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目的构建能高效干扰Trop2基因的si RNA,转染人喉癌HEP2细胞,并初步鉴定其干扰效果。方法以人Trop2基因为靶基因,设计合成3条针对不同作用位点的si RNA干扰序列,用脂质体法将各小干扰序列瞬时转染人喉癌HEP2细胞,同时设立阴性对照、空白对照,分别命名为W组(未转染组)、NC组(阴性对照组)、T1组(S1序列组)、T2组(S2序列组)及T3组(S3序列组),每组各5例。荧光显微镜下观察绿色荧光蛋白表达,计算转染效率。转染24 h后,收集稳定后细胞,采用实时荧光定量PCR法检测各干扰序列对Trop2基因表达的抑制效果。结果荧光显微镜下观察,见HEP2细胞表达绿色荧光蛋白,证实si RNA已转入细胞,转染率达90%。RT-PCR法结果显示,与未转染组、阴性对照组相比,转染si RNA的人喉癌HEP2细胞中Trop2表达均受到抑制,与T2、T3组相比,T1组对Trop2 m RNA的抑制作用最明显,差异有统计学意义(0.470±0.063 vs 0.749±0.024,0.824±0.027,P<0.05)。结论成功构建并筛选出了能高效、特异沉默人喉癌HEP2细胞Trop2基因的si RNA序列,为进一步研究Trop2基因在喉癌中的作用机制及其基因治疗奠定了基础。
Objective To construct si RNA that efficiently interferes with Trop2 gene and transfected it into human laryngeal carcinoma HEP2 cells. Methods Trop2 gene was used as target gene to design and synthesize three si RNA interference sequences targeting different sites of action. The small interference sequences were transiently transfected into human laryngeal carcinoma HEP2 cells by liposome method. At the same time, a negative control and blank control , Named as W group (untransfected group), NC group (negative control group), T1 group (S1 sequence group), T2 group (S2 sequence group) and T3 group (S3 sequence group) . Fluorescent microscope to observe the expression of green fluorescent protein, calculate the transfection efficiency. After 24 h of transfection, the cells were stained and the inhibitory effect of each interfering sequence on Trop2 gene expression was detected by real-time fluorescence quantitative PCR. Results Fluorescence microscopy showed that HEP2 cells expressed green fluorescent protein and confirmed that si RNA had been transferred into cells with a transfection rate of 90%. The results of RT-PCR showed that Trop2 expression was inhibited in HEP2 cells transfected with si RNA compared with untransfected and negative control groups. Compared with T2 and T3 groups, Trop2 mRNA (0.470 ± 0.063 vs 0.749 ± 0.024, 0.824 ± 0.027, P <0.05). Conclusion The siRNA sequence of Trop2 gene, which can efficiently and specifically silence human laryngeal carcinoma HEP2 cells, was successfully constructed and screened, which laid the foundation for further study on the mechanism and gene therapy of Trop2 gene in laryngeal carcinoma.