Zhou Jia-Jia, Chen Ru-Fu, Li Zhi-Hua, Zhou Quan-Bo, Tang Qi-Bin, He Xiao-Yu, Lu Hong-Wei, Guo Ning
Department of General Surgery, Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China.
Zhonghua Yi Xue Za Zhi. 2009 Mar 31;89(12):795-9.
To investigate the inhibitory effect of nanoparticle-mediated endostatin gene therapy on hepatocellular carcinoma xenografts combined with local hyperthermia utilizing heat-inducible promoter.
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 BglII/HindIII and EcoRI/SalI. 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 degrees C, 39 degrees C, 41 degrees C, 43 degrees C, and 45 degrees C 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 ECV304 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.
The nanoparticles encapsulating recombinant plasmid were of round or elliptical shape 90 approximately 120 nm in diameter. The efficiency of gene transfection mediated by nanoparticles was about 30.65%. The expression of Endo/EGFP gene in the HepG2 cells was up-regulated along with the increase of temperature, peaked at 43 degrees C (with the EGFP expression level 3.3 times as that at 37 degrees C). The concentration of endostatin protein in the supernatant of the 43 degrees C group was (177 +/- 28) microg/L, significantly higher than that of the 37 degrees C group [(41 +/- 10) microg/L]. MTT results indicated that endostatin inhibited the growth of ECV304 cells with a inhibition rate of 96.3% at the time point of 72 h in the 43 degrees C group, however, it did not show influence on HepG2 cells no matter what was the temperature The tumor inhibition rate in the mice of endostatin with thermal induction group was 58.5%, significantly higher than that of the 37 degrees C group (34.9%, P < 0.05).
Low temperature thermal induction enhances the expression and secretion of endostatin in hepatocellular cells transfected by nanoparticles, and inhibits the growth of hepatocellular carcinoma xenografts.
探讨纳米颗粒介导的内皮抑素基因治疗联合利用热诱导启动子的局部热疗对肝癌异种移植瘤的抑制作用。
将热诱导型HSP70B启动子和Endo/EGFP融合基因克隆到pcDNA3.1(+)质粒中，使用限制性内切酶BglII/HindIII和EcoRI/SalI获得重组质粒pcDNA3.1(+)/HSP70-Endo/EGFP。采用有机溶剂乳化蒸发法制备包裹重组质粒DNA的纳米颗粒聚乳酸接枝葡聚糖共聚物（DEX-g-PLA），用透射电子显微镜观察纳米颗粒的表面形态。培养人肝癌细胞系HepG2和ECV304，并用纳米颗粒转染重组质粒。在37℃、39℃、41℃、43℃和45℃热诱导30分钟后，用荧光显微镜和流式细胞术检测增强型绿色荧光蛋白（EGFP）的表达。用ELISA法检测上清液中内皮抑素蛋白的浓度，用MTT法检测对HepG2和ECV304细胞的生长抑制作用。将Balb/c裸鼠接种HepG2细胞，然后随机分为2组，进行瘤内注射纳米颗粒（加热或未加热）、Lipofectamine 2000。未进行瘤内注射的小鼠作为对照。4周后处死小鼠，观察肿瘤抑制率。
包裹重组质粒的纳米颗粒呈圆形或椭圆形，直径约90至120nm。纳米颗粒介导的基因转染效率约为30.65%。HepG2细胞中Endo/EGFP基因的表达随温度升高而上调，在43℃时达到峰值（EGFP表达水平是37℃时的3.3倍）。43℃组上清液中内皮抑素蛋白浓度为(177±28)μg/L，显著高于37℃组[(41±10)μg/L]。MTT结果表明，内皮抑素抑制ECV304细胞生长，43℃组在72小时时间点抑制率为96.3%，但对HepG2细胞无论温度如何均无影响。热诱导内皮抑素组小鼠的肿瘤抑制率为58.5%，显著高于37℃组（34.9%，P<0.05）。
低温热诱导增强了纳米颗粒转染的肝癌细胞中内皮抑素的表达和分泌，并抑制了肝癌异种移植瘤的生长。
