The Study On The Improvement Of Skin Keratinocyte Function By MITF-induced Melanocyte Derived Extracellular Vesicles

Animal skin pigment units play an important role in the maintenance of skin color and skin damage repair,which is regulated by the neuroendocrine system,intracellular signal transduction and intercellular signal transduction mediated by extracellular vesicles(EVs).This thesis studies whether EVs synthesis and release of melanocytes can be affected by enhancing melanin synthesis function and explore whether melanocyte derived EVs can be captured by keratinocytes and its effect on keratinocytes to provide a product with EVs for repairing animal skin damage.The test contents and results are as follows:1.The overexpression of MITF and Trp53 melanin cell lines(Melan-α)were successfully constructed by lentivirus technique.The transcriptional expression of MITF and Trp53 melanocytes were 3.18 and 1.4times higher than that of the control group,and the synthesis of melanin was 1.46 and 1.39 times higher,respectively.The result show MITF increased EVs yield by 3.85 times and promote the release of EVs in Melan-α cells.EV synthesis is correlated with the regulation of melanin.2.qPCR and WB showed that MITF may directly or indirectly up-regulate the expression of CD81,VPS36 and VPS37 B to enhance the synthesis of the multivesicle body(MVB).IHC results showed that CD81,VPS36 and VPS37 B were highly expressed in mouse epidermis,sebaceous glands and root sheaths,and showed obvious strip-like distribution in basal layer.3.The transport of EVs between keratinocytes and Melan-α cells was detected by CD63-GFP-labeled Melan-α cells by immunofluorescence staining and flow cytometry.The results show that co-culture of keratinocytes and melanocytes can cause polarization of MVBs along cell processes towards contact site.Fluorescence and real-time cell dynamic detection showed that keratinocytes could capture EVs released by melanocytes and MVBs in melanocytes directly by phagocytosis.4.The diameter of EVs derived from melanocytes was found to be 77.14±16.70 nm by electron microscopy and particle size detection.Flow cytometry and WB analysis showed that EVs contained CD81,CD63 and Alix proteins.Analysis of EVS-miRNA expression profile showed that MITF overexpression caused 76 EVs-miRNA up-regulation and 32 down-regulation.Functional enrichment analysis of target genes with differentially expressed miRNA revealed that they may regulate Ras,Hippo,Hedgehog and other signaling pathways,thus affecting cell proliferation,migration,anchoring and adhesion of plasma membrane.5.Real-time cell function analysis showed that EVs derived from melanocytes and MITF overexpressing melanocytes could promote the growth of keratinocytes.However,the differential expression of miR-127-3p and miR-28-3p in EVs showed no significant influence on the growth trend of keratinocytes.Transcriptome sequencing of keratinocytes transfected with miRNA revealed that the decreased content of miR-28-3p may regulate keratinocytes to resist infection,prevent carcinogenesis and premature apoptosis.Conclusion: EVs derived from melanocytes can be captured by keratinocytes,indicating that EVs plays an important role in signal transduction of the two kinds of cells.MITF can induce the synthesis and release of EVs in melanocytes and change the contents of EVs,thus regulating the biological function of keratinocytes.These results indicate that EVs derived from melanocytes has great potential to be used as a biological product to effectively improve animal epidermal conditions,and lay a foundation for the further development of EVs as a drug to treat animal skin diseases.