Effects Of The PKC-α Antisense Oligodeoxynuleotides Mediated By Polyethylenimine On SMMC-7721 Cell Lines

Objective:To design and chemical syntheses antisense oligodeoxynuleotides(ASODN) of pkc-α, to investigate the biological effect of ASODN mediated by polyethylenimine on hepatocelluar carcinoma SMMC-7721 cells,And to detect it's mechanisms on hepatocelluar carcinoma cells in vitro.Methods:1.The proliferating inhibition of PEI-ASODN and the cytotoxic effects of PEI on SMMC-7721 cell lines:the expression of pkc-αmRNA in cell line of L02,HepG₂ and SMMC-7721 detected by RT-PCR;the proliferating inhibitive effects(also IC₅₀) of PEI-ASODN were separately tested by WST-8 test at various concentrations and by try pan blue dye at continuous 24h points.2.Observe the mechanism of pkc-αASODN delivered by PEI on SMMC-7721:The test of the pkc-αmRNA and protein expression after treated with pkc-α-ASODN by RT-PCR and immunofluorescence;apoptosis tested by Hoechst 33258 staining,PI staining and PI-ANNEXINâ…¤staining;3.The chemotherapeutic drug's sensitivity enhancement on SMMC-7721 cells by PEI-ASODN detected by WST-8 test:SMMC-7721 cells were incubated by 5-fluorouracil(5-FU) or arsenic trioxide(As₂O₃) or hydroxyicamptothecine(HCPT) and their combination of PEI-ASODN at different concentrations for 48 hours respectively,then the Cell proliferation was detected by WST-8 test,finally their IC₅₀and their drug-sensitivity were calculated statistically. Results:1.On SMMC-7721 cell line,the expression pkc-a mRNA is relatively highly than L02;the best quality mixing ratio of PEI and pkc-αASODN was between0.75:1 to 1:1;both in different concentrations and different time,the growth of liver cancer cells treated with PEI-ASODN have been inhibited.2.PEI-ASODN can significantly inhibit the SMMC-7721 pkc-αmRNA and protein expression in high concentration;PEI-ASODN promote SMMC-7721 cell lines to apoptosis3.Compared with 5-FU,As₂O₃ and HCPT,The combition of5-FU,As₂O₃,HCPT with PEI-ASODN have enhanced their chemotherapeutic effect on SMMC-7721 cells,and the sensitivity enhance to 4.01,1.84 and 2.86 times respectively.Conlusions:1.It is certificated that PEI is a low-toxicant and high transient vector for ASODN,the best quality mixing ratio of PEI and pkc-a ASODN was between0.75:1 to 1:1(for the molar concentration ratio of 1/5) in vitro.2.pkc-αantisense oligonucleotides mediated by PEI can inhibit the proliferation of SMMC-7721 cells.3.PEI-ASODN can significantly inhibit the SMMC-7721 pkc-αmRNA and its' protein expression.4.PEI-ASODN can significantly promote SMMC-7721 cell lines to apoptosis.5.0.25μmol/L PEI-ASODN can enhance the chemotherapeutic drug's sensitivity of SMMC-7721 cells to 5-FU,As₂O₃ and HCPT.