载pVAX1/Tat PTD-endostatin壳聚糖纳米基因载体的构建与体外活性评价  

Development and evaluation of chitosan gene nanoparticles loaded with recombinant plasmid pVAX1/Tat PTD-endostatin

作　　者：张新科[1] 陈洪元[2] 杨莹[1] 李妍[1] 王凤山[1] ZHANG Xinke CHEN Hongyuan YANG Ying LI Yan WANG Fengshan(School of Pharmaceutical Sciences, Shandong University, Jinan 250012, Shandong, China Department of General Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong, China)

机构地区：[1]山东大学药学院,山东济南250012 [2]山东大学附属省立医院普外科,山东济南250021

出　　处：《山东大学学报（医学版）》2017年第7期43-48,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金青年基金(81302686);山东省重点研发计划(2016GSF201083)

摘　　要：目的构建载内皮抑素(Es)重组表达质粒的壳聚糖纳米基因载体并评价其抑制内皮细胞增殖的活性。方法选用真核表达质粒p VAX1为重组载体,采用PCR及双酶切法制备能够表达Tat PTD-Es融合蛋白的真核表达载体p VAX1/Tat PTD-Es;离子交联法制备壳聚糖载基因纳米粒,琼脂糖凝胶电泳筛选处方,检测纳米粒的形态、Zeta电位及粒径;采用最佳处方比例制备载重组质粒纳米载体,转染人脐静脉内皮细胞HUVEC,采用ELISA法测定分泌型Tat PTD-Es蛋白的表达量,CCK-8法测定对HUVEC的抑制作用,以上两个实验均以LipofectaminTM2000为阳性对照。结果 p VAX1/Tat PTD-Es的真核表达载体构建成功,壳聚糖纳米粒Zeta电位(14.3±1.6)m V,粒径(145±12)nm,纳米粒转染HUVEC后细胞上清中Tat PTD-Es的浓度为(182.5±16.3)ng/m L,壳聚糖基因载体转染HUVEC后对细胞有明显的抑制作用,72 h的抑制率达(53.4±5.4)%(Z=-2.611,P=0.008)。结论构建的壳聚糖纳米基因载体能够成功转染HUVEC并能明显地抑制内皮细胞的增殖。Objective To develop chitosan gene nanoparticles loaded with recombinant plasmid which contains endosta- tin（Es） gene and evaluate its in vitro inhibitory effects on HUVEC cells. Methods pVAX1 was taken as the expres- sion vector. PCR combined with double restriction enzyme digest method was used to construct recombinant plasmids pVAX1/Tat PTD-Es. Chitosan nanoparticles loaded with plasmids were prepared by ion cross-linking method. Agarose gel electrophoresis experiment was employed to screen the best prescription of nanoparticles. The shape, Zeta potential and size distribution of nanoparticles were evaluated. After nanoparticles were transferred into HUVEC cells, ELISA method was used to detect the expression of Tat PTD-Es, and CCK-8 was explored to determine the inhibitory effect on HUVEC. In ELISA and CCK-8 experiments, LipofectamineTM2000 loaded with pVAX1/Tat PTD-Es was used as posi- tive control. Results The recombinant plasmid pVAX1/Tat PTD-Es was constructed successfully. The Zeta potential of chitosan nanoparticles loaded with recombinant plasmid was （ 14.3 ± 1.6 ） mV, the size distribution was （ 145 ± 12） nm, The concentration of Tat PTD-Es in HUVEC supernatant was （182.5 ± 16.3） ng/mL. CCK-8 results showed that chitosan nanoparticles loaded with pVAXI/Tat PTD-Es had inhibitory effects on HUVEC cells and the inhibitory rate in 72 h was （53.4 ±5.4）% （Z= -2. 611, P=0.008）. Conclusion Chitosan nanoparticles loaded with recombinant plasmid pVAX1/Tat PTD-Es can be transferred into HUVEC ceils, and have distinct inhibitory effects on the prolifera- tion of HUVEC cells.

关 键 词：TAT PTD-内皮抑素 壳聚糖 纳米基因载体 基因治疗 

分 类 号：R318.08[医药卫生—生物医学工程]