敲除NOR1基因对人肝癌裸鼠移植瘤的影响及作用机制  

Effect of NOR1 gene knockout on nude mice xenograft tumor of human liver cancer and its mechanism of action

作　　者：游焜[1] 王大军[1] 王亮[1] 王建国[1] YOU Kun;WANG Dajun;WANG Liang(Department of Hepatobiliary Surgery, The First Affiliated Hospital of Xinxiang Medical College, Xinxiang, Henan 453100, China)

机构地区：[1]新乡医学院第一附属医院肝胆外科

出　　处：《临床肝胆病杂志》2020年第2期381-386,共6页Journal of Clinical Hepatology

基　　金：河南省教育厅(20007320017)

摘　　要：目的 探究敲除NOR1基因后对人肝癌裸鼠移植瘤生长、存活率及器官损伤的影响。 方法 SPF级雄性BALB/C裸鼠30 只随机分为Control组(n=10)、Scramble组(n=10)、siNOR1组(n=10),构建阴性对照质粒 (Scramble)和 siNOR1质粒转染至HepG2细胞并接种至裸鼠构建移植瘤裸鼠模型。连续30 d每5 d检测裸鼠移植瘤组织体积;检测移植瘤重量;免疫组化检测Ki-67、Caspase-3、Notch、NOR1表达情况;Western Blot检测Survivin、Notch1、NICD、Hes1、Hey1蛋白表达量情况,HE染色观察肝、肾、肺、脑病理损伤程度。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验;生存分析采用Kaplan-Meier法,生存率的比较采用log-rank检验法。结果 从建模第20天开始,与Control组相比,siNOR1组第20、25、30 天移植瘤体积显著减小[(418.71±78.24)mm3 vs (149.6.16±60.05)mm3、(864.22±125.66)mm3 vs (239.83±100.51)mm3、(1468.45±199.78) mm3 vs (446.54±147.09) mm3,P值均<0.05];第30天时取出移植瘤,与Control组相比,siNOR1组移植瘤质量显著减小[(0.45±0.07)g vs (0.12±0.04)g,P<0.05];与Control组相比,siNOR1组移植瘤组织中Ki-67、Notch、NOR1阳性表达率显著降低[Ki-67:(48.98±9.75)% vs (11.51±5.09)%, Notch: (62.51±9.26)% vs (18.75±4.61)%,NOR1:(76.33±8.31)% vs (16.57±3.76)%, P值均<0.05],Caspase-3阳性表达率显著升高[(6.39±4.67)% vs (38.03±9.28)%, P<0.05];siNOR1组模型裸鼠30 d内生存率显著高于Control组[(77.66±6.75)% vs (25.32±4.63)%,χ^2=6.897,P<0.05];Western Blot检测结果显示,与Control组相比,siNOR1组Survivin、Notch1、NICD、Hes1、Hey1蛋白表达水平均显著降低(Survivin: 0.34±0.06 vs 0.02±0.01;Notch1:0.16±0.03 vs 0.03±0.01;NICD:0.26±0.05 vs 0.04±0.02;Hes1:0.35±0.04 vs 0.06±0.02;Hey: 0.29±0.06 vs 0.05±0.02,P值均<0.05),且siNOR1组裸鼠各组织器官损伤情况均得到有效缓解。结论 敲除NOR1基因抑制人肝癌裸鼠移植瘤的生长,提高模型裸鼠生存率,并缓解模型裸Objective To investigate the effect of NOR1 gene knockout on the growth of nude mice xenograft tumor of human liver cancer, the survival rate of nude mice, and organ damage in nude mice. Methods A total of 30 specific pathogen-free male BALB/C nude mice were randomly divided into control group, Scramble group, and siNOR1 group, with 10 mice in each group. The negative control plasmid (Scramble) and siNOR1 plasmid were transfected into HepG2 cells, and then the HepG2 cells were inoculated into nude mice to establish a nude mouse model of xenograft tumor. The volume and weight of nude mice xenograft tumor were measured every 5 days for 30 consecutive days. Immunohistochemistry was used to measure the expression of Ki-67, caspase-3, Notch, and NOR1, and Western Blot was used to measure the protein expression of Survivin, Notch1, NICD, Hes1, and Hey1. HE staining was used to observe the degree of pathological injury in the liver, the kidney, the lungs, and the brain. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Kaplan-Meier method was used for survival analysis, and the log-rank test was used for comparison of survival rates. Results Compared with the control group since day 20 of modeling, the siNOR1 group had a significantly lower volume of xenograft tumor on days 20, 25, and 30 (day 20: 149.6.16±60.05 mm3 vs 418.71±78.24 mm3, P<0.05;day 25: 239.83±100.51 mm3 vs 864.22±125.66 mm3, P<0.05;day 30: 446.54±147.09 mm3 vs 1468.45±199.78 mm3, all P<0.05). The xenograft tumor was collected on day 30, and compared with the control group, the siNOR1 group had a significantly lower weight of xenograft tumor (0.12±0.04 g vs 0.45±0.07 g, P<0.05). Compared with the control group, the siNOR1 group had significantly lower expression of Ki-67, Notch, and NOR1 in xenograft tumor tissue (Ki-67: 11.51%±5.09% vs 48.98%±9.75%, P<0.05;Notch: 18.75%±4.61% vs 62.51%±9.26%, P<0.05;NOR1: 16.57%±3

关 键 词：肝肿瘤 小鼠 近交BALB C NOR1基因 

分 类 号：R735.7[医药卫生—肿瘤]