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构建HSV1-sr39tk为报告基因,以VEGF165为治疗基因的重组腺病毒载体Ad5-sr39tk-IRES-VEGF165(Ad5-SIV),将其以不同感染复数(MOI=0、10、25、50、75、100)感染大鼠骨髓间充质干细胞,用免疫激光共聚焦技术检测HSV1-sr39tk与VEGF165的表达。用酶联免疫吸附试验(ELISA)监测感染病毒后细胞培养上清中VEGF165蛋白分泌量;细胞摄取131I-FIAU实验监测HSV1-sr39tk报告基因的表达情况,以腺病毒包装增强型绿色荧光蛋白(Ad5-EGFP)为对照。结果显示:免疫共聚焦显微镜可观察到HSV1-sr39tk蛋白与VEGF165蛋白在细胞内成功表达;VEGF165蛋白分泌情况与MOI间呈线性正相关(P<0.05);感染Ad5-SIV后细胞对131I-FIAU摄取率增高与MOI间呈线性正相关(P<0.05)。不同时间摄取实验结果显示:30-150min间细胞摄取率迅速增高,之后增高减慢,进入平台期。MOI相关摄取结果与MOI相关ELLISA结果具有较好相关性(P<0.05)。Ad5-SIV经感染后可在大鼠骨髓间充质细胞中成功表达,并且治疗基因VEGF与报告基因HSV1-sr39tk的表达呈正相关,说明可通过报告基因HSV1-sr39tk监测治疗基因VEGF165的表达,为动物水平报告基因显像提供理论支持。
The recombinant adenoviral vector Ad5-sr39tk-IRES-VEGF165 (Ad5-SIV) with HSV1-sr39tk as the reporter gene and VEGF165 as the therapeutic gene was constructed and transfected into Ad5-sr39tk cells with different multiplicity of infection (MOI = 0,10,25,50,75, 100) infected rat bone marrow mesenchymal stem cells, and the expressions of HSV1-sr39tk and VEGF165 were detected by immunofluorescence confocal technique. The secretion of VEGF165 protein in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The expression of HSV1-sr39tk reporter gene was detected by 131I-FIAU assay. The adenovirus vector encoding enhanced green fluorescent protein (Ad5 -EGFP) as a control. The results showed that HSV1-sr39tk protein and VEGF165 protein were successfully expressed in the cells by immunofluorescence confocal microscopy; the secretion of VEGF165 was positively correlated with the MOI (P <0.05); The expression of 131I-FIAU There was a linear positive correlation between uptake rate and MOI (P <0.05). The results of ingestion at different times showed that the cell uptake rate increased rapidly between 30-150min, then increased and then slowed down and entered the plateau phase. The MOI-related ingestion results correlated well with the MOI-related ELLISA results (P <0.05). Ad5-SIV was successfully transfected into rat bone marrow mesenchymal cells after infection, and the expression of therapeutic gene VEGF was positively correlated with the expression of HSV1-sr39tk, which indicated that the expression of therapeutic gene VEGF165 could be monitored by reporter HSV1-sr39tk Animal level report gene imaging provide theoretical support.