血清浓度对人髓核间充质干细胞生物学活性的影响

Effect of serum concentration on the biological activity of human nucleus pulposus mesenchymal stem cells

目的 :体外观察、研究不同血清浓度对人正常髓核间充质干细胞(nucleus pulposus mesenchymal stem cells,NPMSCs)生物学活性的影响。方法:选取先天性脊柱侧凸矫形患者手术切除的髓核组织(Pfirrmann分级为Ⅰ级或Ⅱ级),体外分离培养NPMSCs并传代培养。利用流式细胞仪分别对P3代细胞的表型[CD105、CD90、CD73、CD45、CD34和人类白细胞抗原(HLA)-DR]进行检测,并观察其向成骨、成脂和成软骨诱导分化能力。取P3代NPMSCs在无血清(0%)及含有5%和10%体积浓度胎牛血清(fetal bovine serum,FBS)的DMEM培养基中进行培养,分别对细胞增殖能力、细胞周期、细胞凋亡率进行检测,并利用实时荧光定量PCR(Quantitative Real-time PCR,QRT-PCR)技术检测八聚体结合转录因子4 (octamer-binding transcription factor 4,Oct4)、Nanog、Jag1、Notch1干性基因和Ⅰ型胶原、Ⅱ型胶原、蛋白多糖和性别决定区Y框蛋白9 (SRY related HMG box-9,SOX-9)的表达量。结果:从正常髓核组织中分离培养的细胞呈纺锤状、贴壁生长,可体外扩增,流式细胞仪检测结果显示细胞高表达CD73(98.9%)、CD90(97.9%)、CD105(98.9%),低表达HLA-DR(3.11%)、CD34(1.83%)、CD45(1.19%),且可以向成骨细胞、脂肪细胞及类软骨细胞分化。根据国际干细胞治疗协会(International Society for Cellular Therapy,ISCT)评定标准,分离培养的细胞为NPMSCs。随着FBS浓度的降低,细胞增殖率显著下降,细胞凋亡率明显增加,而且可以显著延长G1期时长(P<0.05)。QRT-PCR结果显示:随着FBS浓度的下降,干性基因(Oct4、Nanog、Jag1、Notch1)的相对表达量明显减少,Ⅰ型胶原相对表达量增加,Ⅱ型胶原、蛋白多糖和SOX-9的相对表达量减少,差异均具有统计学意义(P<0.05)。结论:血清浓度降低可显著抑制NPMSCs细胞增殖、干性基因及相关功能基因的表达。

Objectives:To observe and research the effects of different serum concentrations on the biological activity of human normal nucleus pulposus mesenchymal stem cells(NPMSCs)in vitro.Methods:The nucleus pulposus tissue of patients with congenital scoliosis correction(Pfirrmann gradeⅠorⅡ)was selected and NPMSCs were isolated and cultured in vitro.First,Flow cytometry was used to detect cells phenotypes(CD105,CD90,CD73,CD45,CD34,and HLA-DR).The ability to induce differentiation into osteogenic,adipogenic and chondrogenic cells was then observed.After that,third passage NPMSCs were cultured in DMEM medium containing different concentrations of fetal bovine serum(FBS)(0%,5%,and 10%)to determine their cell proliferation capacities,cell cycles,apoptosis rates,and stemness genes(Oct4,Nanog,Jag1,and Notch1)and functional genes(collagenⅠ,collagenⅡ,aggrecan,and SOX-9)expressions were detected by Quantitative Real-time PCR(QRT-PCR),respectively.Results:The cells isolated and cultured from normal nucleus pulposus tissues grew in a spindle shape,adhered to the wall,and could be passaged in vitro.The im-munophenotype results showed the cells highly expressed CD73(98.9%),CD90(97.9%),and CD105(98.9%),and lowly expressed HLA-DR(3.11%),CD34(1.83%),and CD45(1.19%).The isolated cells could be differentiated into osteoblasts,adipocytes and chondrocytes.According to the criteria of the International Society for Cellular Therapy(ISCT),the isolated and cultured cells were NPMSCs.With the decrease of FBS concentration,the cell proliferation rate decreased,cell apoptosis rate increased significantly,and the G1 phase length prolonged(P<0.05).The QRT-PCR results showed that with the decrease of FBS concentration,the relative expression level of stemness genes(Oct4,Nanog,Jag1,and Notch1)decreased significantly,the relative expression level of collagenⅠincreased,and the relative expression levels of collagenⅡ,aggrecan and SOX-9 decreased.There were statistically significant differences(P<0.05).Conclusions:Decreased serum concentration can significantly suppress the proliferation of NPMSCs,and inhibit the expression of stemness genes and related functional genes.