Survivin反义寡核苷酸治疗荷瘤小鼠的实验研究

Antisense Oligonucleotide Targeting Survivin Treating Tumor-bearing Nude Mice

目的评价生存素(Survivin)反义寡核苷酸(AS ODN)在荷肝癌小鼠中的治疗作用。方法BALB/nu/nu裸鼠皮下注射人肝癌细胞株hepG2,建立原位移植荷人肝癌模型。将荷肝癌鼠随机分为3组:AS ODN治疗组、空白对照组和脂质体对照组,分别腹腔注射Survivin AS ODN 8μg/kg、等容积的生理盐水和空白脂质体,每日1次,连续作用7d,第8天处死荷肝癌鼠,留取瘤块标本,测定原位肿瘤大小,肿瘤生长指数;瘤组织经苏木精-伊红染色观察组织病理学形态;Western blot法检测Survivin蛋白表达;流式细胞术检测原位肝癌细胞凋亡指数和增殖指数。结果①成功地建立了原位荷肝癌的动物模型。移植瘤大体形态呈现为不规则结节状,与正常肝组织间分界较清楚。②Surrivin AS ODN治疗组肿瘤生长指数为(50.96±24.20)%,显著低于空白对照组(170.22±23.37)%和脂质体对照组(168.02±28.58)%,差异均有显著性意义(均P<0.05)。③苏木精-伊红染色显示3组瘤块标本均有移植肝癌的典型组织学表现,与对照组比较,AS ODN治疗组的原位肿瘤组织中可见到明显的细胞死亡和细胞碎片。④Western blot结果显示ASODN治疗组肝癌组织中Survivin蛋白的表达明显低于空白对照组和脂质体对照组。⑤流式细胞术结果显示AS ODN治疗组肿瘤细胞的凋亡指数为(21.97±2.11)%,显著高于空白对照组(3.21±0.57)%和脂质体对照组(3.07±0.81)%(P<0.05);而其增殖指数为(11.65±1.25)%,显著低于空白对照组(18.72±0.76)%和脂质体对照组(18.32±1.38)%(P<0.05)。结论原位肝癌移植模型能高度模拟人肝癌的形态学特点和生物学特性。Survivin AS ODN腹腔注射治疗能有效下调肿瘤组织Survivin蛋白表达,诱导肿瘤细胞凋亡,抑制细胞增殖,而对正常肝组织和其他器官组织无明显毒副作用。

Objective To evaluate the therapeutic effect of antisense oligonueleotide （AS ODN） targeting Survivin in the treatment of tumor-bearing nude mice. Methods Orthotopie implant model of human hepatocellular carcinoma was developed in BALB/nu/nu nude mouse with hepG2 cell line. Tumor-bearing nude mice were divided into 3 groups： AS ODN group, control group and lipofectin group. After treatment for 7 days, the intact tumor tissues were collected and some objects were examined. The size of tumor in situ was examined and growth index （GI） was calculated. Pathological histology of tumor was observed with HE staining. Western blot was used to detect the protein expression of Survivin. Apoptotic index （AI） and proliferative index （PI） of orthotopie tumor tissues were measured by flow cytometry. Results Orthotopie implant model of human hepatocellular carcinoma was developed in 23 of 25 nude mice. The GI in AS ODN group was （50.96±24.20）%, lower than in other groups （P〈0.05）. Typical histological changes of orthotopic tumors were observed in 3 groups, and more obvious cell death was seen in AS ODN group than in control and lipofeetin groups. No abnormal changes were observed in adjacent normal liver tissues of AS ODN group. Western blot revealed that the expression of Survivin in AS ODN group was decreased as compared with that in control and lipofectin groups. The AI in AS ODN group was （21.97±2.11）%, higher than that in control and lipofeetin groups （P〈0, 05）. The PI in AS ODN group was （11. 65±1.25） %, lower than that in control and lipofectin groups （P〈0.05）. Conclusion The orthotopic implant model of human hepatoeellular carcinoma might be a useful tool in the researches on antieaneer treatment of human hepatocellular carcinoma. The Survivin AS ODN could reduce the expression of Survivin, induce cell apoptosis and inhibit cell proliferation.