Pre-clinical Study On Hepatocellular Carcinoma Therapy By Targeted Gene Drug (ASOR-PLL-hIL12)

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.