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目的:分析ERCC1对H520细胞中Mdr1及SP细胞比例的影响。方法:利用RNA干扰技术构建ERCC1-shRNA重组质粒表达载体,转染并筛选出阳性稳定转染的H520细胞;采用RT-PCR和WesternBlot的方法检测ERCC1对H520细胞多药耐药1(Mdr1)的影响;采用流式细胞仪检测ERCC1对H520细胞SP细胞比例的影响。结果:RT-PCR检测出Mdr1 mRNA在含有ERCC1-shRNA的H520细胞和含有空载体的细胞中和内参的A比值分别为0.423±0.147和0.468±0.02(n=5),两者有显著性差异;Western-Blot检测出Mdr1蛋白浓度在含有ERCC1-shRNA的H520细胞较含有空载体的细胞中明显降低。含有ERCC1-shRNA的H520细胞SP细胞比例达到0.8%,而含有空载体的H520细胞的SP细胞比例则达到2.2%。结论:RNA干扰技术可以构建重组质粒,稳定转染的H520细胞能被筛选出,当ERCC1基因被抑制时,Mdr1基因也会收到抑制,同时使H520细胞SP细胞比例明显降低。
Objective: To analyze the effect of ERCC1 on the proportion of Mdr1 and SP cells in H520 cells. METHODS: ERCC1-shRNA recombinant plasmids were constructed by RNA interference technique and transfected into H520 cells. The expression of ERCC1 was detected by RT-PCR and Western Blot. The effect of ERCC1 on multidrug resistance 1 (Mdr1) The effect of ERCC1 on the proportion of SP cells in H520 cells was detected by flow cytometry. Results: RT-PCR results showed that the A ratio of Mdr1 mRNA in ERCC1-shRNA-containing H520 cells and empty vector-containing cells was 0.423 ± 0.147 and 0.468 ± 0.02 (n = 5), both of which were significantly different ; Western-Blot detected Mdr1 protein concentration in cells containing ERCC1-shRNA H520 cells containing empty vector was significantly lower. The proportion of SP cells in H520 cells containing ERCC1-shRNA reached 0.8%, while the percentage of SP cells in empty vector-containing H520 cells reached 2.2%. CONCLUSION: Recombinant plasmids can be constructed by RNA interference. Stably transfected H520 cells can be screened out. When ERCC1 gene is inhibited, Mdr1 gene is also inhibited, and the proportion of SP cells in H520 cells is significantly reduced.