RNA干扰对人神经母细胞瘤VEGF—A表达的抑制作用

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

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、psc（11）2，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-SY5Y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SHSY5Y细胞后能显著抑制VEGFA的表达，但没有引起显著的细胞凋亡（P〉0．05）。

Objective To construct the short hairpin RNA（shRNA） vector of VEGF-A and down-regulate the expression of VEGFA through RNA interference in neuroblastoma cell line SH-SY5Y. Methods Two pairs of oligonucleotide sequences were designed and synthesized with human VEGFA 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 ceils 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 fragments were correctly inserted 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 VEGFA expression in SH-SY5Y ceils without leading to apoptosis in the transfected cells.