慢病毒介导的Canstatin基因转移对脐静脉血管内皮细胞增殖与凋亡的影响

摘要

目的:构建携带Canstatin基因的慢病毒载体,体外转染脐静脉血管内皮细胞ECV304,观察其体外培养的脐静脉血管内皮细胞增殖及凋亡的影响.方法:应用基因重组技术构建慢病毒载体表达质粒pGC-FU-Canstatin,通过酶切、测序验证Canstatin基因后,将pGC-FU-Canstatin质粒和包装质粒pHelper 1.0,pHelper 2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Canstatin基因重组慢病毒GC-FU-Canstatin,并测定病毒滴度.将重组慢病毒转染靶细胞人脐静脉血管内皮细胞ECV304,通过Western blotting检测靶细胞中Canstatin的表达.采用 MTT法观察 Canstatin基因对人脐静脉血管内皮细胞 ECV304的体外细胞增殖的影响.TUNEL染色、流式细胞仪 AnnexinV /碘化丙啶双染法,检测慢病毒介导的 Canstatin基因诱导脐静脉血管内皮细胞的凋亡.结果:pGC-FU-Canstatin中携有正确的Canstatin基因序列; pGC-FU-Canstatin和包装质粒pHelper 1.0,pHelper 2.0共转染包装细胞293T产生重组病毒GC-FU-Canstatin;检测病毒滴度为1×109TU/ml;Western blotting检测到Canstatin蛋白在靶细胞中持续表达.Canstatin(感染复数分别为25和50)作用72h后,ECV304细胞增殖数目 显著少于PBS组和空载体组(P<0.01);TUNEL染色,以MOI为25的重组慢病毒GC-FU-Canstain作用于人脐静脉血管内皮细胞ECV304细胞72h后,出现典型的凋亡形态学改变.以感染复数为25的GC-FU-Canstatin处理ECV304细胞72h后,细胞凋亡率为(21.63±1.32)%,PBS组为(2.87±0.76)%,空载体组为(2.66±0.69)%,转基因组与空载体组、PBS组相比差异均有显著统计学意义(P<0.01).结论:慢病毒介导的Canstatin基因可显著抵制人脐静脉血管内皮细胞ECV304的体外增殖,同时对人脐静脉血管内皮细胞的凋亡具有促诱导作用.%Objective : To investigate the effect of Canstatin mediated by Lentivirus( pGC - FU - Canstatin )on the proliferation and apoptosis of vascular endothelial cells in vitro. Methods : Canstatin gene was amplified from plasmid pSPORTl - Sfi by PCR. The plasmid of pGC - FU and PCR products of Canstatin gene were double - digested by with restrictive endonucleases Age I and EcoR I to generate the lentiviral expression vector pGC - FU - Canstatin by gene recombination technique. The corrected Canstatin was confirmed by endoenzym digestion, sequencing. Recombinant lentiviruses were produced by 293T cells following the co - transfection of pGC - FU - Canstatin, with the packaging plasmids pHelper 1. 0 and pHelper 2. 0. The viral titer was detected by Western - blotting. The resulting combinant lentiviruses which carrying Canstatin were then used to transfect huaman umbilical vein endothelial cells. Canstatin protein expression in huaman umbilical vein endothelial cells was dectected hy Western hlotting analysis. The effects of Canstatin gene on the proliferation of ECV304 cells were measured by MTT assay. Morphological change of apoptosis of ECV304 cells was determined by TUNEL staining, and by FCM with AnnexinV/P1 costaining. Results :In the cellular proliferarion assay in vitro, the number of ECV304 cells in GC - FU - Canstatin group was much smaller than that in GC - FU group and PBS group ( P ＜0. 01 ). After 72h of treatment with GC - FU - Canstatin, the ECV304 cells had characteristics of apoptosis revealed by TUNEL staining. The apoptosis rate in GC - FU - Canstatin group ( 21. 63 ±1. 32 )％ was much higher than that in GC - FU group ( 2. 66 ±0. 69 )％ and PBS group ( 2. 87 ±0. 76 )％ ( P ＜ 0. 01 ). Conclusion : The Canstatin gene mediated by lentivirus has an inhihitory effect on the proliferation of human umbilical vein endothelium cells ECV304 in vitro , and could induce the apoptosis of human umbilical vein endothelium cells in vitro.