Biological effects of human urine derived stem cell exosomes on degenerated nucleus pulposus cells

English Keywords:Urine-derived stem cells  Nucleus pulposus cells  Exosome  Extracellular matrix

English Abstract:

  【Abstract】 Objectives: To investigate the biological effects of human urine stem cell-derived exosomes(USC-Exos) on degenerated nucleus pulposus cells. Methods: Human urine-derived stem cells(USCs) were obtained from healthy adult urine and cultured in vitro. The extracted cells were identified by osteogenic, chondrogenic, adipogenic differentiation and Western Blot(WB) techniques. The USCs complete medium was used to culture USCs for 48h, and the exosomes were extracted from the medium by differential centrifugation. Under the electron microscope, particle size analysis and WB technology were used to detect the morphology, diameter and surface markers of USC-Exos. NPCs were extracted from the nucleus pulposus which was removed from patients with lumbar disc herniation during operations, and the cells were cultured to P6. P6 NPCs intervened with 50μg/ml USC-Exos were used as the experimental group, and P6 NPCs intervened with 50μg/ml centrifuged sediment of USCs complete medium without cultured cells were used as the control group. CCK-8 method was used to detect the proliferation of NPCs 1-6 days after intervention. USC-Exos was labeled with PKH26 fluorescent dye, and P6 NPCs were intervened with the labeled USC-Exos. After 12h, the uptake of USC-Exos by NPCs was observed under a laser confocal microscope. Both groups were tested for β-galactosidase aging staining 48h after intervention to detect the proportion of aging NPCs, and immuno-fluorescence staining was used to observe the expression of proteoglycan(ACAN) and type II collagen(COL2). The expressions of ACAN, COL2, matrix metalloproteinase tissue inhibitory factor 1(TIMP1), multi-tumor suppressor gene P16(P16) mRNA and corresponding proteins in NPCs of both groups were detected by RT-PCR and WB at 3d, 5d and 7d after intervention. To compare the relative expression levels of ACAN, COL2, TIMP1, P16 genes between the two groups, with P<0.05 being considered statistically significant. Results: Cells extracted from human urine had the characteristics of somatic stem cells. After subculturing, elliptical vesicle structures with a particle size of 50-100nm could be extracted. CD63 and Tsg101 were expressed, instead of Calnexin protein, which was consistent with the characteristics of Exos. After the intervention of USC-Exos, the cell proliferation speed of P6 NPCs became faster. USC-Exos labeled with PKH26 could be taken up by NPCs. 48h after intervention, the proportion of senescent cells in the experimental group decreased[(13.8±1.4)% vs (19.6±2.4)%], and the fluorescence intensities of ACAN and COL2 were higher than those of the control group. At 3d, 5d, and 7d after intervention, the relative expression levels of ACAN, COL2, TIMP1 mRNA and corresponding proteins in cells of the experimental group were significantly higher than those of the control group(P<0.05), and the relative expression levels of P16 gene mRNA and corresponding proteins were significantly decreased in the control group(P<0.05), but no significant difference was seen in the same group at different time points. Conclusions: USC-Exos can promote the proliferation of degenerated NPCs in vitro condition, increase the expression of ACAN, COL2, TIMP1 mRNA and corresponding proteins in degenerated NPCs, and reduce the expression of P16 mRNA and corresponding proteins.