【作者】 詹平；

【导师】 戴闽；

【作者基本信息】 南昌大学 ， 外科， 2009， 硕士

【摘要】 目的:探讨化疗药顺铂和阿霉素损伤人U2骨肉瘤细胞(U2-OS)后p53、bcl-2、c-myc基因表达情况的变化及其诱导细胞周期阻滞及凋亡的情况,为骨肉瘤化疗耐药的解决提供理论依据。方法:分别使用0.3、3.0、30μg/ml顺铂、0.06、0.6、6.0μg/ml阿霉素作用于U2-OS细胞2h后继续培养0、6、12、24、48h,采用噻唑兰比色法(MTT法)检测U2-OS细胞生长抑制率。流式细胞术、逆转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹(Western Blot)检测顺铂和阿霉素损伤后继续培养6、24h后细胞细胞周期变化、凋亡率及p53、bcl-2、c-myc基因表达情况。结果:不同浓度顺铂、阿霉素损伤U2-OS细胞后继续培养6h,流式细胞术检测各组G1期细胞分别为67.97%、79.64%、78.09%、80.21%、78.25℅、79.13℅、79.92℅,而p53基因mRNA和蛋白表达和空白对照组比较均增加,bcl-2、c-myc的表达和空白对照组比较无变化,细胞生长抑制率无明显统计学差异;继续培养24h,②、③、④、⑤、⑥、⑦组细胞生长抑制率分别为31.58%、44.12%、59.46%、23.40%、40.31%、57.56%,②、③、④组之间,⑤、⑥、⑦组之间有明显组间差异;流式细胞术检测④、⑦组细胞凋亡率分别为10.04%、14.55%,细胞周期以G2期细胞为主。②、③、④组p53、bcl-2和c-myc mRNA及蛋白表达有组间差异,⑤、⑥、⑦组p53、bcl-2和c-myc mRNA及蛋白表达有组间差异。继续培养48h,各组细胞生长抑制率增高。结论:不同浓度的顺铂和阿霉素短时间(2h)损伤U2-OS细胞仍有细胞增殖抑制作用,这种作用与细胞DNA损伤后p53、bcl-2、c-myc基因表达变化有关;U2-OS细胞DNA损伤后6h即表现出明显的p53基因的表达增强并诱导G1期细胞周期阻滞;继续培养24h,p53、bcl-2、c-myc基因表达变化与药物浓度有关联且诱导G2细胞周期阻滞及凋亡发生。更多还原

【Abstract】 Objective: To investigate the expression of p53﹑bcl-2 and c-myc and its effects on cell cycle arrest and apoptosis induced by cisplatin and adriamycin, the chemotherapeutics inU2-OS cells.Methods:After the treatment of 0.3μg/ml, 3.0μg/ml, 30.0μg/ml cisplatin and 0.06μg/ml, 0.6μg/ml, 6.0μg/ml adriamycin for 2h, we keep on cultivate U2-OS cells with fresh medium in 0, 6, 12, 24, 48h. Then MTT were used to evaluate cell proliferation in vitro. The expression of wild-type p53, bcl-2,c-myc were detected by RT-PCR and Western blot, Flow Cytometry (FCM) was employed for examining the cell cycle after cultivate for 6h, 24h.Results: After damage by different dencity of cisplatin and adriamycin, U2-OS cell were cultivated for 6h, FCM show that cell in G1 phase were incresed obviously, the ratio were 67.97%, 79.64%, 78.09%, 80.21%, 78.25℅, 79.13℅ and 79.92℅.The expression of p53 were up-regulate, bcl-2 and c-myc expression were unchangeable, the inhibite of cell grow were not manifest in statistics. After cultivate for 24h, the inhibitory rate of cell growth of group②③④⑤⑥⑦were 31.58%, 44.12%, 59.46%, 23.40%, 40.31% and 57.56%. Group②③④were different in statistics, Group⑤⑥⑦were different in statistics by contrast with each other. The ratio of apoptosis by FCM were 10.04%, 14.55%, cell cycle arrest in G2 phase. The expression of p53, bcl-2, c-myc was different obviously in statistics of group②③④, the expression of p53, bcl-2, c-myc was different obviously in statistics of group⑤⑥⑦. After cultivate for 48h, the inhibitory rate of cell growth was increased..Conclusions:The U2-OS cells were damaged by different dencity of cisplatin and adriamycin in short time(2h), the inhibition of cell grow was also manifest. The effection were related with the diversify of the expression of p53, bcl-2, c-myc after DNA damage. The diversify of p53 induced G1 cell cycle arrest after 6h, and after 24h, the diversify of p53, bcl-2, c-myc induced G2 cell cycle arrest and apoptosis, the change was related with drug concentration.更多还原