| Objective:As my country’s aging process continues to accelerate,the number of people with brain aging is also increasing year by year.The decline in cognitive function caused by brain aging has seriously affected the quality of life of the elderly population.Therefore,we urgently need to conduct more in-depth research on brain aging and find effective therapies to delay brain aging.This paper aims to study the role and potential mechanism of urinary stem cell-derived exosomes in delaying brain aging,and provide new ideas for effectively delaying brain aging.Methods: 1.Firstly,we extracted urine stem cells from the urine of cesarean section pregnant women,and performed flow cytometry identification on the urine stem cells.When the primary urinary stem cells were subcultured to passage 3-5,the serum-free medium cultured for more than 24 hours was collected,and the exosomes of urinary stem cells were extracted from the collected medium using a total exosome isolation kit and carried out Transmission electron microscopy and WB experiments identified and quantified urinary stem cell exosomes.2.We screened out the optimal concentration of D-galactose to construct neural stem cell aging model in vitro by CCK-8 experiment.Then,small RNA transcriptome sequencing was performed on the extracted USCs exosomes,and bioinformatics methods such as GO and KEGG were used to analyze the sequencing data to screen candidate miRNAs related to cell proliferation,migration and apoptosis pathways.Then,the candidate miRNA mimics were transfected into aging neural stem cells,and verified by CCK-8,scratch experiment and flow cytometry experiments,and the miRNAs that play an anti-aging role in exosomes were screened out.3.We transfected the miRNA mimics screened from USCs exosomes into urine stem cells to obtain exosomes overexpressing the miRNA.The 20-month-old naturally aging mice were randomly divided into three groups,namely the exosome group,the miRNA overexpression exosome group and the PBS group,and the amount of exosome injected by each tail vein injection was about 200 ug.Inject once a week for a total of four weeks.Then,the cognitive function of aged mice was evaluated by behavioral testing methods such as water maze,open field,and Y maze.After the assay was completed the brains of each group of mice were sampled and the cortices of the mice were subsequently immunofluorescently stained with frozen sections of NeuN and GFAP.The number of NeuN-positive cells and GFAP-positive cells were counted to assess the therapeutic effect of USCs exosomes on brain senescence.Results: 1.We successfully extracted and cultured urine stem cells from the urine of cesarean section women.The results of flow cytometry test confirmed that the expression of CD90,CD44,CD73,and CD105 in USCs was positive,and the positive rates were 97.89%,99.25%,,98.76%and 98.23% respectively.The expressions of HLA-DR,CD45,CD11,and CD34 were negative,and the positive rates were 0.06%,0.07%,0.05%,and 0.08%,respectively,which was consistent with the expression of mesenchymal stem cells reported in the literature.Exosomes were successfully extracted from urine stem cell culture medium and identified by transmission electron microscopy and WB experiments.The results of transmission electron microscopy identification showed that the exosomes we extracted had a typical "cup holder-like structure",and the results of WB strips showed USCs Exosomes expressed CD9 protein,and the identification results were consistent with the description of exosomes in the literature.Then pkh26 staining results showed that exosomes could enter neural stem cells and play a role.2.The optimal concentration of D-galactose to construct senescent stem cell model is30mg/ml through CCK-8 experiment.Combined with small RNA sequencing results,GO analysis and KEGG analysis,we screened out seven candidate miRNA molecules from 133 up-regulated differential miRNAs and 267 down-regulated differential miRNAs,namely miR-9-5p,miR-93-5p,miR-126-3p,miR-145-5p,miR-182-5p,miR-183-5p,miR-330-3p.After USCs exosomes and 7 miRNA mimics were co-cultured with aging neural stem cells for 48 hours,they were verified by CCK-8experiments,scratch experiments,and flow cytometry experiments.We found that miR-183-5p could significantly enhance the proliferation,migration and anti-apoptosis abilities of senescent neural stem cells,and the difference was statistically significant.3.The results of in vitro behavioral and morphological experiments indicated that USCs-derived exosomes enhanced cognitive function and cortical neuron survival in senescent mice and reduced the number of GFAP-positive cells.Overexpression of miR-183-5p-treated exosomes further enhanced the improvement of cognitive function and cortical neuron survival in senescent mice by exosomes.Conclusion: Exosomes derived from urinary stem cells can enhance the cell viability of aging neural stem cells,and may optimize the function of endogenous aging neural stem cells through the miR-183-5p pathway to achieve the purpose of anti-aging brain.Also urinary stem cell exosomes are effective in improving cognitive decline and cortical neuronal loss after brain aging via the miR-183-5p pathway,and this is likely to be related to the functional optimization of endogenous neural stem cells by urinary stem cell exosomes.In summary,urinary stem cell-derived exosomes exert their anti-aging effects through miR-183-5p,which may be achieved by optimizing the function of endogenous neural stem cells. |
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