人髓核细胞诱导人尿源性干细胞向髓核样细胞分化作用的研究

Studies on the differentiation of urine-derived stem cells into nucleus pulposus cells induced by nucleus pulposus cells

中文关键词:  尿源性干细胞  髓核细胞  共培养  分化

英文关键词:Urine-derived stem cells  Nucleus pulposus cells  Co-culture  Differentiation

中文摘要:

  【摘要】 目的：探讨人髓核细胞（NPCs）诱导对人尿源性干细胞（USCs）向髓核样细胞分化的作用。方法：手术时获取腰椎间盘突出症患者L4/5的髓核组织，并采用贴壁法体外分离培养NPCs；从健康成年人尿液中获取USCs并进行体外培养，通过倒置显微镜观察细胞形态，并采用成骨、成脂、成软骨三系诱导分化及蛋白免疫印迹技术（Western blot，WB）对所获取的USCs进行形态、分化潜能及细胞表面标志蛋白鉴定。使用Transwell小室将P3代NPCs与USCs培养以建立共培养体系，设实验组、对照组及NPCs组，实验组为NPCs与USCs共培养组，对照组为单独USCs培养，NPCs组为单独NPCs培养；培养14d后应用倒置显微镜观察细胞形态；应用定量实时聚合酶链反应（qRT-PCR）及WB分别检测实验组USCs、对照组USCs与NPCs组NPCs中蛋白多糖（ACAN）、SOX-9（SRY-related high mobility groupbox gene 9）、Ⅱ型胶原（COL2）及缺氧诱导因子1α（HIF-1α）的mRNA及蛋白的表达情况；免疫荧光染色观察3组 COL2A1及ACAN荧光表达。结果：培养的USCs成骨、成脂、成软骨三系分化实验结果均为阳性；USCs中干细胞阳性标志物CD29、CD44、CD73和CD90呈高表达，未检出干细胞阴性标志物CD34和CD45。培养14d后倒置显微镜下对照组及NPCs组细胞形态无变化，实验组USCs向髓核样细胞分化，形态变化明显。经共培养诱导14d后实验组及NPCs组中的ACAN、SOX-9、COL2A1及HIF-1α基因mRNA及蛋白表达均显著高于对照组（P<0.05），ACAN及COL2A1荧光强度明显高于对照组；实验组上述各种mRNA及蛋白表达与NPCs组比较均无统计学差异（P＞0.05），实验组荧光强度与NPCs组比较无明显差异。结论：在体外实验中，人NPCs可通过共培养的方式诱导人USCs分化为髓核样细胞，可为椎间盘组织工程研究提供NPCs来源。

英文摘要:

  【Abstract】 Objectives: To investigate the role of human nucleus pulposus cells(NPCs) in inducing the differentiation of human urine-derived stem cells(USCs) into nucleus pulposus cells. Methods: The nucleus pulposus tissue of L4/5 from patients with lumbar disc herniation was obtained during surgery, and NPCs were isolated and cultured in vitro by adherent method. The USCs were obtained from the urine of healthy adults and cultured in vitro. Cell morphology was observed by inverted microscope. Morphology, differentiation potential and cell surface marker proteins of USCs obtained were identified by osteogenic, lipogenic and chondrogenic differentiation induction and Western blot (WB). The Transwell chamber was used to culture P3 NPCs with USCs to establish a co-culture system. The experimental group, control group and NPCs group were set up. The experimental group was the co-culture group of NPCs and USCs, while the control group was of single USCs and the NPCs group was of single myeloid nucleus cells. After 14 days of culture, the morphology of the cells was observed by inverted microscope. The mRNA and protein expressions of aggrecan (ACAN), SOX-9 (SRY-related high mobility groupbox gene 9), collagen type Ⅱ (COL2) and hypoxia-inducing factor 1α (HIF-1α) were detected by fluorescence quantitative PCR (qRT-PCR) and WB, respectively. Immunofluorescence staining was used to observe the fluorescence expressions of COL2A1 and ACAN in the 3 groups. Results: The results of osteogenic, lipogenic, and chondrogenic differentiation of USCs were all positive. The CD29, CD44, CD73 and CD90 were highly expressed in USCs, while CD34 and CD45 were not detected. After 14 days of culture, the morphology of USCs group and NPCs group remained unchanged under inverted microscope, while the morphology of USCs cells in the experimental group differentiated into nucleus pulposus cells, and the morphological changes were obvious. The mRNA and protein expressions of ACAN, SOX-9, COL2A1 and HIF-1α genes in NPCs group and experimental group of USCs cells were significantly higher than those in the control group after 14 days of coculture induction(P<0.05). Immunofluorescence showed that the fluorescence intensity of ACAN and COL2A1 was significantly higher in the NPCs group and the experimental group than in the control group. There was no statistical difference between the experimental group and the NPCs group in terms of mRNA, protein expression and fluorescence intensity. Conclusions: In in vitro experiments, human NPCs can induce human USCs to differentiate into nucleus pulposus cells by co-culture, which can provide a source of NPCs for disc tissue engineering research.