肝癌细胞靶向基因药物分子构形与表达量关系的体外研究

Relationship between the expression quantity and molecular conformations of gene drugs targeted at hepatoma cells in vitro

目的 通过调整靶向基因药物分子构形来提高外源基因的表达量 ,以解决外源基因在靶细胞中表达效率低的难题。方法 应用PCR方法扩增出人白介素 1 2P4 0 、P35亚基的cDNA全长并克隆至真核表达载体pcDNA3 .1 (± )中 ,构建P(+) /IL 1 2、P(+) /P4 0 和P(- ) /P35三种质粒 ,分别与脱唾液酸黏蛋白 多聚左旋赖氨酸 (ASOR PLL)结合成两种靶向基因药物 [ASOR PLL P(+) /IL 1 2和ASOR PLL P(+) /P4 0 、ASOR PLL P(- ) /P35] ,透射电镜观察它们在不同辅料浓度中的分子构形 ;不同构形药物分子转染HepG2细胞 ,48h后用半定量RT PCR和ELISA法检测hIL 1 2的表达量。 结果 靶向基因药物分子构形为直径 2 5～ 1 50nm的颗粒状或圆环状时 ,hIL 1 2表达量最高 ;ASOR PLL P(+) /P4 0 、ASOR PLL P(- ) /P35共转染者较ASOR PLL P(+) /IL 1 2转染者hIL 1 2表达量普遍要高。结论 靶向基因药物分子构形对外源基因的表达有重要影响 ,以直径 2 5～ 1 50nm的颗粒状或圆环状构形为最佳分子构形 。

Objective To increase exogenous gene expression quantity by modulating molecular conformations of targeting gene drugs to solving the problem of their low expression efficiency in targeting cells. Methods The full length of cDNAs of both P 40 and P 35 subunits was amplified through polymerase chain reaction and cloned into embryonic expressing vectors pcDNA3.1(±) to construct plasmids of P(+)/IL 12, P(+)/P 40 and P(-)/P 35 , and then these plasmids were combined respectively with asialoorosomucoid poly L lysine (ASOR PLL) to formed two targeting gene drugs[ASOR PLL P(+)/IL 12 and ASOR PLL P(+)/P 40 , ASOR PLL P(-)/P 35 ] in optimal ratios. All the conformations of two drugs in various adjuvant concentrations were observed under electron microscope(EM) and transfected into HepG2. In order to detect the expression quantity of hIL12, the total RNA of cells was extracted to do semi quantitative reverse transcription polymerase chain reaction(RT PCR) while the cultured supernatant was conducted by enzyme linked immunoabsorbent assay(ELISA) 48 hours after the drugs were transfected into HepG2. Results Targeting gene drugs got the highest hIL 12 expression quantity when their structures were granular and circle like, and their diameters ranged from 25 nm to 150 nm; The hIL 12 expression quantity in co transfecting group of both ASOR PLL P(+)/P 40 and ASOR PLL P(-)/P 35 was commonly higher than that of ASOR PLL P(+)/IL 12 transfecting group. Conclusions The molecular conformations of targeting gene drugs play an important role in exogenous gene expression, among which the best structures are granular and circle like and diameters range from 25 nm to 150 nm. The sizes and link styles of exogenous genes also have some effects on their expression quantity.