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通过RNA干扰(RNAi)技术靶向耐药性人肺腺癌细胞内的MDR1基因实现多药耐药性的逆转作用。构建聚乙二醇-聚谷氨酸-聚乙烯亚胺(PEG-b-PLG-g-PEIs,GGI)阳离子聚合物载体,通过~1H NMR表征确认结构,动态光散射法测定GGI/si RNA的粒径及电位,并以A549敏感株和A549/DDP耐药株细胞系为模型,采用MTT法考察GGI的细胞毒性,以流式细胞术评价新型聚合物载体GGI递送si RNA的效率和强度,RT-PCR法考察GGI转染A549和A549/DDP(cisplatin)后MDR1 m RNA水平的表达;Western blot法测定A549/DDP细胞中P-gp的表达。同时,以MTT法和Annexin V-FITC/PI双染法考察si RNA干扰后抗肿瘤药物对药物敏感性的变化。结果显示,GGI/si RNA复合物粒径为150~200 nm,电位稳定在16~28 m V。GGI细胞毒性远低于PEI 25K,且具有更高运载si RNA至细胞内的效率,并能极大降低A549/DDP细胞内MDR1 m RNA和P-gp的表达,能更大程度地增强耐药细胞株对顺铂的敏感性,说明GGI有望在基因传递系统中成为一种新型的聚合物载体并广泛应用。
Targeting the MDR1 gene in drug-resistant human lung adenocarcinoma cells by RNA interference (RNAi) technology reverses the reversal of multidrug resistance. The PEG-b-PLG-g-PEIs (GGI) cationic polymer carrier was constructed and confirmed by ~ 1H NMR characterization. The dynamic light scattering method was used to determine the GGI / si RNA And the cell line A549 and A549 / DDP resistant cell lines as a model, MTT assay cytotoxicity of GGI, flow cytometry to evaluate the efficiency and strength of the novel polymer carrier GGI si RNA delivery The expression of MDR1 m RNA in GGI transfected A549 and A549 / DDP (cisplatin) cells was detected by RT-PCR. The expression of P-gp in A549 / DDP cells was detected by Western blot. At the same time, MTT assay and Annexin V-FITC / PI double staining were used to investigate the changes of drug sensitivity of anti-tumor drugs after si RNA interference. The results showed that the particle size of GGI / si RNA complex was 150-200 nm and the potential was stable at 16-28 mV. The cytotoxicity of GGI is much lower than that of PEI 25K, and it has higher efficiency of carrying si RNA into cells and can greatly reduce the expression of MDR1 m RNA and P-gp in A549 / DDP cells and enhance the drug resistance to a greater extent The sensitivity of cell lines to cisplatin shows that GGI is expected to become a new type of polymer carrier in gene delivery system and widely used.