Studies On AHVAC-Ⅰ Inhibiting The Cancer Cells Migration Function Via GCAFs

Objective:The lyophilized powder of Agkistrodon halys venom was selected from southern Anhui,and the antitumor component-I（Agkistrodon Halys venom antitumor component-I,AHVAC-I）was isolated and purified to isolate and purify human primary gastric cancer associated fibroblasts（GCAFs）.),to explore the molecular mechanism of AHVAC-I inhibiting the migration of gastric cancer cells by acting on human primary gastric cancer-related fibroblasts.Methods:1)Isolation and culture the primary GCAFs from gastric cancer by tissue block culture method and trypsin digestion method,which were the basis for subsequent studies.2)Cell viability was evaluated using the CCK-8（Cell Counting Kit-8）assay.Based on the half inhibitory concentration(IC₅₀),concentrations of AHVAC-I were selected for the following exposure tests.3)Incubated GCAFs with AHVAC-I and Collect the culture medium(GCAFs-CM^AHVAC-I).Wound healing assay and Transwell test were used to detect the migration ability of MKN-28,which incubated with the GCAFs-CM^AHVAC-I.4)ELISA method was used to detect the level of level of CXCL12,which secreted by GCAFs.5)Realtime PCR ans wester blot were used to detect the expression level of CXCL12 in each group.Results:1.Primary GCAFs were successfully isolated by tissue block culture method and trypsin digestion method.Primary GCAFs are spindle-shaped,and they have irregular arrangement.The GCAFs are strongly potive for Vimentin andα-SMA.CK-18 as a epithelial marker is negative in GCAFs.2.GCAFs were treated with fresh culture medium containing various concentrations of AHVAC-I（1.5,3,6,12,24,48μg/ml）for 72h.The estimated IC₅₀is 8.133μg/ml.According to the IC₅₀results,the experimental concentration of AHVAC-I was set as three concentration groups of 2,4,and 8μg/ml.3.Wound healing assay and Transwell assay results indicated that the conditioned medium from GCAFs,which incubated with AHVAC-I for 72h,could markedly decreas the migration ability of MKN-28 cells.4.The results of ELISA,real-time PCR and western blot showed that the exprssion of CXCL12 was significantly decreased in GCAFs after incubated with AHVAC-I.