Inducing GPI-PLD Gene Expression In Hepatoma Cell Line HepG2 By Insulin And Analysis Of Its Immunological Effect

Objective To investigate the effect of insulin on the genic expression of mRNA of GPI-PLD and its activity level as well as the change to release CEA anchored by GPI-PLD and to test whether the effect can increase susceptibility of HepG2 cells and transfected HepG2 cells by lymphokine-actived killer (LAK) cell-mediated lysis.Methods The GPI-PLD gene eukaryon expression vector pcDNA3.1(+)/GPI-PLD produced by our laboratory was transfected into HepG2 cells under the cationic polymer mediation, after G418 sieve, we got GPI-PLD stable expression cell line. RT-PCR was used to detect the expression level of mRNA. HepG2 cells and transfected HepG2 cells were induced by insulin for 48h. The expression level of GPI-PLD mRNAs and its activities before and after treating with insulin were detected. The CEA released from cells membrane before and after treating with insulin were detected by ELISA. GPI-anchored CEA on cells before and after treating with insulin were analyzed by flow cytometer. Peripheral blood mononuclear cells from healthy adults were activated with interleukin-2 (IL-2). The morphologic changes of LAK cells and HepG2 cells or transfected HepG2 cells were observed continuously under inverted microscope after 12h of co-incubation of LAK cells with one of these target cells respectively at a LAK:HepG2 ratios of 20:1. Cytotoxic activity of LAK cells against the HepG2 cells and transfected HepG2 cells before and after treating with insulin were measured by MTT assay.Results RT-PCR analysis and GPI-PLD activities showed that the HepG2 cell line with overexpression of GPI-PLD gene were established. After treatment with insulin (10-6 mol/L) for 48h,1. the cellular GPI-PLD mRNA level in HepG2 cells was significantly increased;2. the cellular GPI-PLD activity in HepG2 cells was significantly increased; 3. the CEA released from HepG2 cells membrane was increased and GPI-anchored CEA on HepG2 cells membrane was decreased. GPI-anchored CEA on transfected HepG2 cells membrane was decreased, too. In comparison with HepG2 cells, transfected HepG2 cells,HepG2 cells and transfected HepG2 cells were induced by insulin showed greater number of cells lysed by LAK cells. In MTT colorimetry assay, cytotoxic activity of LAK cells against the HepG2 cells and transfected HepG2 cells after treating with insulin were enhanced.Conclusion1. The stable GPI-PLD overexpression HepG2 cell line is established.2. GPI-PLD overexpression of HepG2 cells by insulin is able to induce the release of GPI-anchored CEA and therefore enhance susceptibility of HepG2 cells by lymphokine-actived killer (LAK) cell-mediated lysis.