【作者】 林安平；

【导师】 胡丽娜；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2007， 硕士

【摘要】 陡脉冲电场对细胞膜的不可逆性击穿导致肿瘤细胞死亡的原理使其有可能成为恶性肿瘤低创或微创物理治疗的新方式。本课题组在前期研究中已证实能量可控陡脉冲能有效的杀死肿瘤细胞,但在实验中也发现由于脉冲电场随着距离的增加而减弱,因此在治疗的肿瘤组织的周边存在着残留的肿瘤细胞。这些残留的肿瘤细胞生物学行为是否有变化,其侵袭转移能力是否可能变化;这对于能否将能量可控陡脉冲安全地用于临床具有重要意义。本实验研究旨在用能量可控陡脉冲作用后的肿瘤细胞作为残留肿瘤细胞的实验模型来研究残留肿瘤细胞的生物学行为变化,尤其是其侵袭转移能力的变化。第一部分能量可控陡脉冲对人卵巢癌SKOV3细胞侵袭转移能力的影响目的:观察能量可控陡脉冲对人卵巢癌细胞SKOV3侵袭、转移能力的影响;分析不同剂量的能量可控陡脉冲对SKOV3侵袭、转移能力的影响及其相关性;尤其是亚致死剂量下SKOV3细胞侵袭、转移能力的变化。方法:体外培养SKOV3细胞,将细胞分为七组;以假电击组作为阴性对照组,秋水仙碱处理组、5-氟尿嘧啶处理组、秋水仙碱和5-氟尿嘧啶联合处理组作为阳性对照组,用三种不同剂量的能量可控陡脉冲处理细胞。用机械法测定细胞粘附能力;用Transwell小室分别测定SKOV3细胞的体外侵袭、趋化运动能力。用倒置显微镜观察Transwell小室背面的细胞数并分区计数细胞个数;取下小室的膜做苏木素一伊红(Haematoxylin and Eosin, HE)染色,在光镜下计数小室背面的细胞数。结果:秋水仙碱和5-氟尿嘧啶联合处理组细胞的侵袭、运动现象完全消失,5-氟尿嘧啶处理组细胞的作侵袭、趋化运动而到达膜背面的数量未见明显降低;秋水仙碱处理组及三种不同剂量的能量可控陡脉冲处理组的细胞侵袭、趋化运动而到达膜背面的的数量较阴性对照组有降低,差异有显著性(P<0.05)。结论:能量可控陡脉冲能够降低SKOV3细胞的侵袭转移能力;其降低SKOV3细胞侵袭、转移能力与其剂量有相关性。第二部分能量可控陡脉冲对人卵巢癌细胞SKOV3细胞骨架作用的实验研究目的:探讨能量可控陡脉冲对人卵巢癌细胞SKOV3细胞骨架的影响。方法:将SKOV3细胞分成5组,用不同参数处理,并用经秋水仙碱处理的SKOV3细胞作为阳性对照组;以未经处理的假电击组作为阴性对照组。以罗丹明-鬼笔环肽直接标记微丝,以免疫荧光法标记微管,用共聚焦荧光显微镜观察。制作电镜标本,用电子显微镜观察细胞骨架的超微结构。结果:免疫荧光图像结果表明,能量可控陡脉冲作用的SKOV3细胞丧失正常的细胞骨架网络;微丝明显解聚、骨架蛋白弥散分布、细胞骨架的网络结构消失;微管解聚,伪足减少且其中的微丝明显解聚,排列紊乱,结构消失。而且这一作用与剂量呈正相关。结论:能量可控陡脉冲对人卵巢癌细胞SKOV3细胞骨架有明显的作用,可以使微丝和微管解聚,细胞的伪足减少,细胞骨架的网络结构消失。第三部分能量可控陡脉冲对卵巢癌基质金属蛋白酶9及其抑制物1蛋白表达的调控目的:基质金属蛋白酶(MMPs)与金属蛋白酶组织抑制物(TIMPs)之间活性的平衡调节破坏是导致肿瘤细胞侵袭和转移的主要原因。研究表明高强度陡脉冲对肿瘤有杀伤和抑制作用,但是否能调控卵巢癌细胞MMPs及TIMPs蛋白的表达,目前尚不明确。本实验的目的在于观察能量可控陡脉冲对卵巢癌细胞MMPs及TIMPs蛋白的表达的影响。方法:以卵巢癌裸鼠皮下移植瘤为模型,随机分为实验组和对照组,采用高强度脉冲电场,电压1000V、频率1000Hz、脉宽250ns于卵巢组织5min,免疫荧光法检测陡脉冲作用后卵巢癌细胞MMP-9及TIMP-1蛋白表达的变化。结果:对照组卵巢癌组织MMP-9及TIMP-1免疫荧光强度分别为140.21±5.16和74.98±3.14,两者比例为1.87。陡脉冲作用于卵巢癌组织后,细胞结构保持完整,MMP-9显著降低,荧光强度50.37±2.61,而TIMP-1表达明显增加,荧光强度116.40±4.70,比例倒置。与对照组比较,两种蛋白表达差异均有显著性(P<0.05)。结论:脉冲对MMP-9及其抑制物有显著的调控作用,对卵巢癌转移和侵袭能力有抑制作用。更多还原

