【作者】 张妍；

【导师】 张维东；

【作者基本信息】 济南大学 ， 病理学与病理生理学， 2012， 硕士

【摘要】 卵巢癌是女性生殖器官常见的恶性肿瘤，近年来其发病率和死亡率有缓慢上升趋势。据最新统计，在女性生殖系统恶性肿瘤中卵巢癌发病率已从第三位上升到第二位，由于大部分患者就诊时已是临床晚期，其死亡率更是高居各类妇科肿瘤的首位。目前，卵巢恶性肿瘤的标准治疗方案为肿瘤细胞减灭术联合以铂类为基础的辅助性化疗，五年生存率仅为20％～25％左右，对女性生命仍存在严重威胁，如何控制卵巢癌的发展以及寻找新的治疗方法，已成为目前重点研究课题。肿瘤内部及周围的血管生成被认为是其增殖、转移的前提，因此，抑制肿瘤血管生成对于控制肿瘤进展、改善预后和提高患者生存率具有十分重要的意义。中医治疗肿瘤病症有着悠久的历史，蝎毒作为一种传统中药已被应用于多种疾病的治疗，它的抗肿瘤作用也受到越来越多的重视。蝎毒多肽是从东亚钳蝎蝎毒中提取的一种多肽组分，本研究组前期实验证实解毒多肽对肝癌、非小细胞肺癌等多种肿瘤具有抗血管生成及抑制肿瘤生长、转移的作用，但目前蝎毒多肽对卵巢癌的具体作用及机制研究较少。因此，本实验通过检测蝎毒多肽对卵巢癌细胞的增殖抑制作用、对细胞周期影响以及血管生成过程中关键因子的表达，进一步探讨蝎毒多肽抑制人卵巢癌细胞的可能作用机制。目的：观察蝎毒多肽提取物（PESV）对人卵巢癌SKOV3细胞增殖的抑制作用和对细胞周期的影响，以及PESV干预后SKOV3细胞中VEGF、TSP-1、Id-1蛋白及mRNA表达水平变化，揭示PESV抗肿瘤作用机制，为PESV抗癌作用提供新的理论依据。方法：1.采用细胞形态学观察、MTT实验检测解毒多肽对SKOV3细胞形态以及增殖活性的影响。2.流式细胞仪检测蝎毒多肽对SKOV3细胞周期分布的影响。3.免疫细胞化学法及Western bolt测定PESV干预后SKOV3细胞中VEGF、TSP-1、Id-1蛋白表达变化。4. RT-PCR检测蝎毒多肽对SKOV3细胞中VEGF-A、TSP-1、Id-1基因mRNA的表达的影响。结果:1.蝎毒多肽对SKOV3细胞形态学的影响对照组细胞形态呈短梭形、多边形，细胞大小均匀一致，胞浆丰富透亮，细胞贴壁良好，生长旺盛。与对照组相比，PESV作用48h后细胞密度减少，细胞变圆且胞内颗粒感增强，贴壁差，细胞脱落呈漂浮状，随着药物浓度加大，上述现象更加明显。2. MTT检测蝎毒多肽对SKOV3细胞的增殖抑制作用蝎毒多肽12.5～200μg/mL对SKOV3细胞均有增殖抑制作用，并随着浓度的增加抑制作用逐渐加强。PESV质量浓度为50、100、200μg/mL时，抑制率分别为38.78%、53.30%、79.22%，作用显著且呈质量浓度相关性，各剂量组间的差异有统计学意义（P＜0.01）。3.蝎毒多肽对SKOV3细胞周期的影响随PESV浓度升高，Go/G1期细胞所占比例由对照组的45.80％±1.05逐渐增高至69.28％±1.03，S期细胞比例由对照组的32.50％±0.98逐渐下降至17.23％±0.31，各组间比较P＜0.01。PESV对细胞周期的影响呈质量浓度效应关系。4.蝎毒多肽对SKOV3细胞VEGF、TSP-1、Id-1蛋白表达的影响4.1免疫细胞化学法蝎毒多肽作用SKOV3细胞48h后，VEGF、Id-1表达较对照组明显下调（P＜0.01），且VEGF表达与Id-1呈正相关关系，r=0.819，P＜0.01；TSP-1表达上调，150μg/mL组呈强阳性（P＜0.01），100μg/mL组呈阳性（P＜0.05），50μg/mL组与对照组相比差异不显著。4.2Western blot法蝎毒多肽100、150μg/mL组VEGF表达减少（P＜0.01），Id-1在各剂量组表达均明显下调（P＜0.01）。TSP-1蛋白表达量随PESV浓度升高而增加，150μg/mL组相对于对照组明显增强(P＜0.01)。统计分析得出，Id-1与VEGF表达呈正相关关系，r=0.762，P＜0.01，与TSP-1表达呈负相关关系，r=-0.741，P＜0.01。5.蝎毒多肽对SKOV3细胞中VEGF、 TSP-1、Id-1mRNA表达的影响随PESV浓度增加，细胞中VEGF与Id-1mRNA表达呈下降趋势，而TSP-1mRNA则表达上调。与对照组相比，150μg/mL组VEGF与Id-1mRNA表达明显减少（P＜0.05），TSP-1表达明显增加（P＜0.05）。结论蝎毒多肽对人卵巢癌SKOV3细胞有显著增殖抑制作用，将细胞阻滞在G0/G1期，阻碍其顺利进入S期，此外还能影响细胞中VEGF、TSP-1、Id-1蛋白及mRNA表达。蝎毒多肽抗肿瘤作用机制可能是通过抑制肿瘤细胞增殖，影响细胞周期分布，调控肿瘤细胞血管生成过程中关键因子的表达来实现的。更多还原

