论文部分内容阅读
目的构建大鼠Jagged1真核表达质粒,分析Jagged1过表达对血管平滑肌细胞(vascular smooth musclecells,VSMCs)增殖和凋亡的影响。方法从带Jagged1全长基因质粒pBOS-SN3T中酶切获得目的基因,用载体pCMV-IRES-GFP构建重组质粒,转染至VSMCs。Western blot检测Jagged1蛋白表达变化,[3 H]-TdR掺入法检测细胞增殖,流式细胞仪(FACS)检测细胞凋亡。结果成功构建大鼠Jagged1真核表达载体并转染至VSMCs,经G418筛选后转染率可达90%以上,转染上调了VSMCs的Jagged1蛋白水平约6倍。转染p-rJagged1后VSMCs的增殖能力明显降低[(10 265±1 004)cpm/孔vs(17 584±1 587)cpm/孔,P<0.01],而凋亡明显增高[总凋亡率:(32.35±2.86)%vs(15.24±1.27)%;早期凋亡率:(22.47±2.54)%vs(10.22±0.98)%;P<0.01]。结论成功构建大鼠Jagged1真核表达质粒,过表达Jagged1可抑制VSMCs增殖并促进其凋亡。
Objective To construct a rat Jagged1 eukaryotic expression plasmid and analyze the effect of Jagged1 overexpression on the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). Methods The target gene was digested with the full-length Jagged1 plasmid pBOS-SN3T. The recombinant plasmid pCMV-IRES-GFP was constructed and transfected into VSMCs. The expression of Jagged1 protein was detected by Western blot. The proliferation of Jagged1 protein was detected by [3 H] -TdR incorporation and the apoptosis was detected by flow cytometry (FACS). Results The rat Jagged1 eukaryotic expression vector was successfully constructed and transfected into VSMCs. The transfection efficiency was over 90% after G418 selection. Transfection up-regulated the Jagged1 protein level in VSMCs by about 6-fold. The proliferation of VSMCs after transfection with p-rJagged1 was significantly decreased [(10 265 ± 1 004) cpm / well vs (17 584 ± 1 587) cpm / well, P <0.01] : (32.35 ± 2.86)% vs (15.24 ± 1.27)%; the early apoptosis rate was (22.47 ± 2.54)% vs (10.22 ± 0.98)%; P <0.01]. Conclusion The rat Jagged1 eukaryotic expression plasmid was successfully constructed. Overexpression of Jagged1 can inhibit the proliferation and promote the apoptosis of VSMCs.