基于iPS椎间盘退行性疾病模型的建立及机理研究

Disc degenerative disease (DDD) has been described as an aberrant, cell-mediated, age-, environment- and genetics-dependent molecular degeneration process. Many studies indicate that degenerative changes are predominately due to a loss of nucleus pulposus (NP) cell functionality, so it is important to study the functional changes of NP cells during disc degeneration. NP cells and NP stem cells isolated from NP tissue are limited, NP cells from DDD are always at the end stage of disease, similar to chondrocyte culture, NP cells cultured as monolayer undergo changes such as aberrant flattened morphology and gene expression in terms of dedifferentiation, and all of these are not benefit for the study of DDD mechanism. Induced pluripotent stem cell (iPS) can differentiated into all kinds of tissue cells in the whole body, besides, iPS cells from patient may have "disease memory", which can be used as a good disease model in vitro. In this study, first, we compare the phenotypes and epigenetics differences between the normal and degenerated NP cells, and then the NP cells were reprogrammed into iPS cells which were differentiated into NP cells, KRT18 and FBLN1 double positive cells were sorted by FACS, the phenotypes and epigenetics were tested, and use double positive cells as DDD cell model. iPS cell from normal tissue as control, the effect of HIF on the functions and epigenetics of iPS-NP cells were studied. In conclusion, we study the mechanism of DDD from hypoxia environment-epigenetics-genes base on iPS cells, which will be a good disease model for drug discovery and drug testing.

椎间盘退行性疾病（DDD）是与年龄密切相关的、多基因和多种环境因素所致的一类疾病，明确椎间盘髓核组织细胞功能变化是研究DDD发生发展机制的关键。我们所能研究的都是疾病末期已经退变的髓核细胞，数量极少，体外培养易失去软骨细胞特性且发生去分化。诱导的多能干细胞（iPS细胞）出现为体外研究椎间盘退行性变发生发展的机制提供了较好的体外细胞模型。我们分别从脊柱侧弯病人和椎间盘退行性变的病人体内获得正常和退变的髓核组织，将分离得到髓核细胞重编程为iPS细胞，并将其定向诱导分化为髓核细胞（iPS-NPC），作为体外研究DDD的细胞模型。以正常组织来源iPS作为对照，研究疾病组织来源的iPS-NPC在分化过程中功能活性和表观遗传学改变，并研究低氧诱导因子（HIF-1）修饰后对iPS-NPC功能活性和表观遗传特征的影响，从环境因素-表观遗传-基因三个方面阐明DDD发生发展的机制，为发现新的药物靶点提供依据。

椎间盘退行性疾病（DDD）是与年龄密切相关的、多基因和多种环境因素所致的一类疾病，明确椎间盘髓核组织细胞功能变化是研究DDD发生发展机制的关键。诱导的多能干细胞（iPS细胞）出现为我们在体外研究椎间盘退行性变发生发展的机制和细胞移植治疗提供了一个较好的细胞来源。我们分别从椎间盘退行性变的病人体内获得正常真皮成纤维细胞和退变的髓核组织细胞，分离重编程为iPS细胞，并将其定向诱导分化为髓核样细胞。以正常成纤维细胞来源iPS作为对照，研究退变髓核组织来源的iPS-NPC在分化过程中功能活性和表观遗传学改变，并研究低氧诱导因子（HIF-1）修饰后对iPS-NPC功能活性和表观遗传特征的影响。将iPS细胞在透明质酸-壳聚糖可注射水凝胶中增殖分化为髓核样细胞，并借助水凝胶将iPS细胞移植到兔椎间盘退变模型治疗椎间盘退变。重编程后得到的iPS细胞经多次传代仍保留正常核型，有干细胞特异性表面标记的表达同时外源基因被沉默掉，畸胎瘤实验可见三个胚层的形成。在我们建立的分化体系下，iPS细胞可成功分化为表达collagenII和aggrecan的髓核样细胞，在三维水凝胶中分化效率更高，表达更多髓核特异性基因和蛋白。分化后的iPS细胞移植治疗椎间盘退变，其修复效果比未分化的iPS细胞更好，功能恢复也更强。从上述结果可知，退变髓核来源的iPS细胞是椎间盘退变机制研究和细胞治疗的一个较好的细胞来源。