腺病毒介导的抗MDR1核酶基因转移联合化疗药物治疗恶性淋巴瘤的研究

Adenovirus-mediated transfer of anti-MDR1 ribozyme in the treatment of multidrug-resistant human lymphoma in SCID mice

目的 探讨腺病毒介导的抗MDR1核酶基因转移在体内外逆转肿瘤细胞中P 糖蛋白(P gp)介导的多重耐药性 (MDR)的作用。方法 构建并纯化制备了含有抗MDR1核酶基因的腺病毒Ad 196MDR1 Rz。在体外 ,用该重组腺病毒转导具有P gp介导的、MDR特性的人淋巴瘤细胞株Daudi MDR2 0 ,并分别用RT PCR、FACS分析和MTT法测定核酶基因转导对Daudi MDR2 0细胞MDR1mRNA和膜表面P gp表达的影响及对长春新碱 (VCR)敏感性的改变 ;在体内 ,通过局部注入重组腺病毒联合VCR全身化疗 ,对接种Daudi MDR2 0细胞的SCID小鼠进行治疗观察。结果 在感染Ad 196MDR1 Rz后 ,Daudi MDR2 0细胞MDR1mRNA的表达被阻断 ,细胞膜表面P gp表达水平显著降低 ,细胞对VCR的敏感性增加。在接种Daudi MDR2 0细胞后 ,采用Ad 196MDR1 Rz联合VCR化疗的治疗组小鼠有6 6 .7% (6 9)未发生肿瘤 ,存活时间超过 12 0d ;而采用空载腺病毒联合VCR化疗或单纯VCR化疗的对照组小鼠 ,其长期存活率分别为 12 .5 % (1 8)和 0 (0 9) ,两组差异有极显著性。结论 Ad 196MDR1 Rz能有效阻断Daudi MDR2 0细胞膜表面P gp表达 ,并恢复肿瘤细胞对VCR的敏感性。体内联合VCR化疗能有效抑制肿瘤的发生。

Objective To evaluate the effect of adenovirus-mediated transfer of anti-MDR1 ribozyme on restoring drug sensitivity of multidrug-resistant human lymphoma both in vitro and in SCID mice. Methods A recombinant adenovirus expressing ribozyme against codon 196 of MDR1 mRNA (Ad-196MDR1-Rz) was developed through cotransfection of shuttle vector pCA14 containing 196MDR1-Rz and rescue vector pJM17 into human embryonic kidney cell line 293. In vitro Daudi/MDR20, a MDR1-mediated drug-resistant human lymphoma cell line, was transduced by Ad-196MDR1-Rz at MOI of 400 pfu/cell. RT-PCR and FACS analyses were used to evaluate the MDR1 expression in both transcriptional and translational levels. MTT assay was used for analysis of drug resistance. In vivo, SCID mice were inoculated subcutanuously by 5×106 Daudi/MDR20 or parental Daudi/wt cells. Adnovirus was injected locally. Vincristine (VCR) was given intraperitoneally. Results In vitro transduction of Ad-196MDR1-Rz to Daudi/MDR20 cells was able to interrupt MDR1 transcription, inhibit P-gp expression and restore drug sensitivity to VCR. Of SCID mice bearing Daudi/MDR20 cells, tumor free rate and long term survival were 66.7% (6/9) and >120 days in the therapeutic group of Ad-196MDR1+ VCR vs 12.5% (7/8) and none survived >120 days in the control groups of Ad-Mock + VCR or VCR alone. The difference was very statistically significant. Conclusion Ad-mediated transfer of 196MDR1-Rz can revert drug resistance of MDR tumor cells both in vitro and in vivo. Ad-196MDR1-Rz may be helpful as an adjuvant in the chemotherapy of P-gp mediated MDR human tumor.