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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,P0.05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(P0.05),P16基因mRNA及对应蛋白的相对表达量较对照组显著性降低(P0.05),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。 相似文献
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目的 :观察皮质骨螺钉(cortical bone trajectory,CBT)加传统椎弓根螺钉(traditional trajectory,TT)结合卫星棒、远端固定至S1治疗成人退变性脊柱侧凸(adult degenerative scoliosis,ADS)的可行性及临床疗效。方法:回顾性分析2014年6月～2018年1月在我院采用长节段融合CBT+TT结合卫星棒、远端固定至S1矫形固定治疗的11例ADS患者的资料。比较患者术前和末次随访时的腰背痛视觉模拟评分(visual analogue scale,VAS)、Oswestry功能障碍指数(Oswestry disability index,ODI),术前、术后即刻和末次随访时的Cobb角、C7铅垂线(C7 plumb line,C7PL)与骶骨中垂线(center sacral vertical line,CSVL)的相对距离(C7PL-CSVL)和脊柱矢状轴(sagittal vertebral axis,SVA)。结果:11例患者均为女性,年龄51～73岁(64.36±7.63岁),手术时间247.64±44.96min,术中出血量1118.18±464.37ml。1例患者术后出现脑脊液漏,延长引流时间、换药后愈合;1例患者术后出现一过性肌力减退,给予口服甲钴胺2周,于术后3个月恢复,无其他严重并发症发生。术后随访13～60个月(33.87±14.36个月),术前VAS评分为7.00±0.89分,末次随访时为0.91±0.70分,差异有统计学意义(P0.05);术前ODI为(51.09±7.83)%,末次随访时为(5.45±1.13)%,差异有统计学意义(P0.05)。术前、术后即刻、末次随访时冠状面Cobb角分别为49.10°±11.51°、12.05°±3.78°、13.06°±3.38°,C7PL-CSVL分别为27.27±17.61mm、12.20±8.04mm、12.40±8.05mm,SVA分别为25.33±18.21mm、8.60±5.31mm、9.75±6.94mm,末次随访时的Cobb角、C7PL-CSVL、SVA与术前比较均有统计学差异(P0.05);末次随访时与术后即刻比较均无统计学差异(P0.05)。随访期内所有患者均未出现内固定失败征象。结论:CBT+TT结合卫星棒矫形远端固定至S1长节段腰骶融合可增强脊柱骨盆固定强度,治疗ADS可取得较好的临床疗效。 相似文献
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BACKGROUND: Intervertebral disc degeneration is the pathological basis of degenerative spinal diseases. Studies on the influential factors of intervertebral disc degeneration contribute to the prevention and treatment of degenerative spinal disease.
OBJECTIVE: To observe the growth and proliferation of nucleus pulposus cells isolated by trypsin plus type II collagenase digestion in complete medium with different glucose concentrations, exploring the optimal glucose concentration for growth of nucleus pulposus cells.
METHODS: Nucleus pulposus cells isolated and cultured by trypsin plus type II collagenase digestion method were observed under an inverted microscope, and the cell number was counted. Morphology of nucleus pulposus cells was observed after hematoxylin-eosin staining and toluidine blue staining. Collagen type II immunoreactivity was detected by immunohistochemical staining combined with immunofluorescent staining. Nucleus pulposus cells were incubated in complete medium containing various glucose concentrations (0, 6.25, 12.5, 17.5, and 25 mmol/L) for 24 hours, and then cell proliferation and apoptosis were determined.
RESULTS AND CONCLUSION: The stained nucleus pulposus cells showed polygonal and short spindle, with one or two nuclei. Cellular pseudopod appeared gradually and then became slim with increased passage numbers. The isolated and cultured nucleus pulposus cells positively expressed collagen type II and aggrecan Proliferative activity of nucleus pulposus cells cultured in medium with 17.5 mmol/L glucose was significantly higher than that in medium with 0 and 25 mmol/L glucose (P < 0.05 or P < 0.01). There was no significant difference in cell apoptosis between these groups except for 0 mmol/L glucose (P < 0.05). These results confirm that a large number of nucleus pulposus cells can be harvested by trypsin plus type II collagenase digestion and the optimal glucose concentration is 17.5 mmol/L.
中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程 相似文献
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