RNA干扰对人神经母细胞瘤VEGF-A表达的抑制作用

目的 构建VEGF-A的短发夹RNA真核表达载体,并通过其介导的RNA干扰抑制人神经母细胞瘤细胞株SH-SY5Y VEGF-A的表达.方法 根据VEGF-A的cDNA序列合成两对寡核苷酸片段,退火后分别插入真核表达载体组成重组质粒psc001、psc002,对重组质粒进行PCR、测序鉴定验证并经Western Blot筛选,然后将筛选所得的质粒转染人神经母细胞瘤细胞株SH-SY5Y.转染48h后行Annexin V和PI双重标记流式细胞仪检测转染细胞凋亡情况,ELISA检测VEGF-A蛋白的表达水平.结果 PCR和测序证实寡核苷酸片段已成功插入质粒psc001、psc002中,流式细胞仪检测结果 显示转染的外源基因未引起显著的神经母细胞瘤细胞凋亡,ELISA结果 显示转染后细胞株SH-SYSY的VEGF-A蛋白表达受到显著抑制(P<0.05).结论 成功构建人VEGF-A的短发卡RNA真核表达载体,转染人神经母细胞瘤SH-SY5Y细胞后能显著抑制VEGF-A的表达,但没有引起显著的细胞凋亡(P0.05).

Knockdown of VEGF-A expression in neuroblastoma cell line SH-SY5Y by RNA interference

Objective To construct the short hairpin RNA(shRNA) vector of VEGF-A and down-regulate the expression of VEGF-A through RNA interference in neuroblastoma cell line SH-SY5Y. Methods Two pairs of oligonucleotide sequences were designed and synthesized with human VEGF-A cDNA. The annealed oligonucleotide fragments were subcloned into plasmids psc001 psc002. After being identified by PCR, sequencing and selection by Southern Blot, the recombinant plasmids were transfected into SH-SY5Y cells. Doubly tagged by Annexin V and PI, the transfected cells were sorted by FCMS for apoptosis after 48 hours. VEGF-A expression in the transfected cells was detected by ELISA. Results PCR and sequencing showed that the oligonueleotide fragrnents were correctly in-serted into plasmids psc001, psc002 respectively. VEGF -A expression in the transfected cells was knocked down significantly. Conclusions The shRNA expression vector of VEGF-A was successfully constructed and could significantly down-regulate VEGF-A expression in SH-SY5Y cells without lead-ing to apoptosis in the transfected cells.