磁场联合抗肿瘤药物对肿瘤细胞杀伤机制的研究

【作者】 艾霞；

【导师】 齐浩；

【作者基本信息】 陕西师范大学 ， 生物物理， 2009， 硕士

【摘要】 目的:研究磁场(static magnetic field,SMF)结合抗肿瘤药物对人源肿瘤细胞K562细胞以及SW480细胞协同杀伤的细胞生物学机制。方法:1.采用MTT法检测SMF和ADM单独及联合作用下,对K562细胞生长活性的影响;以及SMF和顺铂单独及联合作用下,对SW480细胞生长活性的影响。2.通过单细胞凝胶电泳、流式细胞术,检测SMF联合不同种类的抗肿瘤药物对细胞周期时相分布和细胞DNA损伤的影响。3.用HE染色、原子力显微镜、透射电镜、免疫荧光等手段,检测SMF联合抗肿瘤药物对K562细胞和SW480细胞的显微结构、超微结构、P-gp表达的的影响。结果:1.(1)阿霉素和顺铂分别对K562细胞和SW480都有明显的时间与浓度依赖关系(或称量效作用)。随药物浓度增加,细胞生长活性也明显下降。(2)K562细胞经35ng/mL阿霉素单独处理12h即可引起细胞生长抑制(p＜0.05)。(3)SW480细胞经2μg/mL顺铂单独处理12h可引起细胞生长抑制(p＜0.05)。2.(1)K562细胞在25ng/mL阿霉素和SMF单独作用12h时均未表现出杀伤效应;25ng/mL阿霉素和SMF联合作用时,与对照组、阿霉素单独处理组相比细胞活性显著降低(p＜0.05);(2)SW480细胞经1μg/mL顺铂或磁场单独处理12h后,均未表现出杀伤效应;而当1μg/mL顺铂与SMF联合作用12h时,细胞活性显著低于对照组(p＜0.05),且与顺铂单独处理组之间也有极显著差异(p＜0.01)。3.(1)K562细胞经SMF处理12h后,G2/M期细胞的比例明显增加,显示SMF可将细胞阻滞于G2/M期。ADM单独处理K562细胞后,G1期细胞减少,S期和G2/M期细胞比例较对照相比略有增加。SMF与ADM共同作用后,可将细胞阻止于G2/M期。(2)以尾长和尾部DNA含量为DNA损伤参数的彗星电泳检测,发现K562细胞经SMF处理后细胞DNA没有损伤(p＞0.05);ADM可对细胞DNA形成明显损伤(p＜0.05);磁场与ADM联合处理后,细胞DNA的损伤与ADM组之间有极显著差异(p＜0.01),表明磁场促进了ADM对细胞DNA的损伤。(3)人淋巴细胞经SMF与ADM单独或联合处理后,细胞DNA未出现损伤(p＞0.05);表明人淋巴细胞对药物和SMF的联合及单独处理的耐受性显著高于K562细胞。4.(1)SW480细胞曝磁后G2/M期细胞比例略有增加。顺铂作用后SW480细胞G1期比例略有增加,各周期时相的细胞比例与对照相比差异不明显,表明顺铂对SW480细胞周期分布的影响有限。顺铂与磁场共处理细胞后,G1期细胞的比例明显增高,表明磁场与顺铂联合作用时细胞可被阻滞于G1期。(2)SW480经顺铂和/或磁场处理后,彗星电泳检测发现,以尾长、尾部DNA含量、头部DNA含量和头部区域为DNA损伤参数,仅在DNA头部区域这一参数下发现顺铂、顺铂结合磁场会致使细胞DNA头部区域变小,与对照相比差异极显著(p＜0.01)。5.