【作者】 羊东晔；

【导师】 赵水平； 卢放根；

【作者基本信息】 中南大学 ， 内科学， 2003， 博士

【摘要】 目的：为有效地进行肝癌基因治疗，利用靶向治疗载体脱唾液酸粘蛋白-左旋多聚赖氨酸（ASOR-PLL）将外源基因人白细胞介素12（hIL12）转入肝癌细胞株HepG2中，以了解hIL12的表达情况及影响因素，初步探索解决靶向基因药物表达效率低的途径。 方法：先构建pcDNA3.1（+）/P40、pcDNA3.1（-）/P35和双亚基共表达的pcDNA3.1（+）/IL12三种质粒，从基因转染角度确定载体ASOR-PLL的靶向性后，三种质粒与之结合组成两种靶向基因药物[ASOR-PLL-P（+）/IL12和ASOR-PLL-P（+）/P₄₀、ASOR-PLL-P（-）/P₃₅]，以不同的转染体系、方法转染HepG2，并在不同的表达时间检测hIL12蛋白质，以确定最佳转染过程及表达时间。透射电镜观察靶向基因药物在不同辅料浓度中的分子构形。以不同构形靶向基因药物转染HepG2。根据表达量不同，用半定量RT-PCR、ELISA、Western blot方法筛选得到利于表达的最佳分子构形。 结果：1．治疗载体ASOR-PLL对ASGr+细胞有靶向性；2．pcDNA3.1（+）/P40、pcDNA3.1（-）/P35和双亚基共表达的pcDNA3.1（+）/IL12三种质粒均可转入HepG2细胞并表达hIL12蛋白质分子，但脂质体转染和ASOR-PLL转染结果不同；3．在不同辅料浓度中两种靶向基因药物分子构形不同，不同构形药物分子转染后表达水平不一，直径在25～150nm的颗粒状或圆环状表达量最高。 结论：1．治疗载体ASOR-PLL与双亚基共表达的pcDNA3.1（+）/IL12质粒是本研究制备的一种治疗肝癌的靶向基因药物；2．靶向基因药物分子构形对外源基因的表达有重要影响；3．外源基因的片段大小及连接方式对其表达也有一定影响。更多还原

【Abstract】 Objective: To fulfill effectively gene therapy of hepatocellular carcinoma (HCC), human interleukin-12 gene was transferred targeting into hepatoma cell line HepG2 (ASGr+) via ASOR-PLL and was investigated the impactive factors of its expression to explore a way for solving the problem of low expression quantity of gene drug targeting to hepatoma cell.Methods: Constructing plasmids of pcDNA3.1(+)/P40, pcDNA3.1(-)/P35 and double-subunit co-expression plasmid of pcDNA3.1(+)/IL12, then combining them respectively with ASOR-PLL in optimal ratios to form two targeting gene drugs [ASOR-PLL-P(+)/IL12 and ASOR-PLL-P(+)/P40, ASOR-PLL-P(-)/P3,5]; These two drugs were transfected into HepG2 in various conditions and detected the hIL12 protein in different time after ASOR-PLL was testified its character of targeting to ASGr positive (ASGr+) cells in order to confirm the best transfecting condition and expressing time. A transmission electron microscope (EM) was chosen to observe the molecular conformations of two drugs in various adjuvant concentrations and used the methods as following semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunoabsorbent assay (ELISA) and Western blot to detect the expression quantity of ML 12 48 hours after their transfected into HepG2, based on the results the best molecular conformations of these two drugs were selected.Results: 1. ASOR-PLL could transfected pEGFP-N1 targeting to, cells which possessed asialoglycoprotein receptor; 2. The three plasmids could express functional hIL12 protein after transfected into HepG2, but there was difference between the liposome group and ASOR-PLL group; 3. Target gene drugs showed different molecular conformations in various adjuvant concentrations and expressed different levels of hIL12 quantities, the best ones are granular and circle-like and the optimal diameters ranged from 25nm to 150nm.Conclusions: 1. The complex, which target transfecting vector ASOR-PLL combined with double-subunit co-expression plasmid of pcDNA3.1(+)/IL12 electrostaticly, was the right gene drug targeting to HCC in this study; 2. The molecular conformations of targeting gene drugs played an important role in exogenous gene expression; 3. The sizes and link styles of exogenous genes also had some effects on their expression quantities.更多还原