摘要
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 recombinant eukaryotic vector expressing rat Jagged1,and to explore the effect of Jagged1 over-expression on the proliferation and apoptosis of vascular smooth muscle cells（VSMCs）.Methods The full-length fragment of Jagged1 gene was obtained from the plasmid pBOS-SN3T（encoding rat Jagged1） by digestion.A eukaryotic vector expressing rat Jagged1（p-rJagged1） was constructed with the aid of the plasmid pCMV-IRES-GFP,and then the vectors were transfected into VSMCs.The expression of Jagged1 protein was detected by Western blotting.The proliferation of VSMCs was observed by assessing the incorporation of 3H-thymidine.The apoptosis of VSMCs was analyzed by flow cytometry.Results Recombinant plasmid p-rJagged1 was successfully constructed and transfected into VSMCs.The efficiency of transfection was over 90% after G418 screening.The protein levels of Jagged1 were increased by almost 6 folds after transfection.The proliferation of VSMCs was significantly lower[（10 265±1 004） vs（17 584±1 587） cpm/well,P〈0.01],and the apoptotic rate of VSMCs was significantly higher[total rate of apoptosis：（32.35±2.86）% vs（15.24±1.27）%;early rate of apoptosis：（22.47±2.54）% vs（10.22±0.98）%;P〈0.01] in the p-rJagged1 transfected group compared with those in the untransfected group.Conclusion A recombinant eukaryotic vector expressing rat Jagged1 is constructed.Jagged1 over-expression can suppress the proliferation and promote the apoptosis of VSMCs.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2011年第11期1095-1098,共4页
Journal of Third Military Medical University
基金
国家自然科学基金(30872710)~~
