Study On Anti-gastric Cancer Effect Of Neuropilin-2 Monoclonal Antibody And Its Mechanism

Background and PurposeGastric cancer is a common malignant tumor in the digestive system.The incidence and mortality of digestive system is the first place in the treatment of gastric cancer.The majority of patients with gastric cancer have been treated for advanced stage,Efficacy is not satisfactory,many patients can not tolerate chemotherapy drugs.With the development of biomedicine,molecular targeted therapy plays an important role in the treatment of advanced tumors.At present,a variety of molecular targeted drugs have been widely used in the treatment of advanced tumors,such as EGFR、HER-2、VEGF、m-TOR and other molecular targets drugs for anti-cancer treatment,which significantly reduce the side effects of chemotherapy drugs and improve patient tolerance,such as Xituo monoclonal antibody and trash Monoclonal antibody,in the clinical manifestations of a good anti-tumor effect.The study showed that Neuropilin-2(NRP-2)is a single-chain transmembrane glycoprotein,which is widely used in tumor cells as a synergistic receptor of VEGF.NRP2 is widely expressed in tumor cells and tumor angiogenesis.In the other side,tumor development 、 Tumor proliferation 、apoptosis、migration and adhesion in the formation of an important role.At present,the study of NRP-2 is not a lot.Studies have confirmed that high expression in colorectal cancer and the occurrence and development of colorectal cancer play an important role,but few studies related to gastric cancer,this article mainly homemade NRP-2 monoclonal antibody was used to detect the effect of NRP-2monoclonal antibody on the growth of gastric cancer cells and its mechanism,and to lay the foundation for the development of NRP-2 as a new target.Experimental methodWe determined the concentration and purity of the purified antibody by homemade NRP-2 monoclonal antibody(NRP-2 mAb)and SDS-PAGE electrophoresis.The titer was determined by indirect ELISA.The expression of NRP-2 protein in three different gastric tissues was detected by Western blottingand PCR.The antitumor effect of the highly expressed cell lines was selected according to the expression results.The antibody affinity and cell immunofluorescence were detected by flow cytometry The expression of NRP-2mAb was detected by immunohistochemical staining.The expression of NRP-2 protein in gastric cancer tissues and adjacent tissues was detected by CCK8 colorimetric assay.The effect of NRP-2mAb on the proliferation and proliferation of gastric cancer cells was detected by light microscopy.The effect of NRP-2mAb on the apoptosis of gastric cancer cells was observed by flow cytometry.Transwell was used to detect the changes of NRP-2mAb on gastric cancer cells.Western blotting detection of NRP-2mAb can regulate the proliferation of signaling proteins and related apoptosis signaling proteins,inhibit cell proliferation and promote cell apoptosis.ResultsThe results showed that BGC-823,SGC7901 and MKN-28 had positive expression of NRP-2 antigen on the cell membrane,and the expression of BGC-823 was higher on the cell membrane.The expression of NRP-2 protein in gastric cancer The expression of NRP2 was significantly higher than that of adjacent tissues,lymph node metastasis,tumor size,TNM stage,depth of invasion and distant metastasis,but not correlated with gender,age,pathological grade and pathological morphology.NRP-2mAb The effect of BGC-823 on the growth and proliferation of BGC-823 cells was inhibited by NRP-2mAb in BGC-823 cells,and increased the NRP-2mAb antibody concentration in BGC-823 cells.Apoptosis and inhibition of gastric cancer cell BGC-823 migration and invasion;NRP-2 mAb by down-regulating the proliferation of signaling signal and anti-apoptotic signal protein expression,up-regulation of apoptosis-related signaling protein expression,thereby inhibiting cell growth and promote cells Apoptosis.Conclusions1、NRP-2mAb with high purity,high specificity and affinity was successfully prepared in this experiment.2、NRP-2mAb can bind to NRP-2 protein on gastric cancer cell line and gastric cancer tissue,and is localized on cell membrane.3、NRP-2mAb inhibited the proliferation,apoptosis,migration and invasion of gastric cancer cell lines.4、NRP-2mAb inhibited the growth of gastric cancer cell lines and inhibited PI3 K / AKT,MEK / ERK1 / 2 signaling pathway.