volume of tumor cell of treatment group, compared with control, is far smaller. It is very obvious that the cells of control are far bigger than the treatment.2. Those cells of treatment, swell, cell plasma loosen, vast bubble filled with plasm, nucleolus,3. The average time of the treatment beyond 32 Gray Degree is about 12s, in contrast, the controls need 32s to reach the same Gray Degree. Among the controls, there are 8 rabbits that showing some skin canker. The treatment only 1 showed this skin damage.Conclusion:①MF has quite apparent inhibitory effect on tumor cells within special intensive range.②MF is capable of resulting in the end of tumor cell proliferation cycle within some intensive value range.③To some extent, MF with the intensity of 1.0mT has the ability to inhibit the growth and proliferation of VX2; No formation of apoptosis under the observation of microscopy;④the treatment time is shorter than the controls, and the damage to the skin is smaller.更多还原