人尿源干细胞外泌体对退变髓核细胞生物学功能的影响

目的:探讨人尿源干细胞外泌体(USC-Exos)对退变髓核细胞(NPCs)生物学功能的影响。方法 :从健康成人尿液中获得人尿源干细胞(USCs)并进行体外培养,通过成骨、成软骨、成脂诱导分化及蛋白质免疫印迹(Western Blot,WB)技术对所提取细胞进行鉴定。使用USCs完全培养基培养USCs 48h后采用差速离心法从培养基中提取外泌体,应用电子显微镜、粒径分析及WB技术对USC-Exos进行形态、直径及表面标志物检测。从腰椎间盘突出症患者术中摘除的髓核中提取NPCs,并培养至P6。以加入50μg/ml USC-Exos干预的P6NPCs为实验组,以50μg/ml未培养过细胞的USCs完全培养基离心沉淀物干预的P6 NPCs为对照组,分别在干预后1～6d使用CCK-8法检测NPCs增殖情况。使用PKH26荧光染料标记USC-Exos,并用标记后的USCExos干预P6 NPCs,12h后在激光共聚焦显微镜下观察NPCs对USC-Exos的摄取情况。两组在干预后48h进行β-半乳糖苷酶衰老染色检测NPCs衰老比例,免疫荧光染色观察蛋白聚糖(ACAN)、Ⅱ型胶原(COL2)的表达。干...

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

Objectives:To investigate the biological effects of human urine stem cell-derived exosomes(USCExos)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 48 h,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 12 h,the uptake of USC-Exos by NPCs was observed under a laser confocal microscope.Both groups were tested forβ-galactosidase aging staining 48 h 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 RTPCR and WB at 3 d,5 d and 7 d 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 50-100 nm 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.48 h 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 3 d,5 d,and 7 d 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 m RNA and corresponding proteins.