The Anti-vascular Effect Of Recombinant Protein Of Gastric Cancer Vascular Targeted Peptide And Endostatin

Background and purposeThe proposal of antivascular therapy is of landmark significance in the comprehensive treatment of solid tumors.At present,antivascular drugs such as Endu have achieved significant clinical benefits in a variety of solid tumors.However,antivascular therapy for gastric cancer has not broken through the bottleneck,not only failed to significantly improve survival,but also has side effects such as hypertension.The main reason is that the drug targeting is not good,which affects the efficacy and increases the toxicity.Therefore,it is of great significance to improve the current situation to find vascular specific targets for gastric cancer,so as to improve the selectivity and efficacy of antivascular drugs and reduce side effects.In this study,gastric cancer vascular targeting peptide was synthesized as the carrier of "precision guidance",and the recombinant protein was constructed with classical vascular inhibitor endostatin.The study will focus on the targeted peptide and the recombinant protein.Methods1.Establishment of vascular endothelial cell model of gastric cancer;2.CCK-8 assay was used to detect the proliferation of gastric cancer cells,HUVEC and coHUVEC cells treated with targeted peptides,control peptides and recombinant proteins;3.Scratch test was used to detect the damage and repair of the above cell lines after the action of targeted peptide and control peptide;4.Detection of cell cycle distribution by flow cytometry;5.Immunofluorescence detection of targeted peptides,control peptides and recombinant proteins in the above-mentioned cell lines;6.Construction of Recombinant protein targeting Peptide and endostatin;7.Detection of protein bands by SDS-PAGE and Western blot;8.The purity of the protein was detected by SEC-HPLC.Results1.Gastric cancer cell lines were co-cultured with HUVEC in conditioned medium co-culture model to obtain gastric cancer vascular endothelial cells(coHUVEC).The co-cultured cells showed the shape of tumor vascular endothelium.The vascular targeting peptide(CNTGSPYEC)and the control peptide(CNKSPSGNC)of gastric cancer were synthesized successfully,and the amino acid sequence of the synthetic peptide was correct.Immunofluorescence showed that compared with the control peptide and PBS,the targeted peptide could specifically bind to co-HUVEC,but not to gastric cancer cells and HUVEC,which confirmed that the targeted peptide had vascular targeting to gastric cancer.The recombinant protein of targeting peptide and endostatin was successfully synthesized with a purity of more than 95%.It was confirmed that the recombinant protein also had vascular targeting of gastric cancer.Immunofluorescence showed that compared with the control peptide and PBS,the targeted peptide could specifically bind to co-HUVEC,but not to gastric cancer cells and HUVEC,suggesting that the targeted peptide has vascular targeting to gastric cancer.2.Cell proliferation experiment showed that the targeted peptide could inhibit the proliferation of co-HUVEC in a dose-dependent manner,and the highest inhibition rate was about 20%,which was significantly higher than that of the control peptide and PBS group(P<0.01).At the same time,the targeted peptide had no significant effect on the proliferation of gastric cancer cells and HUVEC.3.Cell scratch test showed that the targeted peptide had no significant effect on the migration characteristics of gastric cancer vascular endothelial cells.4.Flow cytometry analysis showed that the targeted peptide had no significant effect on the distribution of cell cycle.5.The recombinant protein of targeting peptide and endostatin was successfully synthesized with a purity of more than 95%.Immunofluorescence showed that the recombinant protein could specifically bind to co-HUVEC,but not to gastric cancer cells and HUVEC,suggesting that the recombinant protein had vascular targeting of gastric cancer.6.Cell proliferation experiment showed that the recombinant protein could inhibit the proliferation of co-HUVEC in a dose-dependent manner,and the highest inhibition rate was about 40%,which was significantly higher than that in the PBS group(P<0.001).At the same time,the recombinant protein had no significant effect on the proliferation of gastric cancer cells and HUVEC.Conclusion1.The conditioned medium co-culture model can successfully simulate the tumor microenvironment in vitro and make the co-cultured vascular endothelial cells have the characteristics of tumor vascular endothelium,which is suitable for the study of tumor vascular heterogeneity molecules.2.In vitro experiments suggest that the targeted peptide has vascular targeting of gastric cancer and has a certain inhibitory effect on the proliferation of co-HUVEC.3.Taking advantage of the selective enrichment ability of vascular targeted peptides in gastric cancer,endostatin can be enriched more specifically in co-HUVEC by constructing fusion protein.4.In vitro experiments show that the recombinant protein of targeted peptide and endostatin has stronger targeted killing ability of tumor vessels,which may reduce the damage of drugs to normal blood vessels,and provide a certain experimental basis for improving the efficacy of antivascular therapy for gastric cancer and reducing side effects.