摘要
声动力学疗法(Sonodynamic Therapy,SDT)是利用超声能够穿透深层组织并聚焦于特定的部位,激活富集在肿瘤组织中的声敏剂[血卟啉及其衍生物(Hemtoporphyrin Derivatives,HpD)]产生协同效应,从而起到抗癌效果的一种肿瘤治疗方法。该方法是在光动力学疗法(Photodynamic therapy,PDT)的基础上建立和发展起来的。
目前,防治肿瘤的常规方法有:手术治疗、放射治疗和化学治疗等。手术治疗是采用手术切除肿瘤,在治疗早期没有转移和与重要脏器及大血管无严重粘连的肿瘤效果较好,但中晚期效果不佳;放射治疗是利用同位素衰变利加速器产生的射线治疗恶性肿瘤,肿瘤一旦扩散或局部侵润广泛,治疗效果较差,且放疗本身也会带来一些副作用;化学疗法是利用化学抗肿瘤药物治疗恶性肿瘤,但化疗药物毒副作用较大,主要有恶心、呕吐、脱发、白细胞减少等。1978年,美国学者Doughtery等人首次提出光动力疗法(Photodynamic therapy,PDT)治疗肿瘤。但由于光的穿透能力差,该方法主要应用于人体表面浅层肿瘤的治疗,对深部和中晚期肿瘤的治疗具有一定的困难。而超声具有较强的穿透力,并且对健康组织伤害较小,同时血卟啉等声敏剂可以特异地聚集于肿瘤细胞,从而使得SDT疗法可以有针对性的杀伤肿瘤细胞,加之超声治疗装置简单,造价低廉,因而该法具有良好的应用前景。自1989年日本学者Umemura.S等人在国际超声会议上首次提出SDT疗法以来,各国学者对这一疗法及其机制进行了广泛而深入的研究,并提出了空化效应、单线态氧理论、羟自由基理论、烷氧自由基理论,但其确切的作用机制尚无定论。
细胞骨架是细胞内重要的细胞器,与细胞的形态维持、细胞增殖、细胞迁移和信号传导等多项生命活动有关。细胞骨架已成为肿瘤治疗的重要靶点。大量的文献报道了PDT可以使细胞骨架受损,从而影响相应的细胞功能。例如:酞菁类光敏剂(ZnPc)-PDT对HeLa细胞微管、微丝、角蛋白有光损伤效应,引起周期阻抑、细胞变形等;ALA-PDT则可以使人腺癌细胞WiDr和神经胶质瘤细胞D54MG的微管利微丝受到破坏,使细胞形态利细胞黏附性发生变化;同时,ALA-PDT也可以影响慢性髓系白血病K562细胞骨架的组装。然而,有关SDT对细胞骨架影响的研究未见报道。因此,本论文就SDT疗法对细胞骨架的影响进行了初步研究,同时比较血卟啉(hematoporphyrin,Hp)介导的SDT疗法与原卟啉Ⅸ(protoporphyrinⅨ,PPⅨ)介导的SDT疗法对细胞骨架的影响。
本论文依托国家自然科学基金资助项目“超声激活血卟啉抗肿瘤的细胞分子生物学机制”利“超声激活血卟啉诱导肿瘤细胞凋亡的分子生物学机制”的支持,采用超声频率为1.34 MHz,强度为1 W/cm~2的处理条件,研究超声激活血卟啉或者原卟啉Ⅸ对EAC细胞细胞骨架的影响,所得实验结果如下:
1.超声激活Hp或PPⅨ对艾氏腹水肿瘤细胞(EAC)表面结构的影响:扫描电镜观察发现,对照组细胞呈圆球形,表面有丰富密集的微绒毛;单独Hp、PPⅨ对细胞表面形态没有明显的影响:单独超声处理后,细胞表面微绒毛随着时间的延长而逐渐减少,2小时后细胞表面趋于光滑,4小时后少数细胞表面出现了小孔:超声结合Hp或PPⅨ组细胞表面微绒毛锐减,随着时间的延长细胞表面结构利形态均有明显的改变,而且超声结合PPⅨ组细胞表面结构的变化比超声结合Hp组的变化更为明显,4小时后超声结合PPⅨ组细胞的表面有泡状的突起。
2.不同强度的超声结合不同浓度Hp或PPⅨ经不同声照时间处理对F-actin的影响:发现随着超声强度和声敏剂浓度以及超声作用时间的增加F-actin受损的程度增大、分布变化明显;流式细胞仪检测SDT疗法处理后不同时间段取材被标记的F-actin的荧光强度变化,发现超声处理组细胞F-actin的荧光强度较对照组有所降低,超声结合Hp组或PPⅨ组细胞F-actin的荧光强度明显降低,并且存在时间依赖性。对照组、Hp利PPⅨ组F-actin的荧光强度均没有明显变化。
3.超声激活Hp或PPⅨ对β-tubulin和vimentin的影响:采用间接免疫荧光技术检测SDT疗法处理后EAC细胞β-tubulin和vimentin的变化。结果表明,对照组、单独Hp、PPⅨ织细胞β-tubulin和vimentin均匀分布于胞质中,4小时后变化不明显;单独超声处理组细胞β-tubulin和vimentin的分布略有变化,标记的β-tubulin和vimentin的荧光强度随着处理后时间的延长而减弱;超声结合Hp或者PPⅨ作用组细胞β-tubulin和vimentin的变化比单纯超声处理组的变化更为明显,处理后0、2小时取材,β-tubulin利vimentin的分布均变得弥散、无序,荧光强度减弱,4小时后超声结合PPⅨ作用组细胞表面有明显的膜发泡现象,两种胞质骨架纤维均存在于膜发泡中。
