氧浓度对人髓核间充质干细胞生物学特性的影响

Biological characteristics of human nucleus pulposus mesenchymal stem cell under different oxygen concentrations

中文关键词:  椎间盘退变  髓核间充质细胞  氧浓度  生物学特性

英文关键词:Intervertebral disc degeneration  Nucleus pulposus mesenchymal stem cell(NPMSCs)  Oxygen concentrations  Biological characteristics

中文摘要:

  【摘要】 目的：分离培养髓核来源的间充质干细胞（MSCs），比较不同氧浓度下细胞的生物学特性，探讨氧浓度对椎间盘退变机制的影响。方法：用胶原酶消化法从手术摘除的4个腰椎间盘（椎间盘Pfirrmann分级为Ⅰ级或Ⅱ级）髓核组织中分离MSCs，体外培养、传代，观察记录细胞形态。取P3代细胞，用流式细胞仪对分离得到的细胞表面抗原CD90、CD73、CD105、CD44和CD31、CD34、CD45的表达情况进行检测；用成骨、成脂、成软骨培养液诱导培养细胞，分别在21d、28d、21d时用茜素红、油红O、甲苯胺蓝对细胞进行染色，观察其成骨、成脂、成软骨能力。在三气培养箱的低氧条件（2% O2、5% CO2、37℃）和常规细胞培养箱的常氧条件（20% O2、5% CO2、37℃）下分别培养P3代细胞。通过细胞计数统计培养1d、2d、3d、4d、5d、6d、7d、8d时的细胞数量，比较不同氧浓度下细胞的生长曲线；使用Architect c8000自动生化检测仪检测培养1d、2d、3d、4d、5d时培养基的pH值和渗透压。实时荧光定量（qRT-PCR）检测不同氧浓度培养下细胞的干性基因POU家族类别5同位序列1（POU5F1，OCT4）、NANOG同位序列（NANOG）、性别决定区Y盒2（SOX2）及扩增基因细胞周期蛋白D1（CyclinD1，CCND1）、MYC（c-Myc）、低氧诱导基因低氧诱导因子2α（HIF2α，EPAS1）、能量基因三磷酸腺苷合成酶（ATP5A1）、线粒体相关基因细胞色素c氧化酶Ⅳ亚基1型同工酶（COX4I1）、线粒体转录因子A（TFAM）、线粒体编码细胞色素c氧化酶Ⅰ（MT-CO1）、MT-CO2的mRNA表达情况。结果：分离培养的细胞呈典型的单层贴壁生长，纺锤样；P3代细胞免疫表型鉴定显示MSCs表面分子标记高表达CD90（80.4%）、CD73（99.9%）、CD105（99.8%）、CD44（95.9%），低表达CD31（5.3%）、CD34（4.4%）、CD45（6.8%）；茜素红、油红O染色、甲苯胺蓝染色证实细胞可向骨细胞、脂肪细胞、软骨细胞分化。根据国际干细胞治疗协会（ISCT）有关MSCs的判定标准，分离培养的细胞为髓核MSCs（NPMSCs）。低氧环境下培养的细胞形态更小，更接近原始MSCs，细胞增殖更快，低氧时倍增期为31.22±1.98h，常氧时倍增期为39.56±2.02h，差异有统计学意义（P<0.05）。不同氧浓度各时间点培养基的pH值、渗透压差异无统计学意义（P>0.05），且皆在适合细胞生存的范围。低氧培养下POU5F1（OCT4）、NANOG、SOX2、CCND1、 MYC（c-Myc）、EPAS1、ATP5A1较常氧培养时显著性升高（P<0.05）；COX4I1、TFAM、MT-CO1、MT-CO2较常氧培养时显著性降低（P<0.05）。结论：低氧条件下培养有利于人NPMSCs的细胞形态、干性基因、增殖能力等生物学活性的维持。

英文摘要:

