| Objective: To explore the effect of His-Agkihpin that is a restructuring snake venom arginine esterase on the cell function of liver cancer cells in vitro,and to study the related genes.To explore the effect of His-Agkihpin on hepatocellular carcinoma in vivo.For find the related pharmacological effects of His-Agkihpin on anti-hepatoma,and provide experimental basis for it.Methods: 1.Cultured liver cancer cells SMMC-7721,HepG2 and hepatocyte cells L02,CCL13 in vitro.(1)CCK-8 was used to detected proliferation of liver cancer cells SMMC-7721,HepG2 and hepatocytes LO2,CCL13 on different concentrations(16.52 ? g / ml,11.68 ? g / ml,8.26 ? g / ml,5.84 ?g / ml,4.13?g / ml)of His-Agkihpin after 24,48 and 72 h,to compare the proliferation of different hepatoma cells and hepatocytes.And calculated the IC50 of hepatoma cells.To determine the optimal time and concentration of His-Agkihpin action hepatoma cells.(2)Used microscope to observe the exchange of morphology about liver cancer cells SMMC-7721,Hep G2 andhepatocytes LO2,CCL13 on His-Agkihpin.(3)The effect of apoptosis and cell cycle of liver cancer cells SMMC-7721,Hep G2 and hepatocytes LO2,CCL13 on His-Agkihpin was detected by flow cytometry(FCM).(4)The effect of migration of liver cancer cells SMMC-7721,HepG2 and hepatocytes LO2,CCL13 on His-Agkihpin was tested by Wound Healing Assay and Transwell Migration Experiment.2.The expression level of Ki-67 and Cyclin B1 in liver cancer cells SMMC-7721,HepG2 on His-Agkihpin was assayed by qRT-PCR(Real time fluorescence quantitative-PCR).3.The effect of His-Agkihpin on liver cancer was observed in nude mice with HepG2 cells.Results: 1.The proliferation inhibiting rate of His-Agkihpin on SMMC-7721 and HepG2 is increased,and showed a certain concentration dependence effect,the higher the concentration of His-Agkihpin,the higher the inhibition rate.The concentration of 11.68 ?g/ml His-Agkihpin to SMMC-7721 and HepG2 have obvious inhibition of proliferation(P<0.05).Except 16.52?g/ml His-Agkihpin has obvious inhibitory effect on CCL13,the rest of concentration had no obvious inhibitory effect.All the concentration of His-Agkihpin have different degree effect to promoting L02 proliferation.By calculation,the IC50 of His-Agkihpin on SMMC-7721 and Hep G2 is 8.88?g/ml and 13.62 ?g/ml respectively.2.His-Agkihpin has no obvious morphological changes in SMMC-7721,HepG2,LO2 and CCL13.3.The effect of His-Agkihpin on apoptosis of cell was detected by flow cytometry.The effects of all concentration of His-Agkihpin and NS on L-02 and CCL13 were consistent.11.68 ?g/ml His-Agkihpin promote apoptosis ofSMMC-7721 at a rate of 38.00 %(P<0.05),higher than that of NS group(6.30%).The promote apoptosis rate of His-Agkihpin on HepG2 was 15.10 %(P< 0.05),higher than NS group(1.60%),and the other concentration of His-Agkihpin had no obvious effect on SMMC-7721 and Hep G2.4.The effect of His-Agkihpin on Cycling of cell was detected by flow cytometry.After the His-Agkihpin on SMMC-7721 and Hep G2,the main cell Cycle arrest were in G2 phase,in which 11.68 ?g / ml of His-Agkihpin was the highest,stand up for 45.7%(P<0.05);The block rate of G2 phase of Hep G2 also reached 45.9 %(P<0.05).The effect of all concentration His-Agkihpin on the cycle arrest of L-02 and CCL13 did not change significantly.5.The effect of His-Agkihpin on the migration ability of SMMC-7721 and HepG2 was tested by Wound Healing Assay.The scratch healing rate of NS on HepG2 after 24 ~ 48 h was 16.79 % ~ 16.99 %,on SMMC-7721 was20.67 %~25.05%;And the scratch healing rate of His-Agkihpin on HepG2 and SMMC-7721 after 24~48 h was 0.65~1.54% and 0.74~8.36% respectively.6.The effect of His-Agkihpin on the migration ability of SMMC-7721 and HepG2 was tested by Transwell Migration Experiment.The number of SMMC-7721,HepG2 on His-Agkihpin through polycarbonate membrane was less than NS(P<0.05).7.The changes of mRNA of Ki-67 and cyclin B1 in SMMC-7721 and HepG2 on His-Agkihpin were detected by q RT-PCR.The results showed that the relative transcription level of Ki-67 mRNA in SMMC-7721 and HepG2decreased(P<0.05),and no significant change in cyclin B1 mRNA.8.Using Hep G2 to model human liver cancer model in nude mice,to observe the growth and weight of tumor in nude mice on His-Agkihpin,5-Fu and NS.The results showed that the growth curve,volume and weight of tumor onHis-Agkihpin were under than NS group and 5-Fu group significantly.Conclusion:1.11.68 ? g / ml His-Agkihpin can inhibited the proliferation and promoting apoptosis of HepG2 and SMMC-7721,and block cells in G2 phase,and had no similar effect on L-02 and CCL13.To inhibit the migration of HepG2 and SMMC-7721.The morphology of HepG2,smmc-7721 and L-02 and CCL13 were not changed.2.11.68 ?g / ml of His-Agkihpin can reduced the mRNA level of Ki-67 in SMMC-7721 and HepG2 obviously,but Cyclin B1 slightly.3.11.68 ?g / ml(11.68mg/kg body weight)of the His-Agkihpin may inhibit the growth of tumor in nude mice bearing HepG2,is expected to be as anti-liver cancer drug in the future. |
PDF Full Text Request