Effects Of Human Urine Derived Stem Cell Exosomes On The Biological Functions Of Degenerated Nucleus Pulposus Cells

Background:Low Back Pain is one of the most common diseases in modern society,which has severely affected people’s lives.Epidemiological studies show that more than 80% of people experience one or more low back pains in their lifetime,and more than 15% of people need hospitalization to get relief.Current etiology studies show that more than 40% of low back pain comes from secondary lesions of Intervertebral Disc Degeneration(IDD).Intervertebral disc degeneration is a natural aging process of the human body,but it can cause a series of pathological changes such as lumbar disc herniation,degenerative lumbar spinal stenosis,degenerative lumbar spondylolisthesis,lumbar instability and degenerative lumbar scoliosis.Intervertebral disc degeneration is a multi-layered and slowly progressing process.Among them,the decrease in the number of nucleus pulposus cells(NPCs)in the degenerative disc and the decrease in the activity of the extracellular matrix are one of the important factors leading to the disc degeneration.Therefore,finding a method that has been able to slow down or even reverse disc degeneration has been one of the research hotspots in this field.Mesenchymal stem cells(MSCs)are adult stem cells with self-renewal and multiple differentiation potentials.In recent years,studies have shown that stem cell transplantation can inhibit the degeneration and apoptosis of nucleus pulposus cells.It has also been reported that mesenchymal stem cells can regulate the expression of proteoglycan(ACAN)and collagen type II(COL2)in NPCs through exosome secreted by them to increase the activity of nucleus pulposus cells.However,stem cell acquisition methods are mostly invasive,and their sources are limited,limiting them.Human urine derived stem cells(Urine-derived stem cells,USCs)are a type of stem cells derived from urine.They are widely sourced,easy to obtain,safe and non-invasive,and do not involve ethics.They are an ideal source of exosomes.Studies have shown that human urine-derived stem cell-derived exosomes(USC-Exos)have the ability to improve the microenvironment,enhance metabolism,promote cell regeneration,and tissue repair.Exosomes have a double-layered phospholipid membrane structure similar to cellmembranes,and carry a large number of cell-derived mRNA,miRNA,proteins,etc.,which can be used as media for information exchange between cells,and regulate target cell functions after entering target cells.The research team found in previous research that non-contact co-culture of USCs and NPCs can promote the proliferation of NPCs and increase the expression of genes such as ACAN,COL2 and corresponding proteins,so we verified the role of exosomes in this process through this experiment.Objective:Based on the above,we used USC-Exos to degenerate nucleus pulposus cells in vitro in this study,in order to explore whether USC-Exos can promote NPCs proliferation and its effects on nucleus pulposus cells ACAN,COL2 and matrix metalloproteinase How does the expression of tissue inhibitor factor 1(TIMP1),multiple tumor suppressor gene P16(P16)gene mRNA and corresponding proteins affect.Methods:Human urine-derived stem cells(USCs)were obtained from the urine of healthy adults and cultured in vitro.The cells were identified by osteogenesis,chondrogenesis,adipogenic differentiation,and WB technology.Exosomes were extracted from USCs complete medium by differential centrifugation.Exosome identification was performed using electron microscopy,particle size analysis,and WB technology.USC-Exos was used to intervene P6 NPCs,CCK-8 method was used to detect cell proliferation,PKH26 fluorescent dye was used to label exosomes,and the uptake of USC-Exos by NPCs was observed under a laser confocal microscope.Subsequently,β-galactosidase aging staining and immunofluorescence staining of proteoglycan(ACAN)and type II collagen(COL2)were performed.RT-PCR and WB were used to detect the expression of ACAN,COL2,tissue inhibitor of matrix metalloproteinase 1(TIMP1),multiple tumor suppressor gene P16(P16)gene and corresponding proteins in NPCs;The relative expression levels of the group were analyzed by one-way ANOVA between groups.P <0.05 was considered statistically significant.Result:The extracted USC-Exos has an oval vesicle structure of 50 ～ 100 nm,which expresses CD63 and Tsg101,but does not express Calnexin protein.USC-Exos labeled with PKH26 can be taken up by NPCs;after the intervention of USC-Exos,the proliferation rate of P6 NPCs becomes faster and the proportion of senescent cells decreases;the relative expression levels of ACAN,COL2,TIMP1 mRNA and corresponding proteins in cells are 3d,5d At 7and 7 days,the experimental group was significantly higher than the control group(P <0.05),and the relative expression levels of P16 gene mRNA and corresponding proteins were significantly decreased at 3,5,and 7 days(P <0.05).Conclution:USC-Exos can promote the proliferation of degenerated NPCs under in vitro conditions,increase the expression of ACAN,COL2,TIMP1 genes and corresponding proteins,and reduce the expression of P16 genes and corresponding proteins.