The Relationship Between Insulin Resistance And Drug-resistance In Hepatocarcinoma HepG2 Cells And The Creation And Characteristics Of Transgenic Multi-drug Resistant HepG2 Cell Model

Objectives:To investigate the relationship between the insulin resistance(IR) and drug sensitivity,and to constuct the transgenic multidrug resistant cell hepatocarcinoma model.Thereby set up the corresponding basis for researching the relationship between IR and multidrug resistance(MDR) in the hepatocarcinoma cells.Methods:1 The insulin-resistant HepG2 cell model(HepG2/IR) was induced with high dose of insulin in vitro.The glucose consumption and the maintaining time of IR in HepG2/IR cells were determined by glucose oxidase-peroxidase method;The expression of insulin receptor protein(InsR) and P-glycoprotein(P-gp) were detected by flow cytometry(FCM),and the expression of InsR and mdr1 mRNA by real-time RT-PCR.The sensitivity of the cells to adriamycin(ADM) and the cellular apoptosis were detected with a MTT assay and Annexin V/PI staining,respectively.2 The eucaryotic express plasmid(pCI-neo-mdr1) carrying the human multidrug resistance 1 gene(mdr1) was transfered into the human hepatoma HepG2 cells using liposome,and the human hepatoma multidrug resistant cell Hepg2/mdr1 was selected with G418.The morphology changes of HepG2/mdr1 cell was observed by the inverted phase contrast microscope;the proliferation activity of HepG2/mdr1 was determined with MTT assay.The flow cytometry was employed to detect the P-gp expression,accumulation and retention of intracellular adriamycin in HepG2/mdr1 cells,and the expression of mdrl mRNA in the HepG2/mdr1 using real-time RT-PCR.Results:1 The glucose consumption decreased by 16.61%～41.78%in insulin-induced HepG2/IR cells in a time and concentration-dependent manner,and the insulin rsistance of the cells maintained for up to 48h.Compared with HepG2 cells,the expression of InsR mRNA is decreased by 43.67%,and InsR protein by 69.31.The expression of mdr1 mRNA and P-gp in HepG2/IR cells increased by 4.21 folds and 1.01 folds,respectively.The expression of mdr1/P-gp was negatively related to the glucose consume and InsR expression.The sensitivity of HepG2/IR cells to ADM was 3.2-time lower than that of HepG2 cells,and HepG2/IR cells were resistant to the apoptosis induced by adriamycin,which was 3.5-time lower than HepG2 cells.2 The transgenic multi-drug resistant HepG2/IR cells established in the study growth a little slowly,and the doubling time was about 20h to 22h.Compared with HepG2 cells,HepG2/mdr1 cells possessed no obvious difference in the morphology. The expression of mdr1 mRNA of HepG2/mdr1 cells was 5.6-time higher over the parent HepG2 cells,and the positive rate and expressive amount(MFI) of P-gp expression in HepG2/mdr1 cells increased by 6.7 times and 23.03%,respectively.The retention of adriamycin in HepG2/mdr1 cells was only 61.12%of that in HepG2 cells, and the excavation of intracellular adriamycin of HepG2/mdr1 cells was 192.48%of that in HepG2 cells.These results suggested mdr1 gene be successfully transducted into HepG2 cells and effectively expressed,and the transgenic HepG2/mdr1 cell model possessed the property of multidrug resistance had been successfully constucted.Conclutions:1 The insulin-resistant HepG2 cell model(HepG2/IR) was successfully induced and constucted with high dose of insulin in vitro.2 The expression of InsR mRNA and receptor protein decreases significantly,and the expression of mdr1 mRNA and P-gp increases markedly.The sensitivity of HepG2/IR cells to adriamycin decreases,and the drug resistance enhanced.It is suggested that the insulin resistance is related to the multidrug resistance in human hepatocarcinoma cells.3 The multidrug resistant cell model of HepG2 cells,which effectively expresses mdr1/P-gp,constucted by transgenic techniques.The work provides a perfect model for the further studying the relationship between insulin resistance and multi-drug resistance in hepatocarcinoma cells.