| Part 1 Biological characteristics of umbilical cord mesenchymal stem cell-derived extracellular vesicles(MSC-EVs)Objective: Extracellular vesicles(EVs)are important carriers of intercellular information transmission and functional regulation,and EVs secreted by MSCs are considered to be key "effectors" for MSCs to exert a variety of biological effects.We isolated and obtained umbilical cord MSC-EVs,studied their general characteristics,the expression pattern and functional orientation of secreted mi RNAs,and compared them with adult bone marrow MSC-EVs,in order to further clarify the biological characteristics of umbilical cord MSCEVs,and provide a basis for further study of their clinical translational application potential.Methods:The morphology and structure of umbilical cord MSC-EVs were observed by transmission electron microscopy.Size distribution of MSC-EVs in umbilical cord was detected by NTA nanoparticle tracking system,and the expression of protein markers was detected by Western Blot.RNA was extracted from umbilical cord MSC-EVs and adult bone marrow MSC-EVs,and the expression of mi RNAs in MSC-EVs was detected by Small RNA-seq.The target genes of mi RNAs in umbilical cord MSC-EVs were predicted by mitarbase and mi Randa databases,and the biological processes involved were analyzed by KEGG enrichment.The differences in mi RNAs expression profiles between umbilical cord MSC-EVs and adult bone marrow MSC-EVs were compared,and the enrichment pattern of mi RNAs in umbilical cord MSC-EVs was analyzed.Results: Small RNA-seq results showed that compared with adult bone marrow MSC-EVs,53 mi RNAs were up-regulated and 26 mi RNAs were down-regulated in umbilical cord MSCEVs.KEGG enrichment analysis showed that the target genes of mi RNAs in umbilical cord MSC-EVs were mainly enriched in biological processes such as cell senescence,oxidative phosphorylation and DNA replication.Further combined with the literature,the differentially expressed mi RNAs of umbilical cord MSC-EVs and adult bone marrow MSC-EVs were analyzed,and it was found that various mi RNAs,such as mi R-106,mi R-93-5p and mi R-17-5p,were highly expressed in umbilical cord MSC-EVs.Meanwhile,the expression of mi R-127-3p and mi R-125b-5p,which can promote cell senescence,was generally down-regulated.In addition,mi R-21 and mi R-500a-3p,which inhibit inflammation and apoptosis,were also highly expressed in MSC-EVs of umbilical cord.Conclusions: Compared with adult bone marrow MSC-EVs,umbilical cord MSC-EVs carry more abundant mi RNAs molecular information,and highly express many mi RNAs with inhibitory function of cell senility,while many mi RNAs that promote cell senility are downregulated.Those results suggest that umbilical cord MSC-EVs selectively enrich senescence related secretory mi RNAs,which may play an important role in cell aging information transmission and senescence phenotype regulation.Part 2 Effects of umbilical cord MSC-EVS on degeneration of tissues and organs in naturally aging miceObjective: With the growth of age,aging and multi-tissue and organ degeneration inevitably occur in the body,and the aging of MSCs is closely related to the aging of the body and the degeneration of multiple tissues and organs.Combined with our previous in vitro study,we found that umbilical cord MSC-EVs can effectively reverse the aging of adult bone marrow MSCs,and we speculated that umbilical cord MSC-EVs can delay or even reverse the degeneration of tissues and organs in vivo.In this part,the application of umbilical cord MSC-EVs in vivo was studied in mice with natural aging,and the effects of MSC-EVs on senescence related secretion phenotype,antioxidant capacity and degeneration of various tissues and organs were observed.Methods:The isolated umbilical cord MSC-EVs was injected into naturally aging mice by caudal vein injection,3 times a week for 4 consecutive weeks,and the basic health status of the mice was recorded.In vivo imaging system and confocal laser microscopy were used to observe the distribution of umbilical cord MSC-EVs in mice with natural aging.The uptake of MSC-EVs from umbilical cord by peripheral blood cells and bone marrow cells was detected by flow cytometry.The levels of SASP and oxidase in mouse plasma were determined by ELISA and conjugate enzyme.The curvature of the spine was measured by X-ray.Micro CT was used to detect the changes of bone in mice.The area of bone trabecula,the number of osteoclasts and the expression of osteopontin OPN were observed by paraffin section of femur of mice.The contents of Cr,BUN,cystatin C and NGAL were detected by ELISA.The ratio of CD3+CD4+T/CD3+CD8+T in peripheral blood of mice was determined by flow cytometry.The motor ability of mice was tested by grasping force,rotating fatigue and bar climbing tests.At the same time,the heart,liver and other tissues and organs of mice were obtained,and the effect of MSC-EVs on the tissue