(1)HE染色后光镜观察的结果发现,曝磁后部分K562细胞的细胞核染色略深,阿霉素处理后细胞核深染,细胞表面变得粗糙,有损伤细胞出现;SMF联合阿霉素处理后,细胞周边出现小泡,部分细胞破损,视野中可见细胞碎片,并出现较大细胞。(2)原子力显微镜观察K562细胞表面的精细结构,发现曝磁后细胞表面出现凹陷。经阿霉素处理的细胞表面出现均匀的粒状突起物,并出现一些形状不规则的凹陷。SMF结合阿霉素处理K562细胞后,细胞表面出现凹陷,并可观测到不规则的突起物。实验结果显示,SMF可协同阿霉素增强对K562细胞表面结构的破坏。(3)透射电镜观察K562细胞分别经阿霉素及SMF处理后超微结构的变化。SMF处理后细胞胞浆内线粒体、内质网和溶酶体等胞内结构增多,细胞表面出现微小凹陷。阿霉素处理后细胞胞浆内溶酶体增多,胞内出现较多的空泡,胞膜处有明显的凹陷和突起产生。SMF与ADM联合处理后细胞表面的凹陷与突起均增多,胞内出现较大的空泡,线粒体结构破坏,核膜内褶卷曲。实验观察表明SMF与阿霉素共作用对细胞表面和内部超微结构有协同损伤效应。(4)流式细胞仪观察免疫荧光染色后K562细胞在SMF与阿霉素单独以及联合处理后,细胞内P-gp表达的变化。实验显示,阿霉素可诱导P-gp的表达。SMF可阻止K562细胞的P-gp表达。SMF与阿霉素联合作用亦可降低细胞P-gp的表达。实验结果提示,SMF与阿霉素对K562细胞的协同杀伤与胞内阿霉素含量相关;SMF对P-gp表达的下调可能造成胞内药物含量的升高。(5)AFM观察了SW480细胞经SMF以及顺铂处理后表面形貌的变化。对照细胞表面比较平滑。经SMF处理后细胞表面出现凹陷与皱褶。经顺铂处理后,细胞表面出现均匀的粒状突起物和微小凹陷。SMF结合顺铂处理SW480细胞后,细胞表面出现突起和不规则的凹陷。实验结果提示,SMF可协同顺铂增强对细胞表面结构的破坏。(6)荧光显微镜观察SW480细胞经SMF以及顺铂处理后,FITC-鬼笔环肽标记的细胞F-actin的变化。实验显示SMF导致细胞内F-actin弥散分布,表明F-actin的分布紊乱;顺铂处理后,细胞膜表面出现类似延展过程的棘状突出,胞内F-actin排布紊乱。SMF与顺铂联合作用导致SW480细胞胞内F-actin解聚,荧光信号减弱;胞内荧光信号弥散状分布提示细胞内微丝排列紊乱。结论:(1)磁场结合阿霉素在一定条件下对K562细胞有协同杀伤效应,磁场结合顺铂在一定条件下对SW480细胞有协同杀伤效应。(2)通过对细胞周期分布、细胞DNA损伤及细胞形态的检测发现:K562细胞在SMF与阿霉素协同作用下的生物学效应主要有:细胞内部结构和细胞膜受损、细胞周期分布改变与细胞DNA损伤。其协同机制可能涉及细胞内药物的含量,SMF能够下调多药耐药蛋白P-gp的表达,而阿霉素能上调P-gp的表达。(3)SW480细胞经顺铂和SMF联合处理,造成细胞内部结构与细胞膜受损、细胞周期分布改变与细胞骨架结构(微丝)的破坏。实验发现,与K562细胞不同,顺铂协同SMF杀伤SW480细胞的机制涉及细胞的粘附功能。顺铂与SMF联合作用阻碍了F-actin的形成,使细胞无法粘附于生长基底,致使生长信号缺失,细胞无法进入增殖周期,导致细胞的G1期阻滞。更多还原

