靶向CrkL基因的siRNA真核表达载体的构建及对H9C2心肌细胞凋亡的作用

摘要

To provide an experimental basis for studying CrkL gene action mechanism in Cardiomyocyte.First, six antisense oligodeoxynucleotide sequences targeting rat's CrkL mRNA were designed and synthesized in vitro, and then directionally cloned into pSR-GFP/neo siRNA expressing plasmid respectively.The recombinant vectors pSR-GFP/neo-CrkLi were constructed and identified successfully by double restriction endonuclease digestion analysis and DNA sequencing.The pSR-GFP/neo-CrkLi were transfected into H9C2 cell line of rat cardiomyocyte, and the transfection efficiency was evaluated with fluorescence microscope.The inhibitive effects of the recombinants on CrkL mRNA were observed by RT-PCR.And the effects of CrkL gene on appoptosis of H9C2 cells was detected by flow cytometry.Six pSR-GFP/neo-CrkLi were transfected into H9C2 cell efficiently.The expression of CrkL mRNA in H9C2 cells transfected with pSRGFP/neo-CrkLi was significantly inhibited in three vectors.Flow cytometry showed that the apoptosis rate of group transfected with pSR-GFP/neo-CrkLi-3 was significantly higher than the other groups(P=0.001 ).%为研究CrkL基因在心肌细胞中的作用机制提供实验基础,首先体外合成6个针对大鼠CrkL基因的特异反义脱氧寡核苷酸序列,定向克隆至pSR-GFP/neo siRNA真核表达质粒中,经双酶切和测序证实重组载体pSR-GFP/neo-CrkLi构建成功.然后将其转染入大鼠H9C2心肌细胞内,用荧光显微镜观察其转染率,RT-PCR法鉴定其对CrkL mRNA表达的抑制作用.流式细胞仪检测转染前后H9C2细胞凋亡率.所有pSR-GFP/neo-CrkLi载体均能高效率的转染H9C2细胞,但只有3个载体能显著抑制CrkL mRNA的表达.其中pSR-GFP/neo-CrkLi3效果最佳,其转染H9C2细胞后,该组H9C2细胞凋亡率明显高于其他两组(P=0.001).