【作者】 陈宏亮；

【导师】 孙红；

【作者基本信息】 复旦大学 ， 妇产科学， 2008， 硕士

【摘要】 在卵巢癌中,P53基因的突变是最常见的基因改变之一。30%～79%卵巢癌中出现了P53基因的突变。目前,野生型P53基因替代疗法是肿瘤基因治疗的研究热点,而重组人P53腺病毒液则是最常用的治疗方式。有研究表明,导入野生型P53基因,能明显抑制肿瘤细胞的生长,促进细胞凋亡,增加肿瘤细胞对化疗的敏感性。因此,本课题利用人卵巢癌细胞株SKOV-3及CAOV-3,研究重组人P53腺病毒液对卵巢癌细胞的生长抑制及凋亡诱导的作用。目的与背景:探讨研究重组人P53基因腺病毒液(rAd-P53)对卵巢腺癌细胞株的生长及化疗敏感性的影响。方法:将rAd-P53携带的外源性P53基因导入卵巢癌细胞株SKOV-3及CAOV-3,并联合应用化疗药物DDP,通过Western blot法分析P53基因在肿瘤细胞中的表达,通过MTT法、流式细胞方法观察肿瘤细胞生长、凋亡及细胞周期的影响。1.利用MTT方法比较不同时间、不同滴度rAd-P53对人卵巢癌细胞株CAOV-3、SKOV-3的体外生长抑制率,比较rAd-P53、DDP以及两者联合用药对卵巢肿瘤细胞的体外生长抑制率,比较联合用药时不同用药顺序对卵巢肿瘤细胞的体外生长抑制率的影响;2.利用流式细胞方法比较rAd-P53、DDP以及两者联合用药作用于卵巢肿瘤细胞72h后肿瘤细胞的细胞周期及细胞凋亡率;3.利用Western blot方法检测rAd-P53作用于卵巢肿瘤细胞72h后P53蛋白的表达。结果:1.rAd-P53对人卵巢癌细胞株CAOV-3及SKOV-3的体外生长抑制作用均呈现明显的时间依赖性、剂量依赖性关系。联合用药与rAd-P53、DDP单药用药相比,对卵巢肿瘤细胞的体外生长抑制作用更显著(CAOV-3:联合用药73.57±0.43%,DDP 52.05±2.13%,rAd-P53 45.53±0.95%,P＜0.01;SKOV-3:联合用药74.70±0.86%,DDP 59.46±1.17%,rAd-P53 50.72±2.04%,P＜0.01)。联合用药时不同的用药顺序对肿瘤细胞体外生长抑制作用无明显差异(P＞0.6)。2.联合用药72h后,肿瘤细胞的细胞周期阻滞于G0/G1期CAOV-3:67.47±1.03%,SKOV-3:67.26±2.37%,与对照组、DDP组比较有统计学差异(CAOV-3:对照组41.58±1.72%,DDP组36.87±6.53%;SKOV-3:对照组41.68±3.51%,DDP组48.36±9.34%,P＜0.01),S期比例明显减少,CAOV-3:32.51±1.06%,SKOV-3:32.53±2.32%,与对照组、DDP组、rAd-P53组比较均有统计学差异(CAOV-3:对照组54.65±2.79%,DDP组45.98±8.38%,rAd-P53组36.10±3.67%;SKOV-3:对照组54.94±0.65%,DDP组44.88±6.11%,rAd-P53组37.11±1.20%,P＜0.01),且细胞凋亡率CAOV-3达到19.39±1.67%,SKOV-3达到38.12±1.83%,与对照组、DDP组、rAd-P53组比较均有统计学差异(CAOV-3:对照组0.19±0.13%,DDP组6.61±0.49%,rAd-P53组2.61±0.20%;SKOV-3:对照组0.12±0.85%,DDP组8.52±0.47%,rAd-P53组5.70±0.47%,P＜0.01)。3.Western blot检测rAd-P53作用于卵巢肿瘤细胞CAOV-3及SKOV-3 72h后,比较未用药物的肿瘤细胞,有明显的P53蛋白表达。结论:rAd-P53能将外源性野生型P53基因导入肿瘤细胞的基因组,使肿瘤细胞表达P53蛋白,从而使肿瘤细胞细胞周期阻滞于G0/G1期,抑制肿瘤细胞体外生长,促进细胞凋亡。rAd-P53与常规化疗药物DDP联合应用能显著增加肿瘤细胞对DDP化疗的敏感性,其抗肿瘤效应较DDP、rAd-P53单药应用更加显著。更多还原

