The Cobra Venom Nerve Growth Factor Induced Apoptosisi In Hepatic Stellate Cells Differences In Protein Expression And Validation

Purpose:Isolated from the venom nerve with bioactivity from cobra venom (Nerve growth growth factor factor, NGF), analysis of snake venom NGF on hepatic stellate cells (Hepatic stellate cells, HSC) apoptosis, proliferation and protein expression, mechanism of anti fibrosis effect for snake venom NGF from cells and protein group level learning.Method:Specific as follows according to the grouping experiment method to design different experimental purposes:(1) The MTT grouping of adherent cells in the culture medium:pure culture method as the blank control group, experimental intervention in cobra venom nerve growth factor (intervention under concentration were1,2,5,10,20μg/ml) method as the experimental group(2) Flow cytometry experiment groups:medium simple training method as the blank control group, experimental intervention in cobra venom nerve growth factor (intervention under concentration were2,5,10μg/ml) method as the experimental groups(3) Electrophoresis experiment groups:the medium simple training method as the blank control group, experimental intervention in cobra venom nerve growth factor (intervention under concentration were2,5,10μg/ml) method as the experimental groupsThe experimental method:(1) The cobra venom NGF were isolated and purified by Shephadex G-75gel column and CM Sepharose gel column two step CL-6B ion chromatography;(2) Each elution peak for the determination of biological activity of PC12cells, NGF activity eluting peak purity and relative molecular mass and SDS-PAGE electrophoresis identification;(3) Influence the detection venom the inhibition rate of NGF on the proliferation of HSC-T6cells by MTT method;(4) The effects of snake venom NGF flow cytometry on apoptosis of HSC-T6cells;(5) Using the differential expression proteins of HSC-T6cells before and after two-dimensional gel electrophoresis of snake venom NGF interventions;(6) The expression of matrix assisted laser desorption ionization time of flight mass spectrometry were identified in1.8times and more protein spots on two-dimensional difference gel electrophoresis;(7) Using bioinformatics methods for identification of proteins with differential expression analysis of protein-protein interaction, GO classification and STRING network;(8) Western blot on the differential protein spots were verified.Result:(1) Glasses crude venom by Shephadex G-75gel column separation has3peaks, second peaks with NGF activity with peak; NGF activity of the second peaks after CM Sepharose CL-6B ion gel column and separated into5peaks;(2) The peak effect on PC12cells, found the III peaks have obvious nerve growth factor activity by SDS-PAGE method, neural venom pure electrophoresis to separate growth factor detection, determination of molecular weight of22.3KD;(3) MTT showed that the proliferation of snake venom nerve growth factor on HSC-T6cells significantly inhibited:the concentration of NGF inhibited lug/mL was (25.1±2.1)%, the concentration of inhibition of5ug/mL was (48.2±1.9)%, significantly higher than that of control group (23.8±1.8inhibition rate)%, the difference was statistically significant (P<0.05);(4) At the flow cytometry experiments, through the detection of venom growth factor can induce apoptosis in HSC-T6cells:apoptosis in2ug/mL was (16.16±3.02)%at5ug/mL, apoptosis rate was (22.15±3.31)%, apoptosis in lOug/mL was (31.28±2.16)%, significantly higher than that of control group (4.36±1.85) apoptosis rate%, the difference was statistically significant (P<0.05);(5) In the2-DE results2-DE experiments, intervention group and control group differences in protein expression of a total of47, a22growth factor intervention in nerve group, down another25expression, some differential protein spots with the concentration of NGF showed significant dose-dependent;(6) Analysis of peptide mass fingerprint on the expressions of18protein spots with more than1.8times difference by mass spectrometry,13proteins were identified.(7) By bioinformatics analysis found that the main physiological role of these proteins is involved in cell proliferation, the biological role and oxidative metabolism process;(8) Western Blot experimental results show high expression, growth factor on TGF-β protein in nervous conditions and, its expression decreased with the increase of nerve growth factor concentration; at the same time, the expression of RHOA protein in nerve growth factor intervention conditions is inhibited, and with the increase of the concentration of nerve growth factor gradually increasingConclusion:(1) With biological activity of nerve growth factor can be isolated and purified from Cobra venom;(2) Venom nerve growth factor on the proliferation of HSC-T6cells to inhibit;(3) Can promote apoptosis of snake venom nerve growth factor on HSC-T6cells;(4) Venom nerve growth factor is the main reason to make the protein differential expression of HSC-T6cells;(5) The expression of protein should be the key factors in the process of liver fibrosis, these proteins may regulate the signal transduction pathways involved in the biological to achieve the purpose of anti liver fibrosis.