In Vitro Experimental Study On Hepatocellular Carcinoma With Synergistic Of Hyperthermia And HSV-TK/GCV Suicide Gene Therapy System Using Cationic IONPs As Nonviral Gene Vector

OBJECTIVE:Research on the synergistic therapy effect of Hyperthermia and HSV-TK/GCV suicide gene therapy system on the Hepatocellular Carcinoma(HCC) in vitro,which used Cationic Iron Oxide Nanoparticles(Cationic IONPs) as nonviral vector.METHORDS:Fe₃O₄ magnetic nanoparticles were synthesized by improved coprecipitation method,modification and characterization of surface-ammonium group.The transfection efficiency of Cationic-IONPs was studied by enhanced green fluorescent protein plasmid pEGFP-C1. Cationic-IONPs mediated pcDNA3.1-TK reorganization suicide gene was transferred into human HepG2 cells.The mRNA expression level of TK gene was tested by RT-PCR,resistance cells were screened by G418, HepG2/TK cells were acquired which can stable express TK gene. Trypan blue exclusionmethod was used to estimated the killing effect of GCV to the HepG2/TK cells at different time points.HepG2/TK cells and non transfection HepG2 cells were treated with different concentrations of GCV for 72h,MTT was used to determine the influence of drugs on the proliferation of cells,the bystander effect was observed.HepG2/TK cells and HepG2 cells were divided into five different temperature groups, 38℃,40℃,42℃,44℃,50℃.The Electrical Heating Thermostat water bath were treated for 15 min,30min and 1 hour,MTT was used to test the effects of different temperatures on the Proliferation of the two cell types.A control group,GCV alone treatment group and hyperthermia synergies GCV treatment group was set.Hyperthermia combined with GCV treatment group was cultured at 40℃,42℃and 44℃separately, hyperthermia time was 1 hour.The control group was under routine culture without any processing.GCV was added into the other groups (terminal concentration:0.5μg/ml).The survival rate was studied 72h after exposure by MTT.Flow cytometry was used to investigate the situation of cell apoptosis.RESULTS:1.Cationic IONPs were successfully synthesized,which have well stability and were monodisperse maganetic nanosphers with diameter of 32.1nm.Zeta potential was +13.5mV when measured in the neutral environment(PH=7.0).The gene transfection efficiency by Cationic IONPs was 30.8%,slightly higher than that of positive control group of Lipofectamine^TM2000(28.8%).However,there is not significant difference between them(P＞0.05).2.Cationic-IONPs mediated pcDNA3.1-TK recombinant plasmid was transferred into HepG2 cells.Following the selection by G418 medium, the resistant cells were screened and the HepG2/TK cells with stable expression of TK genes were acquired.Through the observation of trypan blue exclusion method and MTT,we discovered that the killing effect of GCV to HepG2-TK cells were in a time-dependent and dose-dependent manner,and had prominent bystander effect.The introduction of HSV-TK gene obviously enhanced the sensitivity of HepG2 cells to the GCV.Single therapy by subhyperthermia(38-40℃) could not ideally suppress the proliferation of HepG2 cells in vitro;therapy by hyperthermia(42-50℃) alone could inhibit the proliferation of HepG2 cells.3.A control group,GCV alone treatment group and hyperthermia synergies GCV treatment group was set in the experiment.In the single GCV treatment group,the increment inhibition rate was 34.5%and the cell apoptosis rate was 10.2%.The increment inhibition rate of the hyperthermia synergies GCV treatment group was significantly higher than the single GCV treatment group(P＜0.01).As the temperature increases,the effect was more and more obvious.When the temperature reached by 44℃,the increment inhibitions rate arrived at 75.8%and the apoptosis rate was 45.2%by flow cytometry.CONCLUSION:Cationic-IONPs had high transfer efficiency and low toxicity to the cells.It could successfully transfer recombinant plasmid pcDNA3.1-TK into HepG2 cells and was a good cationic conjugated non-viral polymers gene vector.There is a certain extent of killing effect and bystander effect of HSV-TK suicide gene system to the HepG2 cells.Therapy by hyperthermia(42-50℃) alone could inhibit the proliferation of HepG2 cells.Hyperthermia(temperature≥40℃) can significantly enhance the sensitivity of killing effect of GCV to the HepG2/TK cells.