重组腺相关病毒-胸苷激酶-核糖体插入位点-内皮抑素双靶点基因治疗裸鼠膀胱癌的实验研究

目的 探讨重组腺相关病毒(rAAV)-胸苷激酶(TK)-核糖体插入位点(IRES)-内皮抑素(ES)(简称rAAV-TIE)双靶点基因治疗膀胱癌的动物实验效果.方法 膀胱癌T24细胞悬液接种于27只Balb/c裸鼠右侧肩胛区皮下,观察成瘤情况;取已成瘤裸鼠25只,随机分为对照组、rAAV-多克隆位点(MCS)组、rAAV-TK组、rAAV-ES组、rAAV-TIE组,每组5只,瘤内分别注射生理盐水、rAAV-MCS、rAAV-TK、rAAV-ES、rAAV-TIE,4周后颈椎脱臼处死裸鼠,取出移植瘤,HE染色、计算微血管密度(MVD);采集裸鼠血液标本,双夹心酶联免疫吸附试验榆测血清中ES浓度.结果①T24细胞接种3周后,接种部位肿瘤形成25只,成瘤率93%;②瘤内注射rAAV-ES、rAAV-TK、rAAV-TIE 9 d后,肿瘤生长明显抑制.治疗4周后rAAV-ES、rAAV-TK、rAAV-TIE、rAAV-MCS和对照组肿瘤体积分别为(0.75±0.08)、(0.71±0.11)、(0.52±0.09)、(1.27±0.13)、(1.24±0.17)cm3;rAAV-ES组与rAAV-TK组间比较差异无统计学意义,其余组间比较差异均有统计学意义(P值均<0.05);③5组裸鼠移植瘤MVD分别为(18.72±2.53)、(21.74±4.62)、(12.73±1.78)、(52.38±6.46)和(49.94±7.17)个/HP,治疗组显著低于空白对照组和空病毒组(P<0.05).结论霞组rAAV-TIE腺相关病毒被成功构建,体外实验表明rAAV-TIE能抑制膀胱癌的血管生成和肿瘤生长,可有效双靶点基因治疗膀胱肿瘤.

Study of rAAV-TK-IRES-ES gene therapy of bladder cancer in vivo

Objective To study the therapeutically effect of rAAV-TIE model in vivo. Meth-ods Cell suspensions of T24 cells were injected into the subcutaneously of right scapular region of nude mice. The nude mice were raised under SPF condition and the xenograft tumor growth was ob-served. Bearing tumor nude mice were randomly divided into 5 groups: rAAV-MCS group, rAAV-Tk group, rAAV-ES group, rAAV-TIE group and control group. Four weeks later of treatment, the nude mice were sacrificed. The xenografts tumors were fixed for HE staining. The liver tissue and ne-phridial tissue were also fixed for HE stain. The blood sample and endostatin concentration was as-sayed by ELISA. Results After 3 weeks of injected with T24 cells on nude mice, 25 showed visible tumor on the injected location. The rate of tumor formation was 93%. After 9 days injected by rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volume were: rAAV-ES group(0.75±0.08)cm3 , rAAV-TK group(0.71±0.11)cm3 , rAAV-TIE group(0.52±0.09)cm3 , rAAV-MCS group(1.27±0.13)cm3 and control group (1.24±0.17)cm3. The microvessel density in the different groups were as follow-ings: rAAV-ES group(18.72±2.53)/HP, rAAV-TK group(21.74±4.62)/HP, rAAV-TIE group (12.73±1.78)/HP, rAAV-MCS group(52.38±6.46)/HP and control group(49.94±7.17)/HP. The endostatin concentration in the diffcrent groups were as followings: rAAV ES group(38.52 6.53)μg/L and rAAV-TIE group(40.33±7.48)μg/L. HE staining confirmed the tumor. The liver tissue and kidncy tissue of each group had no obviously cell degeneration or necrosis. Conclusion The rAAV-TIE could inhibit tumor induced angiogenesis and suppress both the initiation and the subse-quent growth of human bladder cancer in nude mice model.