【摘要】 目的运用构建的小发夹状RNA重组质粒pshRNA/H-ras,研究其对卵巢癌SK-OV3细胞株内源H-ras基因的抑制作用。方法应用基因克隆技术,设计含21bp H-ras基因编码序列片段及中间以4~5个bp间隔的反向重复序列,克隆至转录载体pTZU6+1上并行序列分析。转染重组质粒pshRNA/H-ras至SKOV3细胞中,分别采用RT-PCR、Western blot检测细胞内H-rasmRNA和蛋白表达的变化。结果成功构建发夹状RNA载体,经DNA序列鉴定为正确的目的序列。RT-PCR和Western blot结果表明,重组质粒pshRNA/H-ras1和pshRNA/H-ras2均能在基因转录水平和蛋白表达水平上明显抑制SKOV3细胞内源H-ras蛋白的表达,抑制率达67%以上。结论重组质粒pshRNA/H-ras可显著抑制SKOV3细胞内源H-ras基因mRNA的转录和相应蛋白质的表达,为进一步研究H-ras基因在卵巢癌细胞异常增殖中的作用打下基础。更多还原

【Abstract】 Objective To investigate the inhibition of endogenetic H-ras gene expression applying the recombinant plasmid targeting H-ras gene in SKOV3 strains.Methods A 21bp reverse repeated motif of H-ras gene target sequence with 4-5bp spacer were designed and inserted into pTZU6+1(pshRNA/H-ras).The recombinant plasmids were identified by DNA sequence analysis.The pshRNS/H-ras were transfected into SKOV3 with DOTAP lipofectmine and expression of Hras were detected by Reverse Transcriptional PCR and Western blot.Results The recombinant plasmids targeting H-ras gene were constructed successfully.Expression of H-ras were all suppressed after transfected pshRNA/H-ras1 and pshRNA/H-ras2 respectively by RT-PCR and Western blot.The inhibition rate was beyond 67%.Conclusion These results indicated that the siRNA directed against H-ras could inhibit the expression of H-ras in SKOV3 cells and established a foundation to further study the unusual proliferation in cancer cells.更多还原