【作者】 刘洋；

【导师】 张亮；

【作者基本信息】 大连医科大学 ， 外科学（专业学位）， 2019， 硕士

【摘要】 目的通过比较SD大鼠的N-NPMSC(Nucleus Pulposus Mesenchymal Stem Cell Derived From Normal Intervertebral Disc,正常椎间盘来源髓核间充质干细胞)与D-NPMSC(Nucleus Pulposus Mesenchymal Stem Cell Derived From Degenerative Intervertebral Disc,退变椎间盘来源髓核间充质干细胞)在细胞增殖与凋亡及干细胞干性维持等生物学性能上是否存在差异,探讨内源性NPMSC(Nucleus Pulposus Mesenchymal Stem Cell,髓核间充质干细胞)的生物学性能是否会随着椎间盘退变而改变。方法取3月龄清洁级SD大鼠,通过尾椎椎间盘穿刺法建立退变模型,即使用21G穿刺针、穿刺深度为5mm穿刺SD大鼠Co(Coccygeal,尾椎)5/6及Co7/8两个节段作为退变组,未穿刺节段作为正常组,2周后通过MRI(Magnetic Resonance Imaging,核磁共振成像)确认退变模型的建立。随后从正常组及退变组中获得正常及退变髓核组织,提取N-NPMSC及D-NPMSC并进行体外扩增培养。通过流式细胞技术检测细胞表型;RT-PCR技术检测Sox-2(Sex Determining Region Y-box2,性别决定区域Y相关基因2)、Oct-4(Octamer-binding Transcription Factor 4,八聚体结合转录因子4)及Nanog干性基因表达;并进行成骨、成脂及成软骨三系诱导分化,诱导28d后,应用茜素红染色鉴定细胞成骨能力,油红O染色鉴定细胞成脂能力,甲苯胺蓝染色鉴定成软骨能力,最后按照ISCT(International Society for Cellular Therapy,国际干细胞治疗协会)提出的间充质干细胞的判定标准,对分离得到的细胞进行综合评估鉴定。通过观察细胞形态,检测细胞克隆形成能力,CCK-8法检测细胞增殖,流式细胞仪检测细胞凋亡,caspase-3试剂盒检测caspase-3表达活性,RT-PCR及Western Blot检测HIF-1α(Hypoxia-inducible Factor-1α,低氧诱导因子1α)、GLUT-1(Glucose Transporter 1,葡萄糖转运蛋白1)、VEGF(Vascular Endothelial Growth Factor,血管内皮生长因子)、SIRT1(Silent Information Regulator Protein 1,沉默信息调节蛋白1)及SIRT6(Silent InformationRegulator Protein 6,沉默信息调节蛋白6)的mRNA及蛋白表达等方法,比较不同来源NPMSC的生物学特性。结果不同椎间盘来源NPMSC均高表达干细胞相关阳性表面抗原分子,低表达干细胞相关阴性表面抗原分子,均可向骨、脂肪及软骨细胞诱导分化,但D-NPMSC干性基因表达明显下降(P<0.05)。两组细胞在形态学上无显著差异,原代细胞均呈贴壁生长,早期可形成葵花样细胞集落,传代后增殖明显加快,细胞形态以纺锤形为主。D-NPMSC细胞克隆形成能力减弱,细胞增殖速度减慢,而细胞凋亡率及caspase-3表达活性显著增加(P<0.05)。RT-PCR及Western Blot显示退变椎间盘来源NPMSC中HIF-1α、GLUT-1、VEGF、SIRT1及SIRT6中mRNA及蛋白表达均明显低于正常椎间盘来源NPMSC(P<0.05)。结论正常及退变IVD(Intervertebral Disc,椎间盘)中均存在内源性NPMSC,D-NPMSC表现出增殖能力减弱、干细胞干性维持能力下降及细胞凋亡增加的生物学特性,而N-NPMSC具有更好的细胞代谢活性及更强的细胞外分泌能力。我们推测,HIF-1α介导的信号通路可能参与NPMSC增殖的调控。更多还原

【Abstract】 Objective To compare the biological characteristics of nucleus pulposu mesenchymal stem cells(NPMSC)derived from normal and degenerative intervertebral disc(IVD).Methods NPMSC were isolated from normal and degenerative IVD and cultured in vitro.Flow cytometric analysis was performed to detect the expressions of cell surface markers including CD73,CD90,CD105,CD45 and CD11b/c.The expression of the stemness genes sex determining region Y-box 2(Sox-2),octamer-binding transcription factor 4(Oct-4)and Nanog was determined RT-PCR.NPMSC from normal(N-NPMSC)and degenerative IVD(D-NPMSC)were induced to differentiate toward osteogenic,adipogenic and chondrogenic linages.The biological characteristics of the NPMSC,including the cell colony formation,cell proliferation cell apoptosis,caspase-3 activity,mRNA and protein expressions of HIF-1α,GLUT-1,VEGF,SIRT1 and SIRT6 were compared.Results NPMSC from both groups highly expressed CD105,CD90 and CD73(characteristic cell surface marker of MSC),and lower expressed CD45 and CD11b/c.The original cells from both normal and degenerative IVD could form sunflower-like colonies and the third passage NPMSC became homogeneous and exhibited spindle-like morphology.The cell morphology of two groups showed no significant difference.N-NPMSC and D-NPMSC had a comparable capacity for osteogenic,adipogenic and chondrogenic multipotent differentiation,but N-NPMSC showed a lower expression of stemness gene(P < 0.05).D-NPMSC showed lower clonogenic capacity and proliferative activity than N-NPMSC,while an increased rate of cell apoptosis and expression of caspase-3(P < 0.05).D-NPMSC also showed lowerexpression of HIF-1α,GLUT-1,VEGF,SIRT1 and SIRT6 in the mRNA and protein level than N-NPMSC(P < 0.05).Conclusions NPMSC can be isolated from both normal and degenerative IVD.However,NPMSCs derived from degenerative IVD showed different biological behaviors including lower proliferative activity,lower expression of stemness gene and higher cell apoptosis rate.The HIF-1α mediated pathway may be involved in the mechanism of cell proliferation.更多还原