人椎间盘髓核来源的诱导多能干细胞重编程及功能的研究

发布时间：2018-04-16 08:05

本文选题：椎间盘退行性疾病 + 髓核　；参考：《生物化学与生物物理进展》2017年07期

【摘要】：椎间盘退变始发于髓核组织,获得足够有功能的髓核细胞是研究及治疗椎间盘退变的关键.而人诱导多能干细胞(induced pluripotent stem cell,iPSC)不仅为建立疾病模型以研究疾病发生发展机制开辟了道路,还在再生医学领域展现出了广阔的应用前景.我们首先从椎间盘退变患者微创手术获得的髓核组织内分离髓核细胞,将携带OCT3/4、SOX2、KLF4和c-MYC的仙台病毒(Sendai virus,Se V)转染髓核细胞,重编程获得iPSC.通过检测多能细胞特异性标志、体内成瘤实验、甲基化及核型分析对所获得的iPSC进行鉴定.并以皮肤成纤维细胞来源iPSC作为对照,在二维和三维水凝胶中对iPSC进行定向分化,检测髓核细胞相关蛋白和基因的表达,比较分析2种iPSC向髓核细胞的分化效率.结果显示,iPSC能表达多能细胞特异性标志,具有正常的二倍体核型,畸胎瘤实验显示三个胚层的出现.诱导分化后的iPSC表达髓核相关基因和蛋白,在水凝胶中诱导培养后,iPSC表达更多的髓核相关基因和蛋白.髓核来源的iPSC与成纤维细胞来源的iPSC相比,可表达更多的髓核相关基因和蛋白.本研究首次将患者退变髓核细胞重编程成iPSC,并在水凝胶内将其诱导分化为髓核样细胞,为椎间盘退变个体化细胞治疗奠定基础.
[Abstract]:Disc degeneration originated from nucleus pulposus tissue and the key to study and treat disc degeneration is to obtain enough functional nucleus pulposus cells.Human induced pluripotent stem cell induced pluripotent stem cell sci not only opens the way for the establishment of disease models to study the mechanism of disease occurrence and development, but also shows a broad application prospect in the field of regenerative medicine.We first isolated the nucleus pulposus cells from the nucleus pulposus tissue obtained from minimally invasive surgery in patients with disc degeneration. We transfected the nucleus pulposus cells with OCT3 / 4 SOX2KLF4 and Sendai virusSe V, and reprogrammed to obtain iPSCs.The iPSC was identified by detecting specific markers of pluripotent cells, tumorigenesis in vivo, methylation and karyotype analysis.IPSC derived from skin fibroblasts was used as control. IPSC was differentiated in two-dimensional and three-dimensional hydrogels to detect the expression of related proteins and genes in nucleus pulposus cells. The differentiation efficiency of two kinds of iPSC into nucleus pulposus cells was compared and analyzed.The results showed that iPSC could express pluripotent cell specific markers and had normal diploid karyotype. Teratoma test showed the appearance of three embryo layers.After induction and differentiation, iPSC expressed more genes and proteins of nucleus pulposus, and more genes and proteins of nucleus pulposus were expressed after induction and culture in hydrogel.Compared with iPSC derived from fibroblasts, iPSC derived from nucleus pulposus could express more genes and proteins associated with nucleus pulposus.In this study, the degenerative nucleus pulposus cells were reprogrammed into iPSCs for the first time, and induced to differentiate into nucleus pulposus like cells in hydrogel, which laid the foundation for individualized cell therapy of degenerative intervertebral disc.
【作者单位】：深圳大学医学部;深圳大学附属第一医院脊柱外科;
【基金】：国家自然科学基金(81301597) 深圳新兴战略产业发展专项资金(JCYJ20150525092940984/JCYJ20160422090807181)资助项目~~
【分类号】：R681.5

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