摘要
目的探索非梗阻性无精症(NOA)患者尿液中尿源干细胞(USCs)的分离培养与鉴定技术。方法收集北部战区总医院2018—2019年收治的3例NOA成年男性患者的尿液。通过离心提取、贴壁培养等方法扩增USCs,观察细胞形态;通过CCK-8实验、流式细胞术、免疫荧光技术等检测USCs的增殖能力、细胞周期、表面标志物表达情况及其向骨、软骨及脂肪细胞分化的能力。结果NOA患者USCs具有米粒样外观,呈对数生长;造血干细胞系表面标志物(CD31、CD34、CD45)表达呈阴性,间充质干细胞系表面标志物(CD44、CD133)、胚胎干细胞表面标志物(SSEA4)、肾周细胞表面标志物(CD146、PDGFRB、NG2)表达呈阳性;经骨、软骨及脂肪细胞定向诱导培养基诱导培养后,分别表达骨、软骨及脂肪细胞表面标志物hOsteocalcin、hAggrecan、mFABP4。结论本研究成功获得NOA患者USCs分离培养与鉴定的方法,为进一步探索USCs治疗NOA患者提供了实验基础。
Objective To investigate the method of isolation and identification of urine-derived stem cells(USCs)in patients with non-obstructive azoospermia(NOA).Methods Urine was collected from 3 adult male NOA patients admitted in 2018—2019.The USCs were amplified by centrifugation and adherent culture to observe the cell morphology.Proliferation,cell cycle,expression of surface markers,and differentiation of USCs into bone,cartilage and adipocytes were determined by CCK-8 assay,flow cytometry,and immunofluorescence.Results The USCs in NOA patients had a rice-grain appearance and showed logarithmic growth.The expression of surface markers of hematopoietic stem cell lines(CD31,CD34,CD45)was negative,while the expression of surface markers of mesenchymal stem cell lines(CD44,CD133),surface markers of embryonic stem cells(SSEA4),and surface markers of perirenal cells(CD146,PDGFRB,NG2)were positive.The surface markers of bone,cartilage and adipocyte were expressed after induction culture by bone,cartilage and adipocyte directed induction medium.Conclusion The present study successfully established the method of isolation and culture of USCs from NOA patients,providing an experimental basis for exploring the clinical study of USCs in the treatment of NOA patients.
作者
王映龙
陈婧
毛杨兴
骆梦兰
范连慧
张乾
WANG Ying-long;CHEN Jing;MAO Yang-xing;LUO Meng-lan;FAN Lian-hui;ZHANG Qian(Department of Anesthesiology,University-Town Hospital of Chongqing Medical University,Chongqing 401331,China;Department of Urology,the Second Affiliated Hospital of Army Medical University,Chongqing 400037,China;Department of Urology,General Hospital of Northern Theater Command,Shenyang 110016,China)
出处
《临床军医杂志》
CAS
2020年第9期995-999,1002,共6页
Clinical Journal of Medical Officers
基金
辽宁省博士科研启动基金(20170520334)
关键词
非梗阻性无精症
尿液
尿源干细胞
分离
培养
鉴定
Non-obstructive azoospermia
Urine
Urine-derived stem cells
Isolation
Culture
Identification