【Abstract】 Purpose:To investigate the synergistic anticancer effects of static magnetic fields with adriamycin(ADM)、cisplatin(DDP) on K562 cells or SW480 cells,and the different cytological mechanism of the synergistic anticancer effects.Methods:1.The survival of suspension cancer cells(K562 cells) exposed to SMF or/and adriamycin were detected by MTT test.The survival of adherent cancer cells (SW480) exposed to SMF and cisplatin were detected by MTT test.2.The cell cycle distribution and DNA damage of K562 cells or SW480 cells induced by SMF or/and ADM、DDP were investigated by flow cytometer(FCM) and single cell gel electrophoresis(SCGE),respectively.3.After exposured to SMF or/and ADM、DDP,The cell membrane structure and internal structure were observed by optical microscope,atomic force microscope(AFM) and transmission electron microscope (TEM).The expressions of P-glycoprotein(P-gp) were analyzed by immunofluorescence of FCM.Resuits:1.(1) The anticancer effect of ADM or DDP on K562 cells/SW480 cells was concentration- and time-dependent.(2) The growth of K562 cells were inhibited after cells were incubated with 35ng/mL ADM for 12hr(p＜0.05).(3) The growth of SW480 cells were inhibited after cells were incubated with 2μg/mL DDP for 12hr (p＜0.05).2.(1) Combination of 9mT SMF and 25 ng/mL ADM for 12h could inhibit the cell activity of K562 cells significantly(P＜0.05),while neither ADM nor SMF could influence cell activity;(2) The activity of SW480 cells can not be inhibited after cells were treated with 1μg/mL DDP or 9mT SMF alone for 12 hr.But the cell activity of combined treatment group(1μg/mL DDP+SMF) in SW480 was significantly reduce, compared with controls or DDP group(p＜0.05 or p＜0.01).3.(1)When K562 cells were treated with only 9mT SMF for 12hr,the proportion of cells in G2/M phase were increased compared to controls;when K562 cells were treated with only 25ng/mL ADM for 12hr,the proportion of cells in S and G2/M phase increased compared to control,respectively.K562 cells were treated with the combination of ADM and SMF for 12hr,cells were almost blocked in G2/M phase;(2) DNA damage could not be detected by SCGE when K562 cells only exposed to 9mT SMF for 12 hr;DNA damage of K562 cells treated with 25ng/mL ADM for 12hr was significantly different controls(p＜0.05);If treated with the combination of ADM and SMF for 12hr,DNA damage was increased significantly compare with other three groups(p＜0.01),respectively;The survival of SW480 cells can not be inhibited after treatment with 1μg/mL DDP for 12hr or exposure to 9mT SMF for 12 hr.(3)However, there were no DNA damage when primary human lymphocyte were treated with ADM or/and SMF.4.(1)If SW480 cells were treated with only 9mT SMF for 12hr,the number of cells in G2/M phase increased compared to control;If treated with only 1μg/mL DDP for 12hr,the distribution of cell cycle was relatively average;If treated with the combination of DDP and SMF for 12hr,the most of cells were blocked in G1 phase;.(2) For SW480 cells,1μg/mL DDP or/and 9 mT SMF have no detectable effect on Tail Length,Tail DNA%,and Head DNA%.But,after treated with DDP or combination of DDP and SMF,the area of head was significantly reduced(p＜0.01).5.(1)Light microscope was used to observe the cell morphological modification. The cells exposed to SMF had no detectable changes.The edge of the cells treated with ADM became rough.Exposure to SMF and ADM lead to cell death,many big vacuoles appeared in the cytoplasm and plasma membrane broke down,and the cell debris also could be observed.(2) The modification of cell surface was observed by AFM.For K562 cells,the surface of cells exposed to SMF became rough,hollows appeared on the cell surface.The damage of the cell surface treated with ADM aggravated.The surface structure of cells treated with SMF plus ADM was injured severely,deeper hollows were detected under AFM.(3) The same results could be observed under SEM.After treatment with 25ng/mL ADM for 12hr,many small vacuoles appeared in K562 cells cytoplasm,and hole-like structures on membrane;After exposure to 9mT SMF for 12 hr, hollows appeared on membrane;After treatment with ADM and SMF,the number and volume of vacuoles increased,the damage of membrane became more serious.(4) Immunofluorescence results demonstrate that treated with 25ng/mL ADM for 12hr can improve the expression of P-gp,but exposure to 9mT SMF for 12 hr can repress the expression of P-gp.Meanwhile treatment with ADM and SMF also repress the expression of P-gp.(5) For SW480 cells,the surface of cells exposed to magnetic field became rough,hollows appeared on the cell surface.The damage of the cell surface treated with DDP were aggravated.The surface structure of cells treated with SMF plus DDP was injured severely,anomalous hollows were detected under AFM.(6)For SW480 cells,after exposure to 9mT SMF for 12 hr,F-actin became more confused; After treatment with 1μg/mL DDP for 12 hr,F-actin congregate along cell membrane which form many protuberance;After treatment with DDP and SMF,the protuberance on the surface of membrane disappeared,but F-actin disassociated in some extend and more confused.Conclusion:(1) Magnetic fields and ADM have synergistic anticancer effect on K562 cells under certain condition.Magnetic fields and DDP have synergistic anticancer effect on SW480 cells under certain condition.(2) For K562 cells,the cytological mechanism of the synergistic anticancer effects maybe induced by the damage of structure of cell membrane,changes of cell internal ultrastructure,cell cycle arrest in G2/M phase and DNA damage.Meanwhile,SMF exposure can reduce the expression of P-gp,but treatment with ADM can improve the expression of P-gp.(3)For SW480 cells,the cytological mechanism of the synergistic anticancer effects maybe induced by the damage of structure of cell membrane,changes of cell internal structure,especially for cytoskeleton,cell cycle arrest in G1 phase. Different from K562 cells,the synergistic mechanism also was induced by adhesion function.After treatment with DDP and SMF,F-actin disassociated and confused,As a result,cells lost the ability to adhere to substrate and lack growth stimulative signal, resulting in cell cycle arrestment in G1.更多还原