携pAd—EGFP／SDF1α和pAd—RFP／BMP2双基因微泡的制备及其特性检测  

Experimental study on construction of ultrasound microbubbles co-carrying pAd-EGFP/SDF-1α and pAd-RFP/ BMP2

作　　者：杨灵洁[1] 刘丽云[1] 穆玉明[1] 

机构地区：[1]新疆医科大学第一附属医院心脏超声诊断科,乌鲁木齐830054

出　　处：《中华超声影像学杂志》2016年第11期1002-1007,共6页Chinese Journal of Ultrasonography

基　　金：国家自然科学基金（81460266）;省部共建国家重点实验室培育基地-新疆重大疾病医学重点实验室开放课题（SKKIB-XJMDR-2014-13）;新疆医科大学研究生创新创业项目（CXCY007）;新疆维吾尔自治区研究生科研创新项目

摘　　要：目的制备携基质细胞衍生因子1（SDF-1α）和骨形态发生蛋白2（BMP2）重组腺病毒（pAd）的超声微泡，检测微泡最大载基因率并探讨双基因与微泡结合的最佳配比。方法采用“生物素-亲和素”桥接法将Targestar—SA微泡分别与共表达绿色荧光蛋白（EGFP）和SDF-1α的重组腺病毒（pAd—EGFP／SDF1a）以及共表达红色荧光蛋白（RFP）和BMP2的重组腺病毒（pAd—RFPjBMP2）相结合，检测微泡最大载基因率；依据结果将两种pAd按不同配比加入微泡制备成三组携双基因微泡，并从物理特性、荧光显微镜下观察、流式细胞仪测定及载基因率等方面评价携双基因微泡。结果携基因微泡PH值、平均粒径和浓度与裸微泡差异无统计学意义（P〉0．05）。微泡载基因效率随病毒投入量增加而增大，达到饱和后继续增大病毒量导致负载率降低。双基因与做泡结合的配比为1：1时，微泡携SDF1a与BMP-2的负载率大致相当，流式散点图示双阳性荧光微泡为（65．6±0．5）％；2：1组双阳性荧光微泡为（59．0±2．3）％，显著低于其他两组（P〈0．05）；荧光显微镜可见携基因微泡表面均匀分布绿色或红色荧光。结论成功制备携EGFPjsDF-1α和RFPjBMP2的双基因超声微泡，双基因与微泡结合的最佳配比为1：1，此时微泡双基因负载率均达到较大水平。Objective To construct the uItrasound microbubbIes co-carrying recombinant adenovirus containing stromal cell derived factor 1 （SDF lc0 and bone morphogenetic protein 2 （BMP2）, and to study the maximum efficiency of carrying adenovirus and the optimum proportion of double gene combined with ultrasound contrast agents. Methods Microbubbles were combined separately with recombinant adenovirus co-expression of enhanced green fluorescent protein and SDF-la （pAd EGFP/SDF-1α） as well as red fluorescence protein and BMP2 （pAd RFP/BMP2） via ＂biotin-streptavidin method＂, and the maximum efficiency of carrying DNA in microbubbles was detected. Three microbubbles with binary vectors were prepared by blending the two above-mentioned pad at different ratio （ 1： 1, 1 ： 2, 2 ： 1 ） into the microbubbles. The microbubbles with binary vectors were evaluated though physiochemical properties, fluorescence microscope and flow cytometry to test the carrying rate of DNA in microbubbles. Results There was no significant difference in PH, average diameter and concentrations between targeted microbubbles and control group （ P〉0.05）. The carrying efficiency of DNA increased with virus loads in microbubbles, but lowered if further increasing virus amount after reaching saturation. When the proportion of binary vectors and microbubbles was 1 ： 1, its efficiency of carrying SDF-1α gene and BMP 2 was approximately equal, and flow cytometry demonstrated that the positive rate of microbubbles labeled by both fluoresceinisothiocyanate（FITC） and rhodamine was （65.6±0.5）%. However, it was （59.0±2.3）% when their proportion was the 2 ： 1, which was significantly lower than those when other two proportions （1 ： 1 and 1 ： 2）. Under the fluorescence microscope, the targeted microbubbles were equally surrounded by bright green or red fluorescence. Conclusions Ultrasound microbubbles of double genes carrying EGFP/ SDF 1α and RFP/BMP2 is made successfully via combining microbubbles with double

关 键 词：微气泡 腺病毒载体 基质细胞衍生因子-1 骨形态发生蛋白-2 

分 类 号：R450[医药卫生—治疗学]