Purification Of Natrin From Guangxi Cobra Venom And The Study Of Anti-Tumor Function

Objective:In order to increase the yield of Natrin and purify it, chromatography was used to purify Natrin from Guangxi Cobra Venom,which was subsequently investigated about its growth inhibitory function on human tumour cells.Methods:(1) The crude snake venom was separated and purified by gel filtration on a Sephadex G-75colum and CM-Sepharose CL-6B cation-exchange column, and the Mono S column chromatography on FPLC system was also used, then the relative molecular mass of the obtained Natrin was identified through sodium-dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).(2) The cytotoxicity and the growth inhibition ratio of four kinds of human tumor cell lines, BEL-7721、HEPG2. SKOV3、 SIHAwere treated with Natrin at different final concentrations of0.625,1.25,12.5,125,250μg/ml for24h were assayed by MTT colorimetric method.(3) The function and morphology of SKOV3cells treated with Natrin at different concentrations for24h,and then was observed by Electron microscope.(4)The function and morphology of SKOV3cells treated with Natrin at different concentrations for24h were observed by acridine orange/ethidium bromide (AO/EB) double staining.(5) The rate of apoptosis of SKOV3cells treated with Natrin at different concentrations for24h was detected by flow cytometer (FCM) which doublely tagged by Annexin V-FITC/PI.Results:(1) Natrin was purified successfully from Guangxi Cobra Venom by chromatography. The relative molecular mass of the product was determined to be25Ku by SDS-PAGE, and to be24.937Ku by MS. And its N-terminal sequence was NVDFNSESTRRKKKQKEIVD by Measured.(2) Natrin had obvious cytotoxicity on these cell lines, which shows to be dose-dependent, especially for SKOV3,its IC50value at the24th hours is1.47μg/ml. The main features of apoptosis of SKOV3under electron microscope were Nuclear chromatin the solid shrinkage, edge set, folds and nuclear membrane cytoplasm and tight, electronic density increases, and apoptotic body phenomenon, and the SKOV3under fluorescent microscope include reduction in volume,nuclear chromatin condensation, intensification under jacinth fluorescence. The apoptosis rate of SKOV3which detected by FCM increases within a certain dose increase with increasing concentration of Natrin,but after reaching the maximum,the apoptosis rate decreases with the Natrin increased.Furthermore,the early apoptosis rate of each concentration significantly differ from the control(P<0.05).