The Notch Signal Path Is Involved In The Protection Of Radiation-induced Apoptosis Of C17.2Neural Stem Cells By Exogenous Basic Fibroblast Growth Factor

RIB(Radiation injuries, brain)is a direct damage and subsequent secondary injurycaused by radiation on brain tissue after exposure in the special environment. RIB is adamage to the nervous system after radiotherapy in patients who has head and(or)neckcancer, that is one of the most serious complications among the long-term survivors.The pathogenesis of RIB is no conclusive current, focused on several aspects ofvascular injury, glial cell injury, neurons and neural stem cells(neural stem cells, NSCs)damage and central nervous system, immune response.No single factor can fully explain all the variation of radiation brain injury, thus,most scholars believe that pathogenesis of radiation brain injury is the result of thecombined effects of multiple factors.The diagnostic criteria of RIB include：dose threshold, clinical manifestations,indexing criteria, treatment methods etc. Currently the clinical treatment is mainly ondehydration, neurotrophic and other symptomatic therapy, however, the traditional drugtreatment is limited and lack of effective treatment for the cause. In recent years, thecarried out of Cytokines, gene therapy and other methods provides a new means for thetreatment of RIB.Notch is a class of highly conserved receptor protein which is widely present in thecell surface and mediated signal transduction between cells, to regulate the growth anddevelopment of the organism through the interaction between cells; Notch signalingpathway is conserved.In the development process of the vertebrates and invertebrates, the Notch signalingpathway has an important role in decision on cell fate, development of the nervous system,organs formation and somites occur.Notch pathway downstream molecules including transcription factor CBF/RBp-Jk and target genes with regulation E (spl)/HES and BLBp and other transcription factor,theycombined into a major effector by binding to a given sequence of Notch intracellular.Notch signaling pathway determines neuronal differentiation through lateral inhibition.Notch signaling is a pathway downstream molecules, such as Hes5, MAML1etc, whocan interact with FGF2signaling pathway, thereby inhibiting oligodendrocyte precursorcells differentiate into oligodendrocytes. In addition, the present study has directinstructions the existence of a link between Notch and Ras/Raf/MEK/ERK signalingpathway.DAPT as a synthetic γ-secretase inhibitors, which can specifically block the Notchsignaling pathway. DAPT studies mostly concentrated in the central nervous systemdiseases, there are a lot of studies have confirmed DAPT can inhibit the proliferation ofcells in vitro, and there is a clear dose-dependent manner.HES1Notch receptor protein isone of the target gene, Studies have confirmed after the DAPT treated HeLa cells, candetected HeLa cells in Notch1mRNA and its downstream target genes H ES1mRNAdownregulated. In the Notch signal transduction process, γ-secretase is a key enzymerelease NICD this critical step, the application of γ-secretase inhibitors can effectivelyinhibit its activity.bFGF is a potential, not yet fully confirmed neurotrophic factor, Its angiogenesis,promote wound healing and tissue repair and promote tissue regeneration plays animportant role, In the nervous system, mainly for the neuronal and glial proliferationactivity has a strong pro-proliferative activity, strong promote regeneration of peripheralnerve injury.Currently on bFGF signal transduction pathway mechanism of action has been studiedmore clearly, Including: Ras/Raf/MEK/ERK, PI3K, PKC, JAK/STAT four ways.Studies have found that ERK1/2signaling pathway plays an important role in cellproliferation prolactin bFGF-mediated process, their participation in the protective effectof bFGF on endothelial cell damage after radiation has been demonstrated. Zhou Haihongother confirmed exogenous bFGF on C17.2neuronal apoptosis inhibit radiation-induceddry and there is a dose-dependent manner.To better define the mechanism of action of exogenous bFGF treatment RIB, tounderstand the relationship between Notch signaling pathway and bFGF antiradiationeffect, After use C17.2neural stem cells(C17.2NSCs)as a tool, to build a radioactive nervestem cells injury model, Using basic fibroblast growth factor (bFGF) and the specificity ofthe Notch signaling pathway inhibitor (γ-secretase inhibitor DAPT) to treate separately C17.2NSCs which received radiation treatment, observe the effect on the growth andproliferation, detection Notch signaling pathway molecules four major nodes Notch1,RBP-κ, Hes1, Hash1altered expression levels, explore the possibility and mechanism ofinteraction between the Notch signaling pathway and bFGF pathway.Not only help to clarify the complex pathology secondary to nervous system damagein the process of radioactive, and there is hope to become the new therapeutic targets toradioactive nervous