壳聚糖纳米粒介导的人类端粒酶反转录酶启动子连接的自杀基因耦联更昔洛韦靶向抗肝癌研究  

Empirical study of hepatocellular carcinoma＇ s gene therapy using hTERTp-TK/GCV in HepG2 which was mediated by chitosan nanoparticles vehicles

作　　者：贾静[1] 邓志华[2] 刘燕[3] 

机构地区：[1]山西医科大学附属太钢总医院消化内科,太原030003 [2]山西医科大学第二医院肝病科 [3]陕西省榆林市第一医院消化内科

出　　处：《肿瘤研究与临床》2011年第7期438-442,共5页Cancer Research and Clinic

基　　金：国家自然科学基金（30672405）

摘　　要：目的比较壳聚糖（Ch）与半乳糖化壳聚糖（GC）纳米载体对肝癌细胞的转染效果；观察转染后的人类端粒酶反转录酶启动子连接的自杀基因（pGL3-hTERTp—TK）对肝癌细胞株HepG2生长及凋亡的影响。方法制备ch及Gc；构建pGL3，hTERTp—TK质粒及ch／DNA和GC／DNA复合物；转染HepG2细胞和正常肝细胞L-02；通过单光子液闪计数仪观察转染效率；流式细胞术（FCM）、Caspase-3万法观察自杀基因对肝癌细胞生长和凋亡的影响。结果质粒经酶切及电泳后在琼脂糖凝胶上出现300bp与1100bp两个清晰条带；在HepG2中，由Gc介导的pGL3-hTERTp．Luc^＋荧光素相对活性表达明显高于ch介导的pGL3-hTERTp—Luc^＋，但比去唾液酸糖蛋白（AF）介导的pGL3．hTERTp—Luc^＋低，而在正常肝细胞荧光素相对活性表达极低；治疗质粒Gc介导的pGL3-hTERTp—TK转染的HepG2细胞抑制率和L-02细胞抑制率，在前体药物更昔洛韦（GCV）的浓度为10μg／ml时差异有统计学意义（t=51．40，P=0．000）。HepG2细胞组GC—pGL3-hTERTp—TK凋亡率为65．28％，明显高于其他组（LSD法，均P〈0．05）。L-02细胞组GC—pGL3-hTERTp—TK凋亡率仅为10．R0％，明显低于对照组Ch—pGL3-control（LSD法，P=0，000）；FCM检测显示Ch—pGL3-hTERTp—TK转染HepG2细胞后其平均荧光强度为168．02±3．68，Gc—pGL3-hTERTp—TK转染后其平均荧光强度为204．45±3．45，两组差异有统计学意义（t=-12．504，P〈0．05）。结论Gc较ch能提高pGL3-hTERTp—TK质粒对肝癌细胞的转染率，GC—pGL3-hTERTp—TK可以靶向攻击肝癌细胞，对正常肝细胞几乎无影响。Objective To compare the transfection efficiency of galactosylated chitosan nanoparticle vehicle with chitosan nanoparticles vehicle, and observe the therapeutic effect of pGL3-hTERTp-TK on HCC cell line HepG2. Methods Preparing the chitosan and galactosylated chitosan. Constructing the pGL3-hTERTp-TK plasmid and the Ch/DNA and GC/DNA complexes. Transfecting the HepG2 and the normal hepatic cell L-02 with chitosan/DNA and galaetosylated chitosan/DNA complexs. Detecting the Fluorescence and the expression of luciferase gene using the fluorescent microscope and the scintillation counter. Detecting the cell growth and apoptosis through the Caspase-3 and the flow cytometry. Results Two clear straps appeared in the agarose gel. The locations were 300 bp and 1100 bp. The relative luciferase activity of pGL3-hTERTp-Luc ＋ mediated by galactosylated chitosan was powerful than which of pGL3-hTERTp-Luc^＋ mediated by chitosan in HepG2 by the scintillation counter. However, the relative luciferase activity was very weak in L-02. The same results were observed by fluorescent microscope. When the eoncertration of the GCV was 10 μg/ml （t = 51.40, P = 0.000）, the HepG2 cell inhibition which was transfected by GC-pGL3-hTERTp-TK was obviously different from the L-02 cell inhibition which was transfeeted by GC-pGL3-hTERTp-TK in the statistics. The significant apoptotic rate was 65.28 % in HepG2 which was transfected with GC/DNA, whereasit was only 10.80 % in L-02. The significant apoptotic rate in HepG2 which was transfected with GC/DNA was very higher than the other groups （LSD, P 〈0.05）. The significant apoptotic rate （10.80 %） in L-02 which was transfected with GC/DNA was very higher than the group which was Ch-pGL3-control （LSD, P =0.000）. The average fluorescence intensity of the HepG2 which is transfected by the Ch-pGL3-hTERTp-TK was 168.02±3.68. The average fluorescence intensity of the HepG2 which is transfected by the GC-pGL3-hTERTp-TK was 204.45±3.45. The two groups had a significant difference in

关 键 词：肝肿瘤 实验性 壳聚糖 靶向基因治疗 端粒酶反转录酶启动子 基因 转基因 自杀 

分 类 号：R735.7[医药卫生—肿瘤]