慢病毒介导沉默食管癌细胞53BP1基因表达对裸鼠移植瘤放射敏感性的研究

Effect of silencing 53BP1 gene on radiosensitivity to esophageal cancer ECA109 cell xenograft in nude mice

背景与目的:p53结合蛋白1(p53 binding protein,53BP1)在多种正常组织和肿瘤细胞中有表达,与放射线照射后细胞周期阻滞有关。体外实验证实抑制53BP1的蛋白表达可有效地消除肿瘤细胞照射后引起的周期阻滞,增加放射敏感性。但体内实验国内尚未见相关报道。该研究旨在探讨沉默食管癌细胞53BP1基因对裸鼠移植瘤放射敏感性的影响。方法:将48只BALB/c/nu裸鼠按随机数字表法分为对照组、单纯照射组、空载体组、空载体加照射组、沉默53BP1组以及沉默53BP1加照射组,每组8只,于裸鼠皮下接种相应食管癌细胞ECA109,制备裸鼠模型,给予15 Gy放射线单次照射,受照后1 h每组处死3只裸鼠,将肿瘤标本按蛋白[质]印迹法(Western blot)及流式细胞分析要求处理,Western blot检测移植瘤组织中CHK1、CHK2和磷酸化CHK2-T68的表达,流式细胞术分析裸鼠肿瘤组织中细胞周期分布和细胞凋亡。观察各组剩余裸鼠移植瘤照射后的生长情况,移植瘤体积变化情况。结果:所有接种裸鼠均成活并有肿瘤形成,各组之间肿瘤体积大小差异无统计学意义(F=0.67,P=0.69);单纯照射组和空载体照射组裸鼠的肿瘤生长速度慢于未照射组(P=0.02),沉默53BP1加照射组裸鼠的肿瘤生长速度最慢(P=0.03),沉默53BP1加照射组的相对生长速率较空载体加照射组和单纯照射组降低,生长抑制率增加(P=0.01)。沉默53BP1加照射组的q值为1.45;沉默53BP1加照射组裸鼠体内CHK1和CHK2的蛋白表达产物未见明显变化(P=0.71),CHK2-T68的磷酸化水平较单纯照射组和空载体照射组明显下降(P=0.03),各组裸鼠的瘤组织中细胞周期分布和凋亡率差异无统计学意义(P=0.45)。结论:体内研究显示沉默53BP1基因可以提高食管癌放射敏感性。

Background and purpose： p53 binding protein （53BP1） expresses in many normal and tumor cells. In vitro experiments have confirmed that inhibition of the protein expression of 53BPI can effectively eliminate cycle arrest of tumor cells, and increase the radiosensitivity after irradiation. However, the in vivo experiment has not been re- ported. This study aimed to explore the effect of silencing 53BP1 gene on the growth and radiosensitivity to esophageal cancer cell ECAI09 xenograft in nude mice. Methods： Forty-eight male BALB/c/nu nude mice were randomly divided into 6 groups： ECA109, ECA109/R, ECAI09/N, ECA109/NR, ECAI09/B and ECA109/BR. Three kinds of prepared cells （ECA109, ECA109AN and ECA109/B） were subcutaneously injected into the paw pads of mice （2×106/100 p L per mouse）. The nude mice in ECA109/R, ECA109/NR, and ECA109/BR groups were irradiated with 15 Gy. Tumor growth was monitored every other day on the 6th day after injection. Tumor volume was measured with calipers. The expression levels of CHK1, CHK2 and phosphorylated CHK2-T68 protein were examined in different groups by West- ern blot. Apoptotic cell and cell cycle distribution were detected by flow cytometry assay. Results： Visible tumors were detectable by day 7 after implantation, and the tumor volumes showed no significant differences among all the groups （F=0.67, P=0.69）. After irradiation with 15 Gy, tumor volumes in ECA109/BR group were smaller than those in other groups （P=-0.03）; the growth inhibition rate increased, but the relative growth rate decreased significantly （P=-0.01）. The q value which reflected the radiosensitizing effect in ECA109/BR group was 1.45. The expressions of CHK1 and CHK2 at protein level in ECA109/13R group were not influenced （P=-0.71）. However, the level of phosphorylated CHK2-T68 expression decreased significantly after irradiation with 15 Gy （P=-0.03）. Cell cycle distribution and apoptosis were not significantly different among all the groups （P=-0.45）. Conclusion： Silencing 53BP1 gene expression could inhibit the growth of esophageal cancer cell xenograft and increase the radiosensitivity to tumors in the nude mice.