| Objective:To isolate and purify phospholipase A2(PLA2) from Guangxi cobra venom, and studied their physical and chemical properties,and then to study its effects on human Hepatocellular carcinoma cell line.Methods:(1) The component I which has phospholipase A2activity was isolated from Guangxi cobra venom using gel filtration chromatography and ion exchange chromatography. To identify its purity and molecular weight, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)、High Performance Liquid Chromatography (HPLC) and MALDI-TOF were used.(2) The automatic potentiometric titration method was usede to study the optimum temperature、optimum pH and thermostability of DEAE Ⅰ.And to investigate the acute toxicity of DEAE I in Kunming mice, experiment about experimental animals with acute toxicity was used.(3) The growth of of human hepatocyte cell line (HL-7702) and Hepatocellular carcinoma cell line (SMMC-7721) after treating different times by DEAE I was detected by MTT method,and then the morphology of the cells were observed under an inverted phase contrast microscope.(4) Using scanning electron microscopy (SEM), the morphologic changes of cells that had treated with DEAE I by different time were observed.(5) To study the mechanism of anti-tumor of PLA2, the cell apoptosis and cell cycle by Flow Cytometry (FCM) were detected, using different dyeing method such as Annexin V/PI double dyeing method and PI dyeing method.Results:(1) By gel filtration chromatography and ion exchange chromatography method, the component I (DEAE I)which had PLA2activity was detected,and specific activity of DEAE I was66.12μmol/(mg*min) by automatic potentiometric titration method.One electrophoretic band whose molecular weight was about14.4KD was detected by SDS-PAGE.Moreover,one peak which almost symmetry was detected using HPLC-TSK2000gel filtration chromatography.Furthermore,the molecular weight of DEAE I which was13282.02Da, was identified by MALTI-TOF.(2) Through automatic potentiometric titration method,we got optimum temperature of DEAE I was about50℃,the optimum pH was about6.0,and the thermostability was excellent. By the acute toxicity test, the lethal dose of50%(LD50) of Kunming mice was13.86±0.057mg/Kg, suggesting that this DEAE I had lower toxicity to Kunming mice.(3) After screening from the dose-dependent and the timing dependency tests,using HL-7702and SMMC-7721cells,the optiumconcentration of DEAE I was200μg/ml,and the optimum time was48h. After treating the cells with DEAE I (200μg/ml), inhibition ratio of HL-7702was66.34±12.62%;but for SMMC-7721, the inhibition ratio was66.34±12.62%.Statistical analysis showed that there was not statistically significant difference between the DEAE I (200μg/ml) and CDDP (10μg/ml)(p>0.05);but compared with cabro venom (3μg/ml)、G50I (40μg/ml) and CM I (80μg/ml), there were statistically significant differences (p<0.05).What’s more,compared the inhibition ratio of SMMC-7721to HL-7702,the inhibition ratio of SMMC-7721was higher (p<0.05).(4) The result of SEM show that:After treating the cells for24h by DEAE I,both SMMC-7721had HL-7702had some apoptotic body shape structure which contained nuclear and cell debris,and they had little death ratio; but after treating48h, for the two cells,the apoptotic body shape structures were increasing obviously,but HL-7702had more necrosis than SMMC-7721.(5) FCM results showed that there were not obvious of the phenomenon of apoptosis after treating24h by DEAE Ⅰ;But prolonging the effect time to48h, the SMMC-7721was induced the apoptosis,and the apoptosis rate was33.03%; but for HL-7702,it was only5.62%,and there were lots of debris.However, after treating48h by CDDP,the apoptosis rate were41.92%and87.37%.(6) Results of the cell cycle showed that, after treating the cells for24h by DEAE Ⅰ,the rate of S phase of HL-7702was increasing,and the same time the others were declined;but for SMMC-7721,the G0/G1phase was increasing.However, after treating the cells for24h by CDDP,there were apoptosis peak on both of the two cells.But after treating48h, the DEAE I could make the G0/G1phase incresing in both of the two cells;What’s more,the CDDP could make the HL-7702necrosis.Conclusion:(1) The DEAE I isolated from Guangxi Cabro venom had the activity of PLA2,and through theSDS-PAGE and MALDI-TOF,we got its molecular weight was detected as13282.02Da.(2) Through automatic potentiometric titration method,the optimum temperature was about50℃,the optimum pH was about6.0,and the thermostability was excellent. By the acute toxicity test, the LD50of Kunming mice was calculated as13.86±0.057mg/Kg suggesting that this PLA2had lower toxicity to Kunming mice.(3) MTT results showed that the DEAE I (200μg/ml) had the same effect with CDDP (10μg/ml).Compared the inhibition of the SMMC-7721with that of HL-7702after treating for48h by DEAE I, inhibition of the SMMC-7721was stronger than HL-7702.(4) The SEM results showed that the DEAE I could induce the SMMC-7721to form the apoptotic body shape structure, and it suggested that the PLA2could induce the apoptosis. But for HL-7702there was no obvious apoptosis.(5) FCM results showed that DEAE I was not only induced the apoptosis but also increased G0/G1phase in the cell cycle after treating the SMMC-7721and HL-7702for48h.Moreover,the apoptosis in SMMC-7721was stronger than that in HL-7702. |
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