structure of umbilical cord was observed by HE staining.Results: MSC-EVs from umbilical cord can enter the liver,spleen,lung,bone marrow and other tissues and organs of mice,and can be absorbed by peripheral blood cells and bone marrow cells.MSC-EVs could significantly decrease the levels of senescence related secreted phenotypes IL-1α and IL-1β,enhance the expression of antioxidant enzyme CAT,and decrease the content of lipid peroxidation product MDA(P<0.05).At the same time,after umbilical cord MSC-EVs treatment,the degeneration of various tissues and organs of aging mice was observed to be improved.It could increase the bone parameters of BS/TV,BV/TV and Tb.N,reduce the number of osteoclasts and enhance the expression of osteopontin OPN in aging mice.The levels of Cr,BUN,Cys C and NGAL in plasma of mice were decreased.Significantly increased the proportion of CD3+CD4+/CD3+CD8+T lymphocytes in peripheral blood and the number of medial cell nuclei(P<0.05).Conclusions: Umbilical cord MSC-EVs could be absorbed by various tissues and organs of naturally aging mice.Continuous application at a certain interval in vivo can significantly reduce the level of senescence related secretion phenotype and enhance the antioxidant capacity of natural aging mice.More importantly,the degeneration of bone,kidney,immune system,blood vessel and other tissues and organs in the naturally aging mice were significantly improved.Based on the safety of clinical application of umbilical cord MSCEVs,it is suggested that it may be a safe and effective new method to delay the degeneration of multiple tissues and organs in the body.Part 3 Umbilical cord MSC-EVS to delay renal degeneration in mice with natural aging and the preliminary study of its mechanismObjective: Aging renal degeneration includes renal structure changes and metabolic function decline,seriously affecting the health of the elderly,but there is no safe and effective means to delay or reverse.In vivo experiments,we found that umbilical cord MSC-EVs significantly improved renal function in aging mice.In order to better apply umbilical cord MSC-EVs transformation to delay age-related renal degeneration,we further systematically observed the delaying effect of umbilical cord MSC-EVs on renal degeneration in mice with natural aging from the aspects of tissue structure,cell and molecule.Methods:The umbilical cord MSC-EVs was injected into naturally aging mice,and renal tissue was obtained after continuous injection for 4 weeks.The changes of renal tubule atrophy and glomerular dilation were detected by HE staining.Masson staining,TUNEL staining and Bcl-2 immunohistochemistry were used to detect the effect of umbilical cord MSC-EVs on renal interstitial fibrosis and renal cell apoptosis in aging mice.The effects of umbilical cord MSC-EVs on the expressions of senior-related secretion phenotypes(IL-1α,IL-1β,IL-6)and senior-related genes(P16,P21,P53)in kidney tissues of aging mice were detected by q PCR and Western Blot.Aging renal tubular epithelial cells(HK-2)were treated with umbilical cord MSC-EVS to detect the effect of umbilical cord MSC-EVS on the aging status of HK-2 cells.CCK8 and flow cytometry were used to detect the proliferation and apoptosis of aging HK-2 after umbilical cord MSC-EVs treatment,and the m RNA and protein expression levels of aging related genes(P16,P21,P53)and apoptosis related genes(Bax,Caspase 3)were detected.Results: Umbilical cord MSC-EVs can significantly reduce glomerular area,reduce the degree of renal tubule atrophy and improve the level of fibrosis in naturally aging mice.Apoptosis rate in renal tissue of aging mice after umbilical cord MSC-EVs treatment(P<0.001),the expression levels of characteristic factors(IL-1α,IL-1β and IL-6)and aging marker molecules(P16,P21 and P53)in renal tissues were significantly decreased.In addition,the expression levels of pro-apoptotic(Bax,Cleaved caspase 3)and pro-fibrosis(Vimentin,αSMA)markers were down-regulated,while the expression of anti-apoptotic(Bcl-2)and anti-fibrosis(E-cadherin)markers were significantly up-regulated.In vitro experiments showed that umbilical cord MSC-EVs could promote the proliferation and inhibit the apoptosis of senescent HK-2 cells,and reduce the expression of senescence related genes(P16,P21,P53)and apoptosis related genes(Bax,Caspase 3)in HK-2 cells.Conclusions: In vivo application of umbilical cord MSC-EVS can significantly delay the degeneration of kidney in natural aging mice from the aspects of organ structure,cell and molecular level.At the same time,in vitro studies showed that umbilical cord MSC-EVS could reverse the aging of renal tubular epithelial cells.It is suggested that umbilical cord MSC-EVs is a new and effective means to delay age-related renal degeneration.The mechanism may be related to the reversal of renal tubular epithelial cell senescence,but further research is needed. |
PDF Full Text Request