Application effects of electroporation and lipofection in HCV RNA transfecting human hepatoma cells

LU Sha; TAN Wenjie; ZHANG Ling; DENG Yao; TAO Gesi; CAI Min; LI Lian; SHEN Xiaoling

doi:10.3969/j.issn.1002-266X.2016.19.005

Abstract：Objective To observe the application effects of electroporation and lipofection in hepatitis C virus ( HCV) RNA transfecting human hepatoma cells .Methods Human hepatoma cells ( Huh7.5-CD81) were cultured and divided in-to groups A, B and control group.Cells in the group A were transfected with HCV RNA of pHebei (E1E2)/JFH1 by elec-troporation, cells in the group B were transfected with HCV RNA of pHebei (E1E2)/JFH1 by lipofection, and cells in the control group were transfected with HCV RNA of replicating defective pJFH 1/GND.Cell morphology was observed using an optical microscope in each group after transfection .Immunofluorescence was employed to detect the transfectional efficien-cy.RNA copies in the supernatant were tested by real-time PCR.TCID50 was applied to detect HCV infectious titer .Re-sults The number of cells used in the group A were 1 ×106 , and more than half were damaged with rough edges one day after transfection;the number of cells in the group B were 2 ×105 and almost no damage was found one day after transfec-tion.Transfectional efficiency of group A was 9.98%±0.83%, and that was 2.58%±0.39%in the group B, P<0.01. Fluorescence positive cells in the control group were not detected .Meantime, HCV RNA copy number in the supernatant of groups A and B first decreased , then increased and eventually reached at 106 copies/mL.HCV infection titer was detected successfully at 21days after transfection in the group A and at 31days in the group B.The highest infectious titer of groups A and B was both 104 ffu/mL.Conclusions Electroporation and lipofection both can be used to establish HCV cell culture system.Lipofection displays less damage to cells .Although electroporation demonstrates higher transfectional efficiency and short time of obtaining HCVcc , but there is no difference in infectious titer of obtained HCVcc .