Preparation Of NGF Gene Engineering Exosomes And Its Antioxidative Effect

Exosomes are nanoscale vesicles,which are released and received by almost all cells in the body.Exosomes can transfer between cells and contain various molecular components closely related to their sources and functions,including protein,lipid and RNA etc.The importance of exosomes in cell communication makes them an important carrier for the delivery of various drugs throughout the body.They are ubiquitous in the circulatory system and can reach the site of injury or disease through various biological barriers.Because of its unique structure and rich contents,they can be used in the diagnosis and treatment of diseases.Nerve growth factor(NGF)is a kind of nerve growth factor,which plays an important role in physiological activities such as cell cycle,cell metabolism,nerve cell injury repair.NGF can reduce the risk of acute or chronic neuropathy.Studies have confirmed that NGF in extracellular vesicles can promote nerve recovery and neoangiogenesis.To study whether umbilical cord mesenchymal stem cell exosomes(hUCMCS-exos)overexpressing NGF gene can resist oxidative stress and promote the repair of nerve related cell tissue injury.In this study,umbilical cord mesenchymal stem cells were transformed by transgenic technology to obtain hucmsc exos overexpressing the NGF gene.Then,the oxidative stress model was established to study its oxidation,so as to provide a basis for the repair of nerve tissue injury by NGF genetically-engineered exosomes.The main research methods and results are as follows:1.hUCMSC-exos overexpressing NGF gene was constructed by transgenic technology.Firstly,the recombinant vector overexpressing NGF gene was constructed.Then,through lentivirus packaging,infection and drug screening,a stable cell line of umbilical cord mesenchymal stem cells(hucmcs)overexpressing NGF gene was obtained and identified by RT-PCR and Western blot.Finally,cell supernatants were collected,exosomes were extracted and the expression of NGF protein was detected.The results showed that NGF mRNA and NGF protein were highly expressed in umbilical cord mesenchymal stem cells,suggesting that hucmsc exos overexpressing NGF gene was successfully constructed.2.hUCMSC exos overexpressing NGF was co-cultured with PC-12.After CoCl2 stimulation,the antioxidant effect of hUCMSC-exos overexpressing NGF on oxidative stress model was verified by experiments including cell viability,intracellular reactive oxygen species content,intracellular LDH release and the expression of apoptosis-related genes BAX and Caspase-3.This shows that hUCMSC-exos overexpressing NGF not only improve the cell viability of PC-12 cells,but also enhance its antioxidant capacity and reduce the expression of apoptosis-related genes.In conclusion,this study constructed a hUCMSC cell line with overexpression of the NGF gene and obtained hUCMSC-exos with overexpression of the NGF gene.The hUCMSC-exos have significant antioxidant effect by the treatment of PC-12 cells stimulated by CoCl2.It is expected to become a new antioxidant agent for injury nerve tissue repair.