Preparation Of NAMPT Gene Engineering Exosomes And Its Anti-aging Effect

Exosomes are nanoscale vesicles with a diameter of 30-150nm.They contain proteins,DNA,miRNAs,lncRNAs and other biological molecules.They participate in material exchange and information exchange between cells through plasma membrane fusion or binding with surface receptors,and play a key role in the occurrence and development of many diseases.Studies have shown that mesenchymal stem cell exosomes,as a new and ideal cell-free therapy tool,have high application value in drug delivery to specific sites through gene editing or chemical encapsulation.NAMPT is the rate-limiting enzyme of NAD+synthesis pathway,which plays an important role in cell cycle,cell metabolism,cell senescence and DNA damage repair.There are two forms of NAMPT in mammals:intracellular and extracellular NAMPT(iNAMPT and eNAMPT).Studies have confirmed that eNAMPT in extracellular vesicles can delay aging and prolong the life span of mice.In order to study whether the exosomes of NAMPT-edited umbilical cord mesenchymal stem cells can enhance the anti-aging effect,we used gene editing technology to modify the exosomes to obtain the exosomes of NAMPT-overexpressed umbilical cord mesenchymal stem cells,and then cocultured with human skin fibroblasts and fed Caenorhabditis elegans to study their anti-aging effect,so as to provide the basis for the anti-aging research of exosomes and NAMPT.The main research methods and results are as follows:1.Construction and identification of exosomes of umbilical cord mesenchymal stem cells with overexpressed NAMPT.In this study,the exosomes of NAMPT-overexpressed umbilical cord mesenchymal stem cells were constructed by gene editing technique.Firstly,the recombinant vector of the NAMPT gene was constructed.After lentivirus packaging,infection and drug screening,the NMAPT-overexpressed umbilical cord mesenchymal stem cell line was obtained and identified by RT-PCR and Western blot.Finally,the supernatant was collected,exosomes were extracted and the expression of NAMPT protein was detected.The results showed that the expression of NAMPTmRNA and protein in umbilical cord mesenchymal stem cells was high,and the expression of NAMPT protein in exosomes was also high,which suggested that the exosomes of umbilical cord mesenchymal stem cells with overexpressed NAMPT were successfully constructed.2.The effect of NAMPT-overexpressed on the senescence of human skin fibroblasts.In this study,human skin fibroblasts were cocultured with the exosomes of NAMPT-overexpressed umbilical cord mesenchymal stem cells.The anti-aging effect of NAMPT-overexpressied umbilical cord mesenchymal stem cells exosomes on human skin fibroblasts was determined by detecting cell growth curve,β-galactosidase staining,cell scratch test,malondialdehyde content,p16 and p21 mRNA and protein.The results showed that NAMPT-overexpressed can not only promote the proliferation and migration of human skin fibroblasts,but also enhance their antioxidant capacity and reduce the expression of aging-related genes.3.The effect of NAMPT overexpression on the senescence of C.elegans.In order to further verify the anti-aging effect of NAMPT-overexpressed exosomes of umbilical cord mesenchymal stem cells,we used NAMPT-overexpressed exosomes of umbilical cord mesenchymal stem cells to feed C.elegans,and tested their longevity,body swing frequency,superoxide dismutase and reactive oxygen species free radicals in vivo.The results showed that NAMPT-overexpressed not only prolonged the life span of C.elegans,but also enhanced their antioxidant capacity and motility.In conclusion,we constructed a NAMPT-overexpressed umbilical cord mesenchymal stem cell line and obtained the NAMPT-overexpressed exosomes.Through coculture with human skin and feeding C.elegans,we found that the exosomes of umbilical cord mesenchymal stem cells overexpressing NAMPT gene have significant anti-aging effect,which is expected to become a new anti-aging agent.