| Objective Pancreatic cancer is one of the common malignant solid tumors of the digestive tract.Its onset is hidden,the prognosis is poor,the fatality rate is high,and it is easy to metastasize and relapse.It seriously threatens the health and safety of humans around the world.Surgical treatment plays an important role in the treatment strategy of pancreatic cancer.Surgical treatment is currently recognized as the best treatment method.However,due to the insidious onset of pancreatic cancer,only a small number of patients can benefit from surgical treatment.Traditional radiotherapy and chemotherapy also play an important role in the treatment of pancreatic cancer.However,traditional radiotherapy and chemotherapy have shortcomings such as insignificant anti-tumor effect,poor specificity,and large toxic and side effects,which limit their further clinical use.Molecular targeted therapy has gradually emerged and played an important role in the treatment of pancreatic cancer in recent years.It has broad research and application prospects.Therefore,it is of great significance to develop more therapeutic targets for pancreatic cancer.In recent years,researchers have discovered neuropilin(Neuropilins,NRPs)include NRP-1 and NRP-2,which can be used as co-receptors of a variety of signaling molecules to play an important role in tumor angiogenesis,proliferation,metastasis and other processes.Studies have shown that NRP-2 is highly expressed in pancreatic cancer tissues,and its expression is closely related to the prognosis of pancreatic cancer patients.Therefore,NRP-2 is expected to become a new therapeutic target in the targeted therapy of pancreatic cancer.This topic aims to study the NRP-2monoclonal antibody(NRP-2 mAb)prepared in our laboratory to observe its effects on the proliferation,migration,invasion,adhesion and apoptosis of pancreatic cancer cells and explore its potential molecular mechanisms Hopefully,it can provide a theoretical basis for the clinical application of NRP-2 mAb as a novel molecular targeted therapy for pancreatic cancer.Methods1.Use hybridoma technology to prepare NRP-2 mAb,and then use protein chromatography column to purify and collect the NRP-2 mAb;gel electrophoresis to detect the purity of the purified antibody;ELISA and BCA protein assay to determine the purified antibody Potency and concentration.After that,the cell immunofluorescence technique was used to observe the binding ability of NRP-2 mAb with the NRP-2 protein on the surface of pancreatic cancer cell lines PANC-1 and MIA-PACA-2.2.Use the GEPIA website to analyze the difference in NRP-2 expression between pancreatic cancer tissue and normal pancreatic tissue,and perform kaplan-miere to analyze the relationship between NRP-2 and the prognosis of pancreatic cancer patients;use the Linked Omics website to analyze NRP-2 mRNA and pancreatic cancer patients The relationship between tissue type,overall patient survival rate,and pathological stage;The Human Protein Atlas website is used to analyze the difference in NRP-2 protein expression between normal pancreatic tissue and tumor tissue.3.Use CCK-8 test and colony formation inhibition test to detect the effect of NRP-2 mAb on the proliferation of pancreatic cancer cell line PANC-1;use Transwell test to observe the effect of NRP-2 mAb on PANC-1 cell migration and invasion;Attached experiments to observe whether monoclonal antibodies can inhibit the adhesion of PANC-1;flow cytometry to detect the effects of different concentrations of antibodies on the apoptosis of pancreatic cancer cell line PANC-1;4.Use the JC-1 test to explore whether NRP-2 mAb induces pancreatic cancer cell line PANC-1 to undergo apoptosis by changing the membrane potential of the mitochondrial surface of pancreatic cancer cell lines,and use Western Blotting to detect different concentrations of NRP-2 After mAb treatment,pancreatic cancer cell line PANC-1apoptosis-related proteins(Caspase-3,Caspase-8,Bax,Bcl-2)and proliferation,migration,invasion,adhesion-related proteins(PI3K,p-PI3 K,AKT,p-AKT,mTOR,p-mTOR).Result1.In this experiment,the purity of NRP-2 mAb prepared by hybridoma technology was90.7%,the titer was about 4×10-5,and the protein concentration was 4.2 mg/m L.And cellular immunofluorescence experiments showed that NRP-2 mAb can specifically bind to the NRP-2 protein on the surface of pancreatic cancer cell lines PANC-1 and MIA-PACA-2.2.GEPIA website analysis showed that the expression level of NRP-2 protein and NRP-2 mRNA in pancreatic cancer was higher than that in normal tissues;the disease-free survival rate of patients with high NRP-2 protein expression was lower than that of NRP-2 protein Patients with low expression.Linked Omics website analysis shows that NRP-2mRNA is correlated with the histological type of pancreatic cancer and the overall survival rate of patients,but has nothing to do with the pathological stage.The Human Protein Atlas database analysis showed that the expression of NRP-2 protein in pancreatic cancer tissues was significantly higher than that in normal tissues.3.CCK-8 and colony formation inhibition experiments show that NRP-2 mAb can effectively inhibit the proliferation of pancreatic cancer cell lines in a dose-dependent manner;the results of Transwell experiments show that NRP-2 mAb can inhibit the migration and invasion of pancreatic cancer cells and also follow The inhibitory effect is gradually enhanced by increasing the concentration of the antibody;the results of adhesion experiments show that NRP-2 mAb can significantly inhibit the adhesion of the pancreatic cancer cell line PANC-1,and the inhibitory effect is also enhanced with the increase of the antibody concentration;flow cytometry Cytometry showed that NRP-2mAb can promote PANC-1 apoptosis.4.The results of JC-1 experiment show that NRP-2 mAb can change the mitochondrial membrane potential of pancreatic cancer cell line PANC-1 in a dose-dependent manner;the results of Western Blotting experiment show that NRP-2 mAb can reduce pancreatic cancer cell line PANC-1 The expression of Bcl-2 protein in the medium,while increasing the expression of Caspase-8,Caspase-3 and Bax proteins.The results of other biological functions and molecular mechanisms indicate that NRP-2 mAb can inhibit the phosphorylation of PI3 K,AKT and mTOR proteins.Conclusions1.This research group successfully prepared NRP-2 mAb with high purity and high titer and can specifically bind to the NRP-2 protein on the surface of pancreatic cancer cell lines.2.Bioinformatics analysis results show that the expression of NRP-2 protein and NRP-2mRNA in pancreatic cancer is higher than that in normal tissues,and is closely related to the prognosis of pancreatic cancer patients.At the same time,NRP-2 mRNA is related to the histopathology of pancreatic cancer patients.Related and not related to pathological staging.3.The results of functional studies show that NRP-2 mAb can significantly inhibit the proliferation,migration,invasion and adhesion of pancreatic cancer cell line PANC-1 and promote its apoptosis.4.NRP-2 mAb can change the membrane potential of the mitochondria in PANC-1 cells to induce apoptosis in PANC-1 cells.At the same time,NRP-2 mAb can also up-regulate Bax,Caspase-3,and Caspase-8 while simultaneously down-regulating Bcl-The protein expression of 2 promotes the apoptosis of pancreatic cancer PANC-1 cells;the inhibitory effect of NRP-2 mAb on the proliferation,migration,invasion and adhesion of PANC-1is through the inhibition of the classical signaling pathway PI3K/AKT/mTOR. |
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