| 周荣耀,郭 柱,苏炜良,秦 明,张国庆,王 岩,吴晓淋,陈五军,邢东明,相宏飞,陈伯华.人尿源干细胞外泌体对退变髓核细胞生物学功能的影响[J].中国脊柱脊髓杂志,2020,(5):427-436. |
| 人尿源干细胞外泌体对退变髓核细胞生物学功能的影响 |
| 中文关键词: 人尿源干细胞 髓核细胞 外泌体 细胞外基质 |
| 中文摘要: |
| 【摘要】 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P6 NPCs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USC-Exos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。干预后3d、5d、7d时用RT-PCR和WB检测两组NPCs中ACAN、COL2、基质金属蛋白酶组织抑制因子1（TIMP1）、多肿瘤抑制基因P16（P16）的mRNA及对应蛋白的表达量，对两组ACAN、COL2、TIMP1、P16基因的相对表达量进行比较，P<0.05为有统计学差异。结果：从人尿中提取的细胞具有干细胞特性，传代培养后可从中提取粒径为50~100nm的椭圆形囊泡结构，其表达CD63和Tsg101，不表达Calnexin蛋白，符合Exos特性。P6 NPCs经USC-Exos干预后细胞增殖速度变快。经PKH26标记的USC-Exos可被NPCs摄取。干预后48h实验组衰老细胞比例[（13.8±1.4）％] 低于对照组[（19.6±2.4）％]，ACAN、COL2的荧光强度高于对照组。干预后3d、５d、７d时，实验组细胞中ACAN、COL2、TIMP1 mRNA及对应蛋白的相对表达量较对照组显著性升高（P＜0.05），P16基因mRNA及对应蛋白的相对表达量较对照组显著性降低（P＜0.05），而同组内不同时间点比较无显著性差异。结论：USC-Exos在体外条件下可促进退变NPCs的增殖，提高退变NPCs中ACAN、COL2、TIMP1 mRNA及对应蛋白的表达，降低P16 mRNA及对应蛋白的表达。 |
Biological effects of human urine derived stem cell exosomes on degenerated nucleus pulposus cells |
| 英文关键词:Urine-derived stem cells Nucleus pulposus cells Exosome Extracellular matrix |
| 英文摘要: |
| 【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. |
| 投稿时间：2019-11-29 修订日期：2020-02-05 |
| DOI： |
| 基金项目:国家自然科学基金资助项目（编号：81802190，81772412）；山东省自然科学基金（编号：ZR2019BH-084）；中国博士后基金（2019M652329）；青岛市应用基础研究计划 （编号：19-6-2-51-cg）；泰山学者青年专家工程资助（编号：tsqn201909190） |
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