半乳糖基多聚赖氨酸携带HSV1-TK对HepG2细胞体外效应研究  

Target delivery of lactose poly-L-lysine combined HSV-TK to human liver cancer cells

作　　者：王炜煜[1] 易继林[1] 邓云华[2] 司进[3] 王从俊[1] 曾建平[1] 曹利民[4] 

机构地区：[1]华中科技大学同济医学院附属同济医院普外科,湖北武汉430030 [2]华中科技大学同济医学院附属同济医院皮肤科,湖北武汉430030 [3]江苏省寄生虫病防治研究所 [4]华中科技大学同济医学院免疫学系

出　　处：《中国医师杂志》2006年第11期1465-1467,共3页Journal of Chinese Physician

摘　　要：目的为肝癌的自杀基因治疗寻找新的靶向分子。方法通过还原氨化法进行乳糖(Lac)与聚赖氨酸(PLL)共价连接;经Sephadex G-10柱分离纯化;以苯酚-硫酸比色法测定n值;与真核表达质粒r-pAs16D r按比例混匀,得到G lanPLL-r-pAs16D r基因转移系统;分别转染不同的细胞株进行体外效应研究:人肝癌细胞株HepG2、人肺癌细胞株549,观察报告基因红色荧光蛋白能否在肝细胞中表达;RT-PCR检测HSV-tk基因的表达情况;观察给予不同浓度的更昔洛韦(GCV)后随时间延长细胞改变情况,用MTT法检测GCV对不同细胞的杀伤作用。结果化学合成得到34个半乳糖基的PLL;转染后48 h在HepG2可见红色荧光蛋白表达,A549细胞未见红色荧光蛋白表达;RT-PCR证实HSV-tk仅在HepG2细胞中表达;体外杀伤实验显示:经G lanPLL-r-pAs16D r基因转移系统处理的两种细胞对GCV表现出不同的敏感性。HepG2细胞对GCV很敏感,低浓度的GCV(1 mg/L)处理3 d,即可使细胞生长抑制率达到70.5%,A549对GCV不敏感。结论半乳糖基多聚赖氨酸能够与ASGPR有效的结合,合成配体来源丰富、制备简单,适合于作为自杀基因HSV-tk肝靶向运送的导向配体。Objective To explore a new molecular target for HSV-tk/GCV system in human liver cancer therapy, Methods The lactose anti poly-L-lysine covalently linked compound （Lac-PLL） were prepared hy using reductive amination methods anti purified by using Sephadex G10 gel filtration, The value of n was determined by methods of phenol-vitriol colorimetry. The plasmid r-pAs16Dr was mixed with the conjugate tu torm a gene delivery complex named GlanPLL-r-pAs16Dr. The GlauPLL-r-pAs16Dr was transformed to different cell lines such as HepG2 and A549 to confirm the expression of RFP. The expression of HSV-tk was confirmed hy RT-PCR, Cells with various concentratiuns of GCV were observed at differeut time points using MTT. Results The PLL modified by 34 Lac was uhtained hy using chemical synthesis. The RFP was expressed in HcpG2 hy 48h after transfeetiou, aud was not expressed in A549. The expression of HSV-tk was only detected in HepG2 usiug RT-PCR. The HepG2 transfurmed with GlanPLL-r-pAs16Dr was sensitive tu GCV and the growth inhihiting rate was 70.5% with the treatment of low concentration of GCV （ 1 mg/L） fur 3 days. The A549 was not sensitive to GCV. Conclusion Lac-PLL, which is easy tu prepare, is an efficient carrier for HSV-tk to he delivered to hepatoma cell lines by hinding to ASGPR.

关 键 词：半乳糖 聚赖氨酸/类似物和衍生物 单纯疱疹病毒属 胸苷激酶 基因疗法 肝肿瘤 

分 类 号：R735.7[医药卫生—肿瘤]