【Abstract】 Ovarian cancer is a common malignant tumor of the female genital organs. In recentyears, the morbidity and mortality of ovarian cancer have a slow upward trend. Accordingto the latest statistics, its incidence rose from third to second place in gynecologic cancer.Due to the majority of the patient are clinical late stage already, the mortality rate is thehighest of the various types of gynecologic oncology. Currently, the standard regimen forovarian malignancy is the Tumor cytoreductive surgery and platinum-based adjuvantchemotherapy, but the5-year survival rate is still hovering at about20%～25%, there isstill a serious threat to the life of women. How to control the development of ovariancancer and to find new treatments, have become a key research project.The premise of tumor proliferation, migration is angiogenesis. Therefore, inhibittumor angiogenesis has great significance for controlling of tumor progression, improvingprognosis and survival rate of patients. Chinese medicine treatment tumor diseases have along history, Scorpion venom has been used in the treatment of many diseases as atraditional Chinese medicine, and its effect of anti-tumor also has draw attention.Polypeptide extract from scorpion venom (PESV) is a polypeptide extracted fromScorpionidae. The early experiments of this study group confirmed that PESV have a goodanti-tumor effect in variety of tumors such as Liver cancer, non-small cell lung cancer andso on. However, the mechanism of PESV effect on ovarian cancer has not been elucidated.So our study group explore the possible mechanism of PESV inhibits human ovariancancer by relational detection methods. ObjectiveThis study was to observe the effects of PESV on the cell proliferation, cell cycle andexpression of VEGF, TSP-1, and Id-1in ovarian cancer cell line SKOV3, to investigate thepossible mechanism of PESV anti-tumor effect, and to provide a new theoretical basis forPESV anti-tumor effects.Methods1. Observation of cellular morphology under microscopy and detection the effect of PESVon proliferation activity in SKOV3cells by MTT assay．2. Detection the effect of PESV on cell cycle distribution in SKOV3cells by flowcytometry.3. Immunocytochemistry and western bolt were applied to detect the effects of PESV onthe protein expression of VEGF、TSP-1and Id-1.4. RT-PCR were applied to detect the effects of PESV on the mRNA expression of VEGF、TSP-1and Id-1.Results1. Effect of PESV on morphology of SKOV3cellCell morphology of the control group showed that: short uniform，polygonal，uniformsize, translucent cytoplasm,well adherence, vigorous growth. After treatment with PESV,contrasting to control group, density of cells was decreased, cells became round and grainyenhanced intracellular, cells shedding and floating. With the PESV concentration increased,the phenomenon is more pronounced.2. Effect of PESV on proliferation of SKOV3cellsPESV remarkably inhibited the proliferation of SKOV3at the range of12.5～200μg/mL, the inhibition gradually strengthen with the increase of the concentration.When the concentration of PESV were50、100、200μg/mL,the growth inhibition ratiowere38.78%、53.30%、79.22%respectively. PESV has a significant inhibitory effect oncell proliferation in a dose-dependent. The differences between the groups were statistically significant (P<0.01).3. Effect of PESV on cell cycle of SKOV3cellsWith the increase concentration of PESV,cell proportion of G0/G1phase wasgradually increased from45.80%±1.05to69.28%±1.03, S phase decreased from32.50%±0.98to17.23%±0.31, There was significant difference between different groups(P＜0.01).The effect of PESV on cell cycle in a dose-dependent.4. Effect of PESV on protein expression of VEGF、TSP-1、Id-1of SKOV3cell4.1immunocytochemistry After treatment with PESV48h, the expression levels ofVEGF、Id-1were significantly decreased compared with that of the control group (p<0.01).There was a positive correlation between the expression of VEGF and Id-1,r=0.819,P<0.01.TSP-1was unregulated by PESV,150μg/mL group was strongly positive(P<0.01),100μg/mL group was positive (P<0.05), The difference between50μg/mL groupand control group was not significant.4.2western blot The expression of VEGF in PESV100,150μg/mL group wasdecreased (P<0.01), Id-1were significantly reduced in all dose group (P<0.01).PESVexhibited a dose dependent up regulatory effect on the expression of TSP-1,150μg/mLwas significantly enhanced (P<0.01). Statistical analysis, the expression of Id-1and VEGFwere positively correlated, r=0.762, P <0.01, while Id-1and TSP-1was negativelycorrelated，r=-0.741, P<0.01.5. Effect of PESV on mRNA expression of VEGF、TSP-1、Id-1of SKOV3cellPESV treatment can down regulate the VEGF、Id-1mRNA, at the same time themRNA expression of TSP-1are up-regulated. Compared with the control group, VEGFand Id-1mRNA expression of PESV150μg/mL were significantly reduced (P <0.05),TSP-1mRNA expression was significantly increased (P<0.05).ConclusionsPESV can inhibit the proliferation on human ovarian cancer SKOV3cells significantly,and arrest the cells stay in G0/G1.In addition, PESV also can affect protein and mRNA expression of VEGF、TSP-1、Id-1. Anti-tumor effects of PESV may be carried out byinhibiting the tumor cell proliferation, affecting cell cycle distribution and impacting theexpression of key factors in the process of tumor angiogenesis.更多还原