  【Abstract】 Objectives: To observe the biological effects of different oxygen concentrations on human nucleus pulposus mesenchymal stem cells(NPMSCs) in vitro, and to explore the mechanism of intervertebral disc degeneration. Methods: MSCs obtained in this study were isolated from non-degenerative nucleus pulposus(Pfirrmann Ⅰ or Ⅱ) which were harvested from 4 patients with congenital scolisis, and then were cultured and passaged in vitro. The immunophenotypes(CD90, CD73, CD105, CD44, CD31, CD34, CD45) of the third generation cells were identified by flow cytometry, and then were induced into osteogenesis, adipogenic and chondroblast cells which were identified by alizarin red, oil red O and toluidine blue staining, respectively. Briefly, the cells should be identified by the criteria of the International Society for Cellular Therapry (ISCT). The 2% oxygen concentration was set to mimic oxygen level of intervertebral disc degeneration and 20% oxygen concentration was used as normoxic oxygen concentration and then the cells were further cultured in different oxygen concentration(2% O2, 5% CO2, 37℃; 20% O2, 5% CO2, 37℃; respectively). The morphology of the cells was observed by inverted microscope at different time points and the growth curve was analyzed by cell counting. The pH and osmotic pressure of the culture medium at each time point were measured by using the Architect c8000 automatic biochemical detector. The mRNA expression levels of stemness-related genes(POU5F1, NANOG, SOX2), proliferation-related genes(CCND1, MYC), hypoxia-induced genes(EPAS1), Energy related gene(adenosine triphosphate synthetase, ATP5A1) and mitochondria -related genes[cytochrome c oxidase Ⅳ subunit 1 isoenzyme(COX4I1), mitochondrial transcription factor A(TFAM), mitochondrial coding for cytochrome c oxidase I(MT-CO1), MT-CO2 were evaluated by QRT-PCR. Results: Nucleus pulposus mesenchymal stem cells(NPMSCs) grew into typical, adherent monolayer cells, with high expressions of CD90(80.4%), CD73(99.9%), CD105(99.8%), CD44(95.9%) and low expressions of CD31(5.3%), CD34(4.4%), CD45(6.8%). Alizarin red staining showed deposition of large amounts of calcium nodules in NPMSCs. Oil red O staining displayed a large amount of lipid droplets deposited in cells. Chondrogenic differentiation was positive for alcian blue staining. Based on the above results, the cells obtained in this study met the criteria of ISCT and these cells were absolute mesenchymal stem cells. NPMSCs that were cultured in a hypoxic atmosphere(2% O2) became comparatively smaller in size, with an original spindle-shaped morphology. The growth curve assays showed that the doubling time of NPMSCs at hypoxia was significantly earlier than that of cells grown at normoxia (31.22±1.98h, 39.56±2.02h, respectively, P<0.05). The pH and osmolality from different conditions were within limits to promote normal cell growth(P>0.05). The major markers of primitive stem cells, POU5F1(Oct4), NANOG and SOX2 increased in NPMSCs grown at 2% O2 compared with NPMSCs cultured in 20% O2(P<0.05). Under the condition of hypoxia, the proliferation-related genes CCND1(CyclinD1) and MYC(c-Myc) significantly increased(P<0.05), and the hypoxia-inducible factor HIFα-related gene EPAS1 was also significantly elevated(P<0.05). The relative mRNA expression levels of ATP5A1 and nuclear-encoded energy-related gene, were significantly elevated under hypoxia(P<0.05). However, nuclear coding mitochondria -related genes, the mRNA expression levels of COX4I1 and TFAM significantly decreased(P<0.05) in hypoxic conditions. Still, the mRNA expression levels of MT-CO1 and MT-CO2 also significantly decreased under hypoxic conditions(P<0.05). Conclusions: Hypoxic atmosphere(2% O2) favored a more primitive morphology, higher NPMSC proliferation rate, and better stemness-related genes expression.