4.超声激活Hp或PPⅨ对EAC细胞核纤层LaminB的影响:用免疫细胞化学方法检测SDT疗法处理后EAC细胞LaminB的平均光密度值变化。研究结果表明:对照组、单独Hp或者PPⅨ组细胞LaminB的平均光密度值没有明显变化:单纯超声作用于EAC细胞后随着时间的延长,细胞中LaminB的平均光密度值逐渐降低;超声结合Hp或PPⅨ协同效应组LaminB的平均光密度值显著降低。
5.观察SDT疗法处理后EAC细胞的凋亡现象:Hoechst 33258核荧光染色显示,对照组细胞核呈均匀的蓝色,随着时间的延长没有明显的变化;单独Hp、PPⅨ组与对照组类似;SDT疗法处理后EAC细胞核随着时间的延长而显著变化,2小时取材时超声结合Hp和超声结合PPⅨ组细胞出现核固缩,染色体凝聚等现象;4小时后超声结合Hp和超声结合PPⅨ组细胞出现明显的核变形,染色体凝聚、趋边等现象,超声结合PPⅨ组细胞还出现明显的凋亡现象。
本论文在前期研究的基础上,初步研究了SDT疗法对EAC细胞细胞骨架的影响,为SDT疗法的研究积累相关资料。
Sonodynamic therapy(SDT) is a approach for cancer treatment basing on ultrasound(Us) could be precisely focused on the target volume,which made it possible to effectively activate the cytotoxicity of sonosensitizers(Hemtoporphyrin and Hemtoporphyrin Derivatives) that preferentially accumulate in tumor sites.
Currently,there are lots of conventional treatments for tumor as surgery,radiation therapy and chemotherapy.However,these methods all have their pros and cons.Surgery,surgical excision of tumor,is better in early treatment but advanced ineffective;Radiation therapy is failed on widely spread or partial tumor invasion,and it also brings some near or long-term side effects; Chemotherapy is the main cancer treatment using chemical means of antineoplastic agents,which have strong side effects,mainly including nausea,vomiting,alopecia,leukopenia losing,and so on. In 1978,American scholar Doughtery put forward Photodynamic Therapy(PDT),which was already applied to the clinical treatment of tumors.Because of the undesirable penetration of the light, photodynamic therapy was used mostly to treat tumors located in human skins,but not those in the deep part of human bodies.Ultrasound which can penetrate deep into the tissue and have little harm to natural tissue,at the same time,hematoporphyrin,can specifically accumulate in tumor cells.So, SDT is a promise therapy for cancer treatment.Since SDT was proposed in 1989,it has been widely studied by many researchers,and they also proposed many cell killing mechanism including sono-cavitation,free radicals,lipid peroxidation,and so on.But until now,we still do not have a unanimous conclusion about SDT.
