Walther Wolfgang, Stein Ulrike, Lage Hermann
Max-Delbrück-Center for Molecular Medicine and Experimental Clinical Research Center, Charité Berlin, Berlin, Germany.
Methods Mol Biol. 2010;629:123-39. doi: 10.1007/978-1-60761-657-3_9.
The use of the RNA interference (RNAi) through the expression of small hairpin RNA (shRNA) is a promising approach for efficient gene silencing for therapeutic applications. In this chapter, we describe the in vivo reversal of the classical MDR1/P-glycoprotein (MDR1/P-gp)-mediated multidrug resistance (MDR) phenotype by shRNA. For local intratumoral delivery of naked shRNA-encoding vector constructs, the nonviral jet-injection was used. This jet-injector system uses compressed air to inject small volumes (5-10 muL) of naked nucleic acid solutions into tumor tissues. Furthermore, the design of the jet-injector allows multiple injections. Under our experimental design, the delivery of plasmid DNA encoding anti-MDR shRNA by jet-injection into human MDR1/P-gp overexpressing MaTu/ADR breast cancer xenografts resulted in a decrease of MDR1 mRNA expression level to more than 90%. Accordingly, the corresponding MDR1/P-gp protein is no longer detectable in the tumors after anti-MDR1 shRNA vector injection. Furthermore, combination of two intratumoral jet-injections of anti-MDR1 shRNA vectors with two intravenous administrations of doxorubicin is sufficient for a complete reversal of the MDR phenotype in association with tumor growth inhibition.
通过表达小发夹RNA（shRNA）来利用RNA干扰（RNAi）是一种很有前景的方法，可用于治疗应用中的高效基因沉默。在本章中，我们描述了通过shRNA在体内逆转经典的多药耐药蛋白1/ P-糖蛋白（MDR1 / P-gp）介导的多药耐药（MDR）表型。对于裸shRNA编码载体构建体的局部肿瘤内递送，使用了非病毒喷射注射法。该喷射注射系统利用压缩空气将小体积（5-10微升）的裸核酸溶液注射到肿瘤组织中。此外，喷射注射器的设计允许进行多次注射。在我们的实验设计中，通过喷射注射将编码抗MDR shRNA的质粒DNA递送至过表达人MDR1 / P-gp的MaTu / ADR乳腺癌异种移植瘤中，导致MDR1 mRNA表达水平降低至90％以上。因此，在注射抗MDR1 shRNA载体后，肿瘤中不再能检测到相应的MDR1 / P-gp蛋白。此外，两次肿瘤内喷射注射抗MDR1 shRNA载体与两次静脉注射阿霉素相结合，足以完全逆转MDR表型并抑制肿瘤生长。
