反义微小RNA-21基因治疗改善膀胱癌化疗敏感性的实验研究及临床应用前景

The empirical study of antisense miRNA-21 gene therapy to imrove chemical sensitivity of bladder cancer and the outlook of clinical application

目的建立裸鼠原位膀胱癌移植瘤模型,探讨反义微小RNA-21(miRNA-21)腺病毒载体+化疗药物丝裂霉素C(mitomycinC.MMC)对裸鼠膀胱癌发生、发展及转移的作用,为评价基因治疗改善膀胱癌化疗耐药性的策略提供实验依据。方法分别用携带反义miRNA-21载体+MMC和单独使用携带反义miRNA-21载体或MMC经尿道膀胱灌注治疗成瘤裸鼠,观察肿瘤生长及盆腔淋巴结转移情况,同时应用二苯基溴化四氮唑蓝(MTT)法检测瘤细胞的增殖,原位末端标记(TUNEL)法测定瘤细胞的凋亡。结果 MTT法检测结果显示,联合用药组,反义miRNA-21组和MMC组细胞的增殖率分别是25.6%±6.9%,50.4%±8.3%,37.9%±2.8%,组间比较,差异具有统计学意义(P<0.05)。TUNEL法测定结果提示:瘤细胞凋亡率依次为:联合用药组(81.5%±6.1%),反义miRNA-21组(49.5%±7.3%),MMC组(56.8%±3.9%),组间比较P<0.05。联合用药组10只成瘤裸鼠中无一只出现盆腔淋巴结转移,转移率为零,而单独应用载体或化疗组各发生4只转移,转移率为40%(4/10),组间差异具有统计学意义(P<0.01)。结论基因治疗+化疗联合治疗人膀胱癌裸鼠原位移植瘤的策略明显优于只用基因或化疗的单一治疗方法。

Objective To set up nude rice model of transplanted blader cancer in situ and discuss the function of an- tisense miRNA-21 adenovirus vector combined with mitomycin C on genesis, development and metastasis of bladder cancer of nude rice. To evaluate whether gene therapy can improve chemical tolerance of bladder cancer or not. Methods The nude rice with bladder cancer was treated with irrigation of bladder. They were divided into 3 groups：antisense miRNA-21 ＇adenovirus vector, mitomycin C and antisense miRNA-21 adenovirus vector with mitomycin C. Tumor growth and transplantion of pelvic lymph nodes were observed. Proliferation of tumor cells was examined by MTT and apoptosis of tumor cells was examined by TUNEL. Results The proliferation rate in combined group,antisense group and mitomycin C group was 25.6% ± 6, 9 %, 50.4 % ± 8.3 % and 37.9 %±2.8 %. The difference of groups was P〈0.05. The apoptosis rate was 81.5% ±6.1%,49.5%±7.3% and 56.8%±3.9%. The difference of groups was P〈0.05. There was no metastasis of pelvic lymph nodes in combined group. But the metastasis rate was 40~ in other groups（4/10）. The difference was significant P（0.01. Conclusion Gene therapy combined chemotherapy on nude rice model of transplanted blader cancer in situ was obviously better than any other method .