CAJ | 학술논문

目的构建大鼠骨桥蛋白(OPN)基因的shRNA慢病毒表达载体,并观察其对大鼠血管平滑肌细胞(VSMC)增殖和凋亡的影响.方法针对OPN基因的不同部位设计3对shRNA的寡核苷酸片段,克隆到慢病毒载体PLKO.1中,构建靶向OPN基因的慢病毒载体PLKO.1-OPN-shRNA,检测并筛选最佳抑制效率的shRNA干扰载体.并将其转染大鼠血管平滑肌细胞,用逆转录-聚合酶链反应(RT-PCR)检测VSMCs OPN mRNA表达水平；蛋白免疫印迹法(Western blot)检测VSMC OPN蛋白表达水平；噻唑蓝(MTT)比色法和流式细胞仪检测沉默OPN基因后对VSMC增殖和凋亡能力的影响.结果靶向OPN慢病毒表达载体构建成功.重组慢病毒载体PLKO.1-OPN-shRNA转染后可显著抑制VSMCs的OPN mRNA及蛋白的表达水平,OPN蛋白表达明显下降,其中以PLKO.1-OPN2-shRNA最为明显,达到90％以上；转染PLKO.1-OPN2-shRNA后72 h的细胞增殖能力[吸光度(A)值=0.365 ±0.011]明显低于未处理组(A值=0.941±0.028)和阴性对照组细胞(A值=0.941±0.040,P＜0.05)；而早期细胞凋亡率(20.44±2.69)％和晚期细胞凋亡率(16.79±1.01)％均明显高于未转染组和阴性对照组(P＜0.01).结论成功构建并筛选最佳抑制效率的靶向OPN慢病毒表达载体PLKO.1-OPN2-shRNA,该载体能有效抑制大鼠血管平滑肌细胞增殖,并促进细胞凋亡.
목적 구건대서골교단백(OPN)기인적shRNA만병독표체재체,병관찰기대대서혈관평활기세포(VSMC)증식화조망적영향.방법 침대OPN기인적불동부위설계3대shRNA적과핵감산편단,극륭도만병독재체PLKO.1중,구건파향OPN기인적만병독재체PLKO.1-OPN-shRNA,검측병사선최가억제효솔적shRNA간우재체.병장기전염대서혈관평활기세포,용역전록-취합매련반응(RT-PCR)검측VSMCs OPN mRNA표체수평；단백면역인적법(Western blot)검측VSMC OPN단백표체수평；새서람(MTT)비색법화류식세포의검측침묵OPN기인후대VSMC증식화조망능력적영향.결과 파향OPN만병독표체재체구건성공.중조만병독재체PLKO.1-OPN-shRNA전염후가현저억제VSMCs적OPN mRNA급단백적표체수평,OPN단백표체명현하강,기중이PLKO.1-OPN2-shRNA최위명현,체도90％이상；전염PLKO.1-OPN2-shRNA후72 h적세포증식능력[흡광도(A)치=0.365 ±0.011]명현저우미처리조(A치=0.941±0.028)화음성대조조세포(A치=0.941±0.040,P＜0.05)；이조기세포조망솔(20.44±2.69)％화만기세포조망솔(16.79±1.01)％균명현고우미전염조화음성대조조(P＜0.01).결론 성공구건병사선최가억제효솔적파향OPN만병독표체재체PLKO.1-OPN2-shRNA,해재체능유효억제대서혈관평활기세포증식,병촉진세포조망.
Objective To construct a recombinant short hairpin RNA (shRNA) lentiviral vector carrying osteopontin (OPN) gene of rat,and to investigate its effects on the proliferation and apoptosis of vascular smooth muscle cells (VSMCs) by silencing OPN.Methods Four oligonucleotides targeting OPN gene were synthesized and cloned into lentivirus vector PLKO.1.The shRNA lentiviral vector with the best transfection efficiency was detected and identified,which was transfected into rat VSMCs.After RNA interference ( RNAi)-mediated knockdown of rat OPN,the mRNA and protein levels of OPN in VSMCs were assessed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting.The effects of silencing OPN on the proliferation and apoptosis of VSMCs were examined by methyl thiazol tetrazolium (MTT) assay and flow cytometry.Results The recombinant lentivirus vector PLKO.1-OPN-shRNA was constructed successfully.The recombinant lentivirus PLKO.1-OPN-shRNA knocked down the expression of OPN mRNA and protein in VSMCs dramatically,especially PLKO.1-OPN2-shRNA,whose transfection efficiency was more than 90％.The proliferation rate of VSMCs transfected with PLKO.1-OPN2-shRNA (A value =0.365 ±0.011 ) was significantly lower than un-transfected group (A value =0.941 ±0.028) and negative control group (A value =0.941 ± 0.040,P ＜ 0.05 ).Meantime the early apoptosis rate (20.44 ± 2.69)％ and late apoptosis rate ( 16.79 ± 1.01 )％ were significantly higher in transfection group than un-transfected group and negative control group ( P ＜ 0.01 ).Conclusion The recombinant lentivirus vector expressing shRNA of targeting OPN is constructed successfully.And shRNA with the best transfection efficiency is PLKO.1-OPN2-shRNA,which can inhibit the proliferation of VSMCs,and promote the cell apoptosis.