靶向载基因及穿膜肽超声造影剂转染缺氧人脐静脉内皮细胞的研究

The feasibility of a targeted ultrasound contrast agent carrying genes and cell-penetrating peptides tohypoxic HUVEC

目的探讨P-选择素靶向载基因及穿膜肽超声造影剂的制备及转染缺氧损伤型人脐静脉内皮细胞（HUVEC）的可行性。方法采用机械振荡法及碳二亚胺法制备P一选择素靶向载绿色荧光蛋白基因及穿膜肽超声造影剂，检测其形态分布、浓度、粒径，采用激光共聚焦显微镜观察基因和穿膜肽的分布，用荧光分光光度法计算基因及穿膜肽的包封率。体外培养HUVEC，用过氧化氢处理制备HUVEC缺氧模型，并分为靶向和非靶向载基因及穿膜肽造影剂组进行转染实验。用荧光显微镜观察绿色荧光蛋白的表达情况，流式细胞仪检测转染率，并用SPSS17．0统计软件进行t检验及直线相关分析。结果制备的P一选择素靶向载基因及穿膜肽超声造影剂平均粒径约（2．15±0．36）u脚，计数为（1．58±0．23）×10’个／ml。激光共聚焦显微镜下观察到基因和穿膜肽均匀分布在造影剂壁上，基因和穿膜肽的包封率分别为28％[Y=0．932x一0．09（r=0．993，P〈0．05）]和25％[Y=5．875x一0．81（r=0．987，P〈0．05）]。转染24h后，荧光显微镜下观察到靶向和非靶向载基因及穿膜肽造影剂组均有绿色荧光蛋白的表达，流式细胞仪检测靶向组和非靶向组平均转染率分别约为（18．74±0．47）％和（15．34±0．22）％（t=10．923，P〈0．001）。结论P-选择素靶向载基因及穿膜肽超声造影剂能够转染缺氧损伤的HUVEC，这为靶向基因投递提供了新的思路。

Objective To prepare an anti-P-selectin targeted ultrasound contrast agent carrying genes and cell-penetrating peptides （CPP） and to investigate its feasibility of delivery to hypoxic human umbilical vein endothelial cells （HUVEC）. Methods Anti-P-selectin targeted ultrasound contrast agent car- rying a green fluorescent protein gene （ pEGFP-N1 ） and CPP was prepared by mechanical vibration and carbodiimide techniques. The appearance, distribution, concentration and diameter of the ultrasound contrast agent were measured. The gene and CPP distribution on the agent was investigated using confocal laser scanning microscopy （CLSM）. The efficiency of the ultrasound contrast agent to carry the gene and CPP was investigated by fluorospectrophotometry. HUVEC were cultured in vitro and hypoxic HUVEC were pre- pared using hydrogen peroxide （H202 ）. Hypoxic HUVEC were randomly assigned targeted ultrasound con- trast agents and non-targeted ultrasound contrast agents for transfcction. The transfection effect of green fluorescent protein in the two groups was observed using fluorescence microscopy and flow cytometry. T-test and linear correlation analysis were used for statistical analysis. Results The average diameter of anti-P-selectin targeted ultrasound contrast agents carrying gene and CPP was （2.15 ：t： 0.36） txm and the concentration was （ 1.58 ＋ 0.23 ） x 107/ml. The results of CLSM showed that gene and CPP were distnbuted on the shell of the agent. The gene encapsulation efficiency was 28% （y = 0. 932x - 0. 09, r = 0. 993, P 〈 0.05 ）, and the CPP encapsulation efficiency was 25% （y = 5. 875x - 0. 81, r = 0. 987, P 〈 0.05 ）. EGFP expressionwas observed using fluorescence microscopy in targeted ultrasound contrast agents and non-targeted uhra- sound contrast agents. The average transfection efficiencies of targeted ultrasound contrast agents and non- targeted ultrasound contrast agents were （ 18.74 ＋ 0.47 ） % and （ 15.34 ＋ 0.22） % after 24 h （ t = 10. 923, P 〈 0.001 ）. Conclusions The in vitro studies showed that the targeted ultrasound contrast agent could sig- nificantly enhance the transfection efficiency in hvDoxie HUVEC. This may be a new idea for gene therapy.