半乳糖基多聚赖氨酸携带HSV1-TK对HepG2细胞体外效应研究

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枉分离纯化；以苯酚-硫酸比色法测定n值；与真核表达质粒r-pAs16Dr按比例混匀，得到GlanPLL-r-pAs16Dr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HepC2、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48h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GlanPLL-r-pAs16Dr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（1mg／L）处理3d．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。

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

Objective To explore a new molecular target for HSV-tk/GCV system in human liver cancer therapy.Methods The lactose and poly-L-lysine covalently linked compound(Lac-PLL) were prepared by using reductive amination methods and 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 to form a gene delivery complex named GlanPLL-r-pAs16Dr.The GlanPLL-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 by RT-PCR.Cells with various concentrations of GCV were observed at different time points using MTT.Results The PLL modified by 34 Lac was obtained by using chemical synthesis.The RFP was expressed in HepG2 by 48h after transfection,and was not expressed in A549.The expression of HSV-tk was only detected in HepG2 using RT-PCR.The HepG2 transformed with GlanPLL-r-pAs16Dr was sensitive to GCV and the growth inhibiting rate was 70.5% with the treatment of low concentration of GCV(1mg/L) for 3 days.The A549 was not sensitive to GCV.Conclusion Lac-PLL,which is easy to prepare,is an efficient carrier for HSV-tk to be delivered to hepatoma cell lines by binding to ASGPR.