Preliminary Study On Pathogenesis Of Intraspinal Schwannomas Based On NGF/TrkA Signaling Pathway

Background:Existing evidence has shown that that the effect of NGF/TrkA signaling pathway on proliferation and differentiation of tumor cells is closely related to PI3K/AKT signaling pathway in human benign and malignant tumors.However,there is little information on the NGF/TrkA signaling pathway in pathogenesis of intraspinal schwannomas.Objective:To investigate the effect of nerve growth factor-beta on the proliferation of interspinal schwannoma cells and to explore on the pathogenesis of NGF/TrkA signaling pathway in interspinal schwannoma.Methods:The first step,specimens of intraspinal schwannoma resected under aseptic condition were collected,tissue digestion and primary cell culture were carried out in super-clean workbench.The purity of intraspinal schwannoma cells was identified by the principle of positive expression of S-100 protein and negative expression of fibroblasts.Cells with purity(?95%)were selected as experimental cells.The second step,single-cell suspension was prepared by trypsinization and centrifugation of intraspinal schwannoma cells,which met the purity requirement.7,000 cells per pore and 200 mu L/pore were inoculated into 96-well plate,respectively.The control group was treated with 200ulvolume fraction of 10%FBS·DMEM medium,and the experimental group was treated with 200ulvolume fraction of 10%FBS-DMEM medium,which The concentration of nerve growth factor beta solution was 15,30,60,120,240 ug/L,respectively.After 24 hours,the cell proliferation was evaluated by MTT method-According to the above scheme,the control group(10%FBS-DMEM complete medium)was set up.The experimental groups were as follows:(1)K252a group concentration was 100,200,300,400,500,600 nmol/L;(2)LY294002 group concentration was 10,20,30,40,50,60 umol/L;(3)nerve growth factor beta(120 ug/L);(4)nerve growth factor beta(120 ug/L)+K252a(400 nmol/L)group;(2)nerve growth factor beta(120,30,40,50,60 umol/L);The OD values of each pore at 570 nm wavelength were measured after 24 hours in the group of ug/L+LY294002(50 umol/L).The third step,on the basis of this experiment,intraspinal schwannoma cells were inoculated into 96-well plates,and 5 plates were inoculated.The control group was given 200 ug/L volume fraction of 10%FBS·DMEMmedium The experimental group was given 200 ug/L nerve growth factor beta solution.After 0,12,24,36 and 48 hours of continuous culture,the intraspinal schwannoma cells in 5 plates were measured at 570 nm wavelength by enzyme labeling instrument.OD value at each time point and growth curve were plotted.The fourth step,the intraspinal schwannoma cells were inoculated into 6 holes of 6 foramen plates with 5×104 cells per foramen,and the volume fraction of 10%FBS-DMEM medium was added to 3 mL.After 12 hours,Abandonment the culture medium,the control group was given 3 mL volume fraction of 10%FBS-DMEM medium.The experimental group was given 3 mL of nerve growth factor beta(120ug/L),nerve growth factor beta(120ug/L)and K252a(400nmol/L),nerve growth factor beta(120ng/L)adn LY294002(50umol/L)medium,respectively.After 24 hours of culture.The expression of TrkA,AKT,p-AKT(Ther308)and p-GSK-3 beta proteins were measured by Western blot,and the corresponding expression of TrkA and AKT proteins was detected by RT-PCR.Results:Compared with the control group,the absorbance value of cells in the nerve growth factor-beta groups increased in a concentration-dependent manner(P<0.05),and increased obviously at the concentration of 120 ?g/L(P<0.01).The absorbance value of cells in the K252a and LY294002 groups decreased continuously(P<0.05),and decreased obviously at the concentration of 400 nmol/L and 50 mol/L respectively(P<0.001).The expression levels of TrkA,p-AKT(Ther308),and p-GSK-3 beta protein were upregulated in the nerve growth factor-beta group(P<0.05),and the expression level of TrkA mRNA was upregulated(P>0.05).In the nerve growth factor-beta(120 ?g/L)plus K252a(400 nmol/L)group,the absorbance value of cells increased(P<0.01),the expression levels of TrkA,p-AKT(Ther308)and expression of p-GSK-3 beta protein upregulated(P<0.05),and the expression level of TrkA mRNA upregulated(P<0.05).In the nerve growth factor-beta(120 ?g/L)plus K252a(400nmol/L)group,the absorbance value of cells decreased(P<0.01),the expression levels of TrkA,p-AKT(Ther308),and p-GSK-3 beta protein downregulated(P<0.05),and the expression level of TrkA mRNA downregulated(P<0.05).In the nerve growth factor-beta(120 ?g/L)plus LY294002(50 ?mol/L)group,the absorbance value of cells decreased(P<0.01),the expression levels of p-AKT(Ther308),and p-GSK-3beta protein downregulated(P<0.05),and TrkA protein and mRNA did not change significantly(P>0.05).There was no significant change in AKT protein and mRNA in each group(P>0.05).Conclusion:Through this study,we can draw the following conclusions:1.Nerve growth factor beta can promote the proliferation of intraspinal schwannoma cells,and with the increase of its concentration,the effect of promoting cell proliferation is increasing.2.When Nerve growth factor beta promotes the expression of TrkA and its corresponding mRNA,the expression of AKT and its corresponding mRNA remains unchanged,but the phosphorylation level of protein AKT increases.3.The expression level of phosphoglycogen synthase kinase 3(p-GSK-3beta)increased when nerve growth factor beta promoted the proliferation of sheath tumor cells.4.The occurrence and development of intraspinal schwannoma may be related to the expression of TrkA,p-AKT(Ther308)and p-GSK-3beta in NGF/TrkA signal transduction pathway.