Cytoskeleton,the important cell component,plays a crucial role in numerous cell functions, such as proliferation,morphology,signal transduction,cancer metastasis,etc.Literatures repoted the photodynamic damages of PDT on cell cytoskeleton which affect the functions of cells.For example, Zinc(Ⅱ)-phthalocyanine can induced photodamage to microtubule,actin,α-actinin and keratin of HeLa Cells,and suppresses cell cycle and change cell shape;ALA-PDT-induced changes in adenocarcinoma WiDr and glioblastoma D54Mg cells cytoskeleton,cell shape,and adhesion; ALA-PDT can also affect the cytoskeleton organization in K562 cells.Cytoskeleton constitutes a very important objective for all cancer therapies.However,so far,the effect of SDT on cytoskeleton has not been reported.So it is very important to identify whether the sonodynamic effect have some impacts on cytoskeleton.and also interesting to compare the effects between protoporphyrin IX (PPIX) and hematoporphyrin(Hp) on cytoskeleton of Ehrlich ascites carcinoma(EAC) cells under the same ultrasound exposure conditions.
Basing on the international development and earlier experiment results,this paper has done some work about the National Natural Science Foundation of China(Grant No.39870240 and No. 30270383).The sonodynamical effect was investigated on EAC cells exposed to the combination of 20μmol/L PPIX and Hp and focused ultrasound at the frequency of 1.34 MHZ,and the present results can be explained as follows:
1.When observed under SEM,untreated cells showed a typical surface morphology,with numerous randomly distributed microvilli.The number of microvilli on cells in ultrasound alone group decreased with time.After 2 h of incubation,the surface of many cells became relatively smooth with no obvious microvilli,several small craters were also seen at 4 h.In the synergistic treatment groups,the extent of cell damage increased in a time dependent manner.In both Uh and Up groups,the number of microvilli remarkably decreased at 2 h after treatment and had apparent deformation 4 h later.Cells in Up group with some irregular blebs were seen in the surface where the cytoplasm seemed to have extruded through the membrane boundary.Hp and Pp groups had only a slight effect on the surface of the cells until 4 h post-treatment and had no obvious difference with CT group.
2.The ultrasonically induced F-actin damage increased rapidly with ultrasound intensity and the sensistizer concentration increased.The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry.The fluorescence intensity of F-actin decreased in ultrasound treated group cells.The fluorescence intensity of F-actin in Hp-SDT and Pp-SDT decreased as time prolonged.
3.The fluorescence study revealed restructuring of microtubule and intermediate filament networks represented byβ- tubulin and vimentin elements.MT and VF polymerization state in Hp and Pp groups cells had no obvious changes as time passed.In Us alone group,cells MT and VF fluorescence intensity partially decreased as time prolonged.Changes of MT and VF were more obvious in Uh and Up groups.Immediately after the treatment,cells MT and VF became diffuse, and the fluorescence intensity of MT and VF decreased.Severe alterations in morphology and distribution of MT and VF could be detected in most Up group ceils 4h later,which exhibited alterations of the plasma membrane,and numerous blebs reacted positively toβ-tubulin and vimentin antibodies could be seen on the surface of cells.
4.The IOD value of nuclear lamina proteins- LaminB was examined by the method of the immunocytochemistry which indicated that the IOD value remained no visible difference among control groups,Hp and Pp groups.In ultrasound alone group,the IOD value of LaminB proteins decreased at 4 h after the sonication.The IOD value of LaminB proteins signifficantly decreased in both Uh and Up groups as time prolonged,which was more obvious in Up groups.
5.Hoechst 33258 staining was used to detect the apoptosis of EAC cells after SDT treatment. The control cells were uniformly blue,and had no visible nuclei changes with time.Hp and Pp groups had no obvious difference with CT group.The apoptotic cells were blue and contain bright blue dots in their nuclei,representing the nuclear fragmentation in both Uh and Up groups as time prolonged.
In this paper,we study the sonodynamic effect of SDT on the cytoskeleton of EAC cells to find out the biology effect of SDT.
引文
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