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目的 研究RNA干扰(RNAinterference)效应对人鼻咽癌低分化上皮细胞株CNE- 2Zbcl- xL基因表达的抑制作用及其对CNE- 2Z细胞增殖抑制和凋亡诱导的作用。方法 使用美国Ambion公司提供的网上设计软件设计针对人bcl- xL基因的小干扰RNA(siRNA)序列,体外转录试剂盒合成siRNA;荧光素标记试剂盒标记siRNA;脂质体法将siRNA转入CNE -2Z细胞株。荧光显微镜下观察siRNA的转染效率;RT- PCR法半定量检测siRNA对bcl- xL基因表达的抑制作用;噻唑蓝法检测siRNA对细胞生长增殖抑制作用;流式细胞术检测siRNA诱导细胞凋亡作用。结果 荧光显微镜下荧光素标记的siRNA转染组可见到细胞内清晰的绿色荧光,而在未转染siRNA对照组未见;各siRNA转染组bcl- xLmRNA表达水平有不同程度的下调,下调范围在10% ~70%之间,而在未转染对照组内bcl xLmRNA表达水平无明显改变;细胞生长增殖抑制率在一定范围内具有剂量依赖性(剂量增加,抑制率增高)和时间依赖性;各浓度siRNA4转染组可不同程度诱导CNE -2Z细胞凋亡。结论体外转录合成的siRNA能特异有效地下调bcl- xL基因的表达,不同序列特异性的siRNA下调bcl -xL基因表达的能力不同;瞬时转染bcl- xLsiRNA4能有效抑制鼻咽癌细胞增殖并诱导其凋亡;siRNA不仅为基因组功能分析提供了强有力的工具,而?
Objective To study the effect of RNA interference on CNE-2Zbcl-xL gene expression in human nasopharyngeal carcinoma (NSCLC) poorly differentiated epithelial cell line CNE-2Z and its effect on CNE-2Z cell proliferation and apoptosis induction. Methods The small interfering RNA (siRNA) sequence targeting the human bcl-xL gene was designed by the online design software provided by the Ambion company in the United States. In vitro transcription kit was used to synthesize siRNA. Fluorescein labeling kit was used to label the siRNA. -2Z cell line. The transfection efficiency of siRNA was observed by fluorescence microscopy. The inhibition of bcl-xL gene expression by siRNA was detected by semi-quantitative RT-PCR. The inhibition of proliferation by siRNA was detected by thiazolyl blue assay. The apoptosis of cells was detected by flow cytometry effect. Results Fluorescein-labeled siRNA transfection group under fluorescent microscope showed clear green fluorescence in cells, but not in untransfected siRNA control group. The expression levels of bcl-xL mRNA in each siRNA transfection group were down-regulated to some extent, Between 10% and 70%, while the expression level of bcl xLmRNA did not change significantly in the untransfected control group. The inhibition rate of cell growth and proliferation was dose-dependent (dose increase, inhibition rate increased) and time-dependent The apoptosis of CNE-2Z cells was induced by siRNA4 transfected cells at various concentrations. Conclusions siRNA synthesized in vitro can down-regulate the expression of bcl-xL gene effectively, and different sequence-specific siRNAs have different ability to down-regulate the expression of bcl-xL gene; transient transfection of bcl-xLsiRNA4 can effectively inhibit the proliferation and induce the proliferation of nasopharyngeal carcinoma cells Its apoptosis; siRNA not only provides a powerful tool for genomic functional analysis,