| Objective:To observe the proliferation and migration ability of human liver cancer cells HepG2 influenced by different final concentration of morphine.To investigate the related mechanism involving protein kinase C（PKC）signaling pathway influenced the proliferation of Hep G2 cells by morphine.Methods:CCK-8 assay and Transwell assay were used to detect the proliferation and migration on the human liver cancer cells HepG2 influenced by morphine.These cells were cultivated in a period of logarithmic phase in25cm2 culture bottles randomly.The liver cells Hep G2 were maintained as a monolayer culture in Dulbecco’s modified Eagle medium（DMEM）supplemented with 10%heat-inactivated fetal bovine serum（FBS）,streptomycin（100μg/ml）and penicillin（100 U/ml）.These cells incubated by different final concentrations morphine（0μmol/L,0.1μmol/L,1μmol/L,10μmol/L,100μmol/L;named C1,M1,M2,M3,M4）,added isopyknic cell culture fluid with 10%fetal bovine serum,and ench group included parallel holes.Counted 2×103/ml cells which were incubated in 96-well culture plates,then the proliferation activity of cells incubated for 24 hours was evaluated by CCK-8assay;Counted 5×104/ml cells which were incubated in 24-well culture plates with these wells of cells incubated by morphine respectively for 24 hours,then the migration activity of cells was detected by Transwell assay;Counted3×106/ml cells which were incubated in 6-well culture plates were incubated respectively for 24 hours,the gene expression of PKC mRNA in HepG2 cells was determined by q RT-PCR and the expression of protein p-PKC was detected by western bloting.The liver cancer cells HepG2 were treated with final concentration of10μmol/L morphine（M）,10μmol/L morphine and 100nmol/L naloxone（M+N）,10μmol/L morphine and 300nmol/L PKA（M+P）,10μmol/L morphine and 25nmol/L Ly317615（M+L）,cells were added isopyknic cell culture fluid to Group C,and ench group included parallel holes.The proliferation activity of cells Hep G2 incubated for 24 hours was evaluated by CCK-8 assay;Counted3×106/ml cells which were incubated in 6-well culture plates for 24 hours,and the expression of protein p-PKC was detected by western blotting.Results:Different final concentrations of Morphine were incubated Hep G2cells for 24 hours,CCK-8 assay showed the absorbance(A490)of C1,M1,M2,M3and M4 were（0.82±0.08）（0.76±0.09）（0.71±0.10）（0.65±0.06）（0.60±0.07）,which showed that morphine inhibit the proliferation of the Hep G2 cells;Transwell experiment displayed the cells migration of C1,M1,M2,M3 and M4were（83.65±5.88）（70.36±2.09）（69.72±3.10）（55.65±1.06）（51.65±2.07）,andthis experient showedmorphine with finalconcentrations1μmol/L,10μmol/L and 100μmol/L inhibited cells HepG2 migration;RT-q PCR showed that the relative expression of PKC mRNA of C1,M1,M2,M3 and M4were（1.61±0.20）（1.53±0.19）（1.36±0.24）（0.95±0.12）（0.90±0.08）,and that Western blotting showed that the expression of protein p-PKC were（0.81±0.10）（0.73±0.09）（0.68±0.12）（0.41±0.06）（0.33±0.07）,the result of qRT-PCR and western bolting showed that the PKC mRNA and protein p-PKC expression of Group M3 and Group M4 were relatively lower than Group C1.Group C,Group M,Group M+N,Group M+P and Group M+L were detected in HepG2 cells for 24 hours,CCK-8 assay showed each group of cells proliferation,the 24-hour absorbance(A490)were（0.83±0.51）（0.46±0.09）（0.79±0.19）（0.81±0.36）（0.43±0.17）,compared with the Group C,the absorbance(A490)of HepG2 cells in Group M and the Group M+L were decreased,while naloxone or phorbol esters,Compared with group M,Group M+N and GroupM+P were increased;HepG2 cells treated with Group C,M,M+N,M+P and M+L,Western blotting showed that the relative expression of protein p-PKC were（0.63±0.51）（0.32±0.09）（0.59±0.19）（0.65±0.36）（0.28±0.07）,compared with Group C,the p-PKC protein was decreased in Group M and GroupM+L,but compared with group M,protein p-PKC in Group M+N and Group M+P increased.Conclusions:Morphine can inhibit the proliferation and migration ability of human liver cancer Hep G2 cells,morphine decreased the PKC mRNA expression and p-PKC protein expression of Hep G2 cells,the influence of morphine may be related to protein kinase C（PKC）signaling pathway. |
PDF Full Text Request