| [Background]Cisplatin plays an important role in the treatment of head and neck malignancies.However,cisplatin has different degrees of toxic and side effects on the body while treating tumors.Progressive or irreversible sensorineural deafness is one of the most common ENT complication of cisplatin.Because mammalian cochlear hair cells are extremely fragile and vulnerable to many factors such as drug-borne,pathogenic and noise exposure,in adult mammals and human inner ears,very few vestibular hair cells can spontaneously regenerate,and current research shows that cochlear hair Cells cannot regenerate spontaneously after injury.The mechanism of cisplatin ototoxic damage and the mechanism of hair cell regeneration are not fully understood.However,hair cell regeneration is reversible after the zebrafish lateral line hair cell damaged.Studying its mechanism has provided a potential target for mammalian hair cell regeneration and the research direction of regeneration mechanism to a large extent.In our previous research,we found that the support cells around the neuromast are in a proliferative state.After cisplatin damage the zebrafish juveniles,the supporting cells were proliferating actively.The new hair cells were mainly derived from the supporting cell proliferation and then differentiated into Hair cells.The Shh signaling pathway plays a positive regulatory role in the normal development and maintenance of the zebrafish lateral line neuromast,and is activated during the regeneration of zebrafish lateral line hair cells.Wnt/?-catenin not only plays an important role in the migration of zebrafish posterior lateral line cells,but also participates in the early auditory substrate and vesicle differentiation of mammalian inner ear.During the development of mouse inner ear,the Shh pathway and the Wnt/?-catenin pathway antagonize each other,playing a decisive role in the formation of the DV axis of the mouse inner ear.In view of this,we used cisplatin to establish a zebrafish ototoxicity model to study the proliferation of zebrafish juvenile lateral neuromast hair cells,and we further studied the expression and the possible relationship of Shh signaling pathway and Wnt/?-catenin in the zebrafish hair cells regenerate process after cisplatin injury.[Objective]To investigate the expression and role of Shh signaling pathway and Wnt/?-catenin in the regeneration of lateral hair cells of juvenile zebrafish after cisplatin injury,and the relationship between Shh and Wnt signals[Method]The first experiment ues cisplatin solution to treat zebrafish fish 5 days after fertilization.Treated juvenile zebrafish with BrdU solution after withdrawal of medicine and then removed the BrdU solution.Labeled of proliferating cells,hair cells and nuclei by FM1-43FX,BrdU and DAPI.In the second experiment,in situ hybridization technology were adopted to observe the expression of genes related to Wnt/?-catenin signaling pathway and Shh signaling pathway in the control group?cisplatin-treated group and co-treatment with cisplatin and drugs group.[Result](1)Hair cells can spontaneously regenerate after cisplatin damage to the zebrafish lateral line neuromast hair cells,and hair cells and proliferative cells increase with time.(2)After co-treatment with cisplatin and the Shh signaling pathway inhibitor(GANT61),the number of hair cells and proliferation of zebrafish lateral line neuromast hair cells was significantly less than that of the control group,while the number of hair cells and proliferation cells of the shh signaling pathway agonist(SAG)group was more than the control group.(3)After co-treatment with cisplatin and Wnt signaling pathway agonists(AZ),the number of hair cells and proliferation of zebrafish lateral line neuromast hair cells was significantly higher than that of the control group.(4)Gli1,Dkk2,Sfrp1,and ?-catenin are all expressed during the development and regeneration of zebrafish hair cells.In addition,Glil,Dkk2,and ?-catenin are expressed in the central area of the zebrafish lateral line neuromast,and Sfrpl is expressed in peripheral support cells of the neuromast.(5)Compared with the cisplatin group and the cisplatin and GANT61 co-treatment the expression of Glil reduced,the expression of Dkk2 increased,and showed no significant change in ?-catenin.The cisplatin and SAG co-treatment compared with the cisplatin group,it was found that the expression of Glil increased,Dkk2 decreased,and ?-catenin did not change significantly.In the cisplatin AZ treatment group,the expressions of Gli and ?-catenin increased,and the expression of Dkk2 decreased.Sfrp1 is expressed in the support cells during the regeneration of zebrafish lateral hair cells.The expression level of Sfrp1 is not increased or decreased,and it is expressed in the central region only when the Wnt signaling pathway is activated.[Conclusion]1.A zebrafish lateral line hair cell regeneration model by 50uM cisplatin-treated was feasible.2.In the process of zebrafish lateral line hair cell regeneration,multiple pathways co-regulate the regeneration process.The Shh signal pathway and Wnt signal pathway are activated and participate in the hair cell regeneration process,playing a positive regulatory role in this process.3.Sfrp1 is expressed in the outermost support cells of the zebrafish lateral line neuromast,which is a specific expression gene of the outermost support cell.During the regeneration process,Sfrp1 is expressed in a small amount in the neuromast,which means that the outermost support cells of the neuromast play a weak role in the regeneration process.Peripheral support cells are not the main source of hair cell regeneration cells.4.Wnt signal as an upstream pathway of Shh signaling pathway co-regulates the regeneration process of zebrafish lateral line neuromast hair cells after cisplatin injury. |
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