RNA干扰A549细胞核干细胞因子基因表达

目的探讨A549细胞株NS基因的表达干扰后，A549细胞株的mRNA表达水平及细胞增殖变化。方法构建NSshRNA真核表达载体，转染A549细胞，G418筛选稳定表达细胞克隆，RT-PCR检测NS基因沉默，MTT检测细胞增殖情况。结果构建的NSshRNA真核表达载体经菌落PCR和测序证实重组表达载体中插入序列与设计一致。RT-PCR检测显示构建的shRNA表达载体转染A549细胞后可显著降低NSmRNA的表达；M1vr结果显示干扰后细胞增殖速率明显减低。结论成功构建了一组针对NS基因的shRNA真核表达载体，转染A549细胞后NS基因表达被特异性沉默，且细胞增殖受到明显抑制，通过RNAi技术实现NS基因沉默可能是一种较有前景的肿瘤治疗方法。

Knocking-down Nucleostemin Gene Expression by RNA interference Technique on the A549 Cells

Objective To investigate the expression level of nucleostemi （NS） and cell proliferation in A549 cell after interfering with RNA interference technique. Methods The shRNA sequences targeting NS gene were inserted into plasmid pSilencer4.1-CMV neo for constructing the recombinant plasmids. After identification by PCR and DNA sequencing, the recombinant plasmids were transfected into A549 cell lines with liposome. The mRNA expressions of NS gene in the cells were determined by RT-PCR and cell proliferation by MTT assay. Results The correct insertion of the designed sequences of recombinant plasmids were identified by PCR and sequence analysis. RT-PCR showed that mRNA expressions of NS gene substantially decreased in the transfected cells. NS-silencer A549 cell lines were slower in the rate of cell proliferation. Conclusion The recombinant plasmid expressing the shRNA targeting NS gene has been successful- ly constructed. NS gene expression was specially silenced and cell proliferation was slower.