【Abstract】 P53 gene is the most commonly mutated gene in ovarian tumors. There is P53 gene mutated among 30%～79% ovarian tumors. At present, wide-type P53 gene replacement therapy is among focus of tumor gene therapy, of which, recombinant adenovirus-P53 is the most commonly used. It has been reported that adenovirus-mediated wide-type P53 gene transfection into tumor cells can inhibit cell growth, promote cell apoptosis, and enhance chemotherapy sensitivity. This research is to evaluate the effects of domestic recombinant adenovirus-P53 (rAd-P53,Gendicine) on growth inhibition and apoptosis of human ovarian adenocarcinoma cell lines SKOV-3 and CAOV-3.Objective: The aim of the study is to evaluate the effects of domestic recombinant adenovirus-P53 (rAd-P53, Gendicine) on growth and chemosensitivity of human ovarian adenocarcinoma cell lines.Methods: Human ovarian adenocarcinoma cell lines CAOV-3 and SKOV-3 (mutant P53) were treated with rAd-P53, cisplatin (DDP) and rAd-P53 + DDP respectively. P53 expression was dectected by Western blot. The cell growth inhibition was assessed by MTT, and cell cycle and apoptosis were dectected by flow cytometry.1. use MTT to compare CAOV-3 and SKOV-3 cell growth inhibition rate when they were treated with rAd-P53, cisplatin (DDP) and rAd-P53 + DDP respectively of different time and dose. Compare tumor cell growth inhibition of different sequence of combination treatment.2. use flow cytometry to compare cell cycle and apoptosis of CAOV-3 and SKOV-3 when treated with rAd-P53 (100MOI), DDP (20uM) and rAd-P53 (100MOI) + DDP DDP (20uM) respectively after 72 hours.3. use Western blot to detect P53 expression of CAOV-3 and SKOV-3 after treated with rAd-P53 (100MOI) 72 hours later.Results: 1. There was a dose-dependent and time-dependent inhibition of cell proliferation by rAd-P53. After combined treatment with rAd-P53 (100MOI) and DDP (20uM) for 72 hours, the growth inhibition rate of CAOV-3 cells was 73.57±0.43%,which was significant higher than that in rAd-P53 group (45.53±0.95%, P<0.01) and DDP group (52.05±2.13%, P<0.01) . The growth inhibition rate of SKOV-3 cells was 74.70±0.86%, which was significant higher than that in rAd-P53 group ( 50.72±2.04%, P<0.01) and DDP group (59.46±1.17%, P<0.01) .The sequence of combined treatment is of no difference to the cellgrowth inhibition of SKOV-3 and CAOV-3.(P>0.6)2. Combined administration of rAd-P53 and DDP remarkably arrested CAOV-3 and SKOV-3 in G0/G1 (CAOV-3: 67.47±1.03 % , SKOV-3: 67.26±2.37%), which was significantly higher than that in control group (CAOV-3: 41.58±1.72%, SKOV-3 : 41.68±3.51 %, P<0.01) and DDP group (CAOV-3: 36.87±6.53%, SKOV-3: 48.36±9.34%, P<0.01).And cells in S phase significantly decreased (CAOV-3: 32.51±1.06%, SKOV-3: 32.53±2.32%), which was significantly lower than that in that in control group (CAOV-3: 54.65±2.79%, SKOV-3 : 54.94±0.65%, P<0.01), rAd-P53 group (CAOV-3: 36.10±3.67%, SKOV-3 : 37.11±1.20%, P<0.01) and DDP group (CAOV-3: 45.98±8.38%, SKOV-3 : 44.88±6.11%, P<0.01).Meanwhile the apototic rate of CAOV-3 cells was 19.39±1.67% in rAd-P53 + DDP group, which was significantly higher than that in rAd-P53 group (2.61±0.20%, P<0.01) and DDP group (6.61±0.49%, P<0.01). The apototic rate of SKOV-3 cells was 38.12±1.83% in rAd-P53 + DDP group, which was significantly higher than that in rAd-P53 group (5.70±0.47%, P<0.01) and DDP group (8.52±0.47%, P<0.01).3. High level P53 expression was dectected in CAOV-3 and SKOV-3 cells by Western blot after administeration of rAd-P53 (100MOI) 72 hours later.Conclusion: rAd-P53 can tranfect wide-type P53 into the genome group of human ovarian tumor cells, arrest tumor cells at G0/G1, inhibit the growth of human ovarian tumor cells, promote tumor cells apoptosis. Its combibation with DDP may significantly enhance the chemosensitivity of human ovarian tumor cells to DDP.更多还原