system damage, to design blocking or activating specific signalingmolecules RIB target new therapeutic drugs,to the benefit of mankind.Purpose:1. After establishing C17.2NSC model of radiation injury to observe the differentialexpression conditions of Notch1, RBP-κ, Hes1, Hash1in different periods.2. In the case of bFGF participation C17.2NSC protection, observe the expression ofmolecules in different periods of the above node, and make comparative analysis.3. Add γ-secretase inhibitor DAPT to observe the apoptosis, necrosis and proliferationof C17.2NSC radioactive model cell in different stages.Research Methods:1. C17.2neural stem cells conventional culture2. C17.2neural stem cells Nestin immunocytochemistry3. MTT cell viability assay4. Radiation-induced neuronal apoptosis dry C17.2and protective effect of bFGF andthe impact of DAPT etc.5. various protein detection:⑴immunocytochemistry staining⑵total protein was extracted and detection levels①Total cell protein extracts②protein detection microplate reader③Western Blot Detection6. Notch signaling pathway is involved in C17.2neural stem cell apoptosis signalingpathway study radiation-induced inhibition of bFGF.Result:1. Normal cell culture: On the morning of the second day after the cells were seededobserved under the microscope, adherent cell growth, most cells triangular, polygonal,irregular shape, and round, cells have long axons, axons can be connected between theadjacent cells, the cells cultured for about2-3days, the cells were covered with cell culture flasks.2. Normal cells seeded in tin flasks with slides, when the cells were grown to coverabout60-70%of the slides, immunohistochemical staining for cells, detection of thepresence of protein nestin in the neural stem cells, the results showed that afterimmunohistochemical staining of cells, more than98%of the cytoplasm was stainedbrown, after hematoxylin blue nuclei, cytoplasm brown. Cells cultured for7-8generations,immunohistochemical staining was still above90%.3. Normal cells were seeded in96well plates, when the cells attached to the wall,adding different concentrations of DAPT, MTT results showed that, DAPT can effectivelyinhibit the growth of neural stem cells, and with the increasing concentration, MTTabsorbance value decreased,that is with the concentration increases of DAPT, increasethe degree of cell growth inhibition, after joining in basic fibroblast growth factor,compared with the simple DAPT group between the groups, the differences werestatistically significant (p <0.05), that is basic fibroblast growth factor capable ofinhibiting the proapoptotic effects of DAPT on neural stem cells, and there is a clearconcentration-dependent manner.4. After irradiation MTT showed control group (with irradiation) has significantly (p<0.05); After adding different concentrations of DAPT cultured, some time later useingirradiation, MTT results showed differences between each group and the control group (p <0.05);Between adding different concentrations of DAPT with ray treatment and pure raytreatment group, there were significant differences (p <0.05)and the stronger DAPTconcentration the more inhibition,Between bFGF+DAPT+irradiation group and sameconcentrations of DAPT+irradiation group, they all have significantly(p <0.05).5. Flow cytometry results showed compared with the normal group, the irradiatedcells of apoptosis and cell death was higher than the control group (p <0.05); Differentconcentrations of DAPT, cells cultured24h and then useing radiation. Flow cytometryresults showed that with DAPT concentration improved, cell apoptosis rate and mortalitywas increased. Between different groups, they have statistically significant (p <0.05).Between bFGF+DAPT+irradiation group and same concentrations of DAPT+irradiationgroup, they all have significantly (p <0.05).6. After immunofluorescence staining showed that neural stem cells have protein ofHes1and RBP-κ.7. Western blot showed with elevated concentrations of bFGF the expression ofRBP-κof the neural stem cells protein decreased, while Hes1protein content increased gradually; The irradiation treated neural stem cells with increased DAPT the express ofRBP-κprotein improved, and Hes1protein content decreased gradually; BetweenbFGF+DAPT+irradiation group and same concentrations of DAPT+irradiation group,RBP-κof the neural stem cells protein decreased, while Hes1protein content increasedgradually.Conclusion:1. DAPT can inhibit the growth of C17.2neural stem cells, and with concentration,inhibition enhanced2. irradiated cells, cell growth and reproduction was inhibited, rays can inhibit thegrowth and proliferation of neural stem cells;3. DAPT can collaborate ray interaction with neural stem cells, inhibit the growth andproliferation of neural stem cells;4. basic fibroblast growth factor can inhibit the injury DAPT and rays on neural stemcells, this protective effect may be through the role of Notch signaling pathway, affectingthe expression of proteins in signaling pathways downstream achieved.