纳米载体介导热诱导启动子调控内皮抑素基因治疗肝细胞癌  

Nanoparticle-mediated endostatin gene therapy targeting hepatocellular carcinoma utilizing heatinducible promoter

作　　者：周嘉嘉[1] 陈汝福[1] 李志花[1] 周泉波[1] 唐启彬[1] 贺晓玉[1] 卢红伟[1] 郭宁[1] 

机构地区：[1]中山大学附属第二医院普外科,广州510120

出　　处：《中华医学杂志》2009年第12期795-799,共5页National Medical Journal of China

基　　金：国家高科技计划“863”基金（2007AA02ZA37;2002AA214061）;国家自然科学基金（30872485）;广东省医学科学研究基金（A2005228）;广州市科委基金（200723-D2061）;广东省自然科学基金（2003A031700;A40094403）

摘　　要：目的探讨加热诱导调控下纳米载体介导内皮抑素（Endo）基因治疗对肝癌细胞HepG2的体内外杀伤作用。方法双酶切法构建热诱导启动子（HSP70B）调控Endo基因的重组质粒pcDNA3．1（＋）／HSP70-Endo／EGFP。溶剂挥发法制备载DNA的葡聚糖接枝聚乳酸纳米粒，透射电镜观察载DNA纳米粒的形态，并介导转染HepG2细胞，转染24h后予以37℃、39℃、41℃、43℃、45℃加热诱导30min，荧光显微镜和流式细胞术检测不同温度下EGFP表达，ELISA检测转染细胞上清Endo蛋白浓度，MTT法检测热诱导后Endo对HepG2和ECV304细胞生长的影响，动物实验观察热诱导后Endo对肝癌移植瘤的抑制作用。结果载DNA纳米粒呈圆形或椭圆形，粒径约90～120nm，转染效率约30．65％。低温加热可增强HepG2细胞中Endo／EGFP表达，43℃组EGFP表达强度达37℃组的3．3倍。43℃热诱导后转染细胞上清中Endo浓度为（177±28）μg／L，高于不加热组（41±10）μg／L。Endo抑制ECV304的生长，72h时热诱导后细胞抑制率为96．3％，对HepG2细胞的生长却无影响。体内实验显示热诱导后Endo显著抑制肝癌移植瘤的生长，肿瘤抑制率为58．5％，高于不加热组（34．9％）。结论纳米载体可介导Endo基因转染HepG2细胞，低温加热可增强HepG2细胞内Endo基因的表达和分泌，抑制肝癌移植瘤的生长。Objective To investigate the inhibitory effect of nanoparticle-mediated endostatin gene therapy on hepatoeellular carcinoma xenografts combined with local hyperthermia utilizing heat-inducible promoter. Methods Heat-inducible HSP70B promoter and fusion gene of Endo/EGFP were cloned into pcDNA3.1 （ ＋ ） plasmid, thus obtaining recombinant plasmid of pcDNA3. 1 （ ＋ ）/HSP70-Endo/EGFP using restriction endonucleases Bgl Ⅱ/Hind Ⅲ and EcoR Ⅰ/Sal Ⅰ. The nanoparticles polylactide-grafted dextran copolymer （DEX-g-PLA） encapsulating the recombinant plasmid DNA were prepared by the method of emulsification and evaporation of organic solvent, and the surface shape of nanoparticles was observed by transmission electron microscope. Human hepatocellular cells of the lines HepG2 and ECV304 were cultured and transfected with the recombinant plasmid utilizing the nanoparticles. Following thermal induction at 37 ℃, 39 ℃ , 41 ℃, 43 ℃ , and 45 ℃ for 30 min, the expression of enhanced green fluorescent protein （EGFP） was detected by fluorescence microscope and flow cytometry. The concentration of endostatin protein in the supernatant was tested by ELISA, and the growth inhibition on the HepG2 and ECV30d cells was tested by MTT method. Balb/c nude mice were inoculated with HeG2 cells and then randomly divided into 2 groups to undergo intra-tumor injection of nanoparticles （ heated or not heated）, Lipofectamine 2000. Mice were used as controls without intra-tumor injection. Four weeks the mice were killed to observe the tumor inhibition rate. Results The nanoparticles encapsulating recombinant plasmid were of round or elliptical shape 90 - 120 nm in diameter. The efficiency of gene trausfection mediated by nanoparticles was about 30.65%. The expression of Endo/EGFP gene in the HepG2 ceils was up-regulated along with the increase of temperature, peaked at 43 ℃（ with the EGFP expression level 3.5 times as that at 37 ℃）. The concentration of endostatin protein in the supernatant of the 43 ℃

关 键 词：肝细胞癌 基因治疗 内皮抑素 热诱导启动子 纳米粒 

分 类 号：R686[医药卫生—骨科学]