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目的探讨hVEGF165及hBMP-7共表达重组腺相关病毒载体(recombinant adeno-associated virus,rAAV)介导基因表达的时效性,为体内应用其进行骨坏死的基因治疗奠定理论基础。方法应用荧光细胞计数法测定rAAV转染兔BMSCs的最佳转染复数。分别采用rAAV-hVEGF165-内部核糖体进入位点序列(internal ribosome entry site,IRES)-hBMP-7(实验组)、绿色荧光蛋白(green?uorescent protein,GFP)标记平行对照rAAV-IRES-GFP(对照组)在最佳转染复数条件下转染体外培养的第3代兔BMSCs。于不同时间点分别行GFP表达检测、RT-PCR检测、Western blot检测明确rAAV体外介导基因表达的时效性关系。将实验组及对照组病毒转染后的BMSCs以5×106个/mL密度,分别注射至9只2月龄纯种雄性新西兰大耳白兔制备的肌袋模型内各1mL(实验组1及对照组1),于不同时间点分别行GFP表达检测、Western blot检测及ELISA检测明确rAAV体内介导基因表达的时效性关系。结果 rAAV转染兔BMSCs的最佳转染复数为5×104v.g/cell。通过体外检测显示rAAV转染BMSCs后1d即有目的基因表达,14d时表达强度明显增强,至28d时仍维持较强表达。通过体内检测显示体内转染2周可检测到目的基因表达,6周时表达强度增强,8周时仍维持较高水平。ELISA法检测实验组1细胞培养上清中hVEGF165和hBMP-7含量分别为(248.67±75.58)pg/mL和(4.80±0.61)ng/mL,对照组1分别为(32.28±8.42)pg/mL和(0.64±0.42)ng/mL,两组比较差异均有统计学意义(P<0.05)。结论双基因共表达rAAV-hVEGF165-IRES-hBMP-7具有较好的转染时效性。
Objective To investigate the timeliness of gene expression mediated by hVEGF165 and hBMP-7 co-expressing recombinant adeno-associated virus (rAAV), and to lay a theoretical foundation for the gene therapy of osteonecrosis in vivo. Methods Fluorescence cytometry was used to determine the optimal transfection complex of rAAV transfected BMSCs. The rAAV-hVEGF165-internal ribosome entry site (IRES) -hBMP-7 (experimental group) and the green fluorescent protein (GFP) -labeled parallel control rAAV-IRES-GFP Control group) were transfected into 3rd generation rabbit BMSCs cultured in vitro under optimal transfection conditions. GFP expression was detected at different time points, RT-PCR detection, Western blot detection rAAV in vitro-mediated gene expression timeliness relationship. The experimental group and the control group were infected with BMSCs transfected 5 × 106 / mL density, were injected into 9 2-month-old male New Zealand white rabbits muscle bags prepared in 1mL (experimental group 1 and Control group 1), GFP expression was detected at different time points, Western blot detection and ELISA detection rAAV in vivo-mediated gene expression timeliness relationship. Results The optimal transfection complex of rAAV transfected BMSCs was 5 × 104v.g / cell. The expression of target gene was detected at 1 day after rAAV transfection in vitro. The expression level of the target gene was significantly increased on the 14th day and remained strong at 28 days. By in vivo test showed that the expression of the target gene can be detected within 2 weeks of transfection in vivo, the expression intensity increased at 6 weeks, still maintained a high level at 8 weeks. The contents of hVEGF165 and hBMP-7 in the culture supernatant of experimental group 1 were (248.67 ± 75.58) pg / mL and (4.80 ± 0.61) ng / mL, respectively, while those in control group 1 were (32.28 ± 8.42) pg / mL And (0.64 ± 0.42) ng / mL, respectively, with significant difference between the two groups (P <0.05). Conclusion Double gene co-expression of rAAV-hVEGF165-IRES-hBMP-7 has good transfection efficiency.