【Abstract】 Steep pulsed electric fields can cause irreversible electrical breakdown on the tumor cell membrane and results in tumor cells death, it would become a new non-invasive or mini-invasive physical approach in the treatment of neoplasm. In our prophas research it were proved that the ECSP can effectly kill the tumor cell. But we discover that there were existing the residual tumour cell aroud the tumor organiz, when pulse electric field was weakened with the distance increasing. The problem that the Biology behavior of the residual tumour cell is whether or not to change and the invasion and metastasis ability of the residual tumour cell is whether or not to strengthen is very significance. In our studies we use the residual tumour cell treated by ECSP as experiment model in order to study it’s biology behavior and change of the invasion and metastasis ability. PART ONE THE EFFECTS OF ENERGY CONTROLLABLE STEEP PULSE ON INVASION AND METASTASIS ABILITY OF HUMAN OVARIAN CANCER CELLS SKOV3Objective: To observe the effects and analysis the different parameter especial sublethal parameter of ECSP on invasion and metastasis ability of human ovarian cancer cells SKOV3.Methods: SKOV3 cells were divided into seven groups . Taking without lightning stroke group as negative control group. The positive control groups were treated by volchicine,5-fluorouracil, volchicine and 5-fluorouracil,; other groups were treated by three different parameter of steep pulse.Using mechanical method to determine cell adhesion ability,Using Transwell cave to determine cell in vitro invision and chemotaxis locomotory ability.Using inverted microscope to observe and zonatize counting cell population on tergal Transwell cave ;Using Haematoxylin and Eosin to dye the membrane of Transwell cave and using light microscope counting cell population on tergal Transwell caveResults: The invasion and movement phenomenon of the groups treated by colchicina combined 5-fluorouracil was completely vanish . The invasion and chemotaxis locomotory ability of the group treated by 5-fluorouracil was not obviously depress. The invasion and chemotaxis locomotory ability of the groups treated colchicines and three different parameter of ECSP was lower than that of negative control group (P<0.05Conclusion: The ECSP can inhibit the invasion and metastasis ability of SKOV3,and has direct correlation with dosage.PART TWOEXPERIMENTAL STUDY ON EFFECTS OF ENERGY CONTROLLABLE STEEP PULSES ON CYTOSKELETON OF HUMAN OVARIAN CANCER CELLS SKOV3Objective:The aim of our study was to determine the effects of energy controllable steep pulse (ECSP) on cytoskeleton of human ovarian cancer cells SKOV3 .Methods: SKOV3 cells were divided into five groups and treated by different parameter of steep pulse; Taking SKOV3 cells treated with volchicine as positive control group; and taking without lightning stroke group as negative control group. Using rhodamine-phalloidine to lable microfilament directly. Using immunofluorescence to label microbule and observing them by using Confocal Laser Scanning Microscope . Making specimen, and using electron microscope to observe the ultramicrostructure of cystoskeleton .Results : the immunofluorescence picture shows that SKOV3 cells whose energy controllable steep pulses treated lose the normal cystoskeleton network strcture. Obvious microfilament disaggregation, diffused skeleton protein , disappearance of cystoskeleton network stucture. Microbule disaggregation; reduction of pseudopodia; obvious microfilament disaggregation, permutation disorder and structrue disappearance.This effects has direct correlation with dosage.Conclusion: energy controllable steep pulse has obvious effect on SKOV3 cells’ cystoskeleton structure . It can cause the disaggregation of microfilament and microbule, the reduction of cells’pseudopodia and the disappearance of network structure of cystoskeleton.PART THREE ELECTRIC PULSES REGULATE THE EXPRESSION OF MMP-9 AND TIMP-1 IN OVARIAN CANCERObjective: researchers have confirmed electric pulses have effects on cancer treatment. To further study the mechanism in cancer metastasis, the balance between matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) are researched as the main reason of tumor metastasis.Methods: ovarian cancer bearing nude mice was subject investigated. And arranged randomly on experiment group and controlled group. Using electric pulses with voltage 1000V, frequency 1000Hz, duration 250ns treated on ovarian cancer tissue 5min, immunofluorscence was taken to detect the expression of MMP-9 and TIMP-1.Results: the fluorescence intensity of MMP-9 and TIMP-1 in controlled group were 140.21±5.16 and 74.98±3.14,the ratio was 1.87. in experiment group the fluorescence intensity respectively were 50.37±2.61 and 116.40±4.70, MMP-9 expressed lowly and TIMP-1 expressed heightened ,the difference was significant statistically(P<0.05), the ratio between MMP-9 and TIMP-1 was inverted to 0.43.Discussion: high intensity electric pulses regulated the balance of MMP-9 and TIMP-1, and indicated that the electric pulses inhibit ovarian cancer from metastasis and invasion, and also taken the evidence for clinical research of electric pulses.更多还原