【作者】 刘静；

【导师】 韩金祥；

【作者基本信息】 济南大学 ， 微生物与生化药学， 2019， 硕士

【摘要】 背景:威尔逊氏症(WD),ATP7B基因突变引起的罕见的铜代谢障碍的常染色体隐性遗传病。除肝脏,神经症状外,较低的骨密度是WD的另一个最常见的临床特征,但其潜在的机制尚未完全了解。诱导多能干细胞(iPSCs),有着自我更新、生长迅速、可分化为身体各组织器官的潜能且与胚胎干细胞相比没有伦理宗教法律的限制等优点。基于这些优点,掺入基因治疗的iPSCs可以通过体外培养建立一些疾病如威尔逊氏症的细胞模型,进而为相应疾病的治疗提供新的思路与方法。目的:探究威尔逊氏症骨密度低的原因;初步探究威尔逊氏症低成骨活性的机制;筛选小分子促进威尔逊氏症的成骨分化。方法:通过拟胚体(EBs)形成以及添加成骨培养基的方法进行正常和WD iPSCs的成骨诱导并通过荧光实时定量以及茜素红染色从基因型以及表型两方面比较正常对照组和威尔逊氏症组之间成骨表达是否有差异;通过转录组测序技术分析威尔逊氏症和正常iPSCs诱导的成骨细胞的基因表达谱,进而比较正常对照组和威尔逊氏症组之间的差异基因并推测威尔逊氏症组的低成骨活性可能是通过β-catenin途径影响成骨细胞分化的,然后进一步通过免疫蛋白印迹和免疫荧光验证上述推测;最后通过筛选小分子发现SKL2001这个小分子化合物可通过破坏β-catenin/Axin之间的相互作用,稳定细胞内β-catenin蛋白。进而我们通过实时定量以及免疫蛋白印记、免疫荧光验证SKL2001是否可以促进威尔逊氏症组的成骨分化。结果:通过拟胚体(EBs)形成以及添加成骨培养基的方法进行正常和WD iPSCs的成骨诱导并得到在成骨分化的所有阶段中威尔逊氏症成骨细胞中的成骨标志物基因的表达显著低于正常对照组,并且威尔逊氏症成骨细胞中的矿化水平也显著低于正常对照组;通过转录组测序分析得到来自威尔逊氏症的成骨细胞和健康对照之间β-catenin途径存在显着差异,推测威尔逊氏症的低成骨活性可能是通过β-catenin途径影响成骨细胞分化的。然后通过免疫蛋白印记和免疫荧光得到在iPSCs阶段以及在成骨诱导各阶段,威尔逊氏症组的β-catenin的总蛋白的表达略低于正常对照组β-catenin的总蛋白的表达,以及在成骨诱导7天以及21天时,威尔逊氏症组的胞核蛋白中β-catenin的表达也都略低于正常对照组,进而可得到威尔逊氏症的低成骨活性可能与异常的β-catenin途径有关;最后通过查阅文献筛选出了一个小分子药物SKL2001,可通过破坏β-catenin/axin之间的相互作用,稳定细胞内β-catenin蛋白。通过荧光实时定量证明了加入小分子SKL2001处理组成骨细胞的成骨标志性基因的表达较未加入小分子SKL2001处理组成骨细胞的成骨标志性基因的表达有升高,通过免疫蛋白印记以及免疫荧光证明了加入小分子SKL2001处理组成骨细胞β-catenin的表达较未加入小分子SKL2001处理组成骨细胞中的β-catenin的表达向细胞核的转移增加,核蛋白的表达升高,进而激活了下游转录。但却没有剂量依赖性。结论:在本课题中,通过拟胚体(EBs)形成以及添加成骨培养基的方法进行正常和WD iPSCs的成骨诱导并通过与正常对照组比较首次发现了WD-iPSCs衍生的成骨细胞表现出比正常对照组更低的成骨活性。进一步通过基因表达谱的分析,总蛋白和胞核蛋白中β-catenin的检测以及β-catenin的核定位情况,我们鉴定并验证了威尔逊氏症组中低成骨活性可能是由于异常的β-catenin途径所致。最后,通过加入小分子SKL2001处理组与未处理组的比较得到小分子SKL2001可以通过破坏Axin/β-catenin复合物的形成,促进β-catenin的表达量以及向细胞核的转移增加,促进威尔逊氏症组的成骨分化,改善威尔逊氏症骨密度低的现状。因此,我们的上述研究结果表明,威尔逊氏症患者骨密度低和骨质疏松可能是由于异常β-catenin信号通路引起的成骨减少,这些威尔逊氏症患者可能受益于β-catenin靶向策略,以改善骨重建的不平衡。更多还原

【Abstract】 Background: Wilson’s disease(WD)is a rare autosomal recessive disorder of copper metabolism caused by an ATP7 B gene mutation.Except for hepatic,neurological symptoms,lower bone mineral density is another most frequent clinical features of WD,but the underlying mechanisms have not been fully understood.Induced pluripotent stem cells(iPSCs)has the advantages of self-renewal,disease specific,and potential to differentiate into various diseases-associated cell types.And it has no restrictions on ethical、 religious and laws compared with embryonic stem cells.Based on these advantages,iPSCs incorporated into gene therapy can establish a cell model of some diseases such as WD by in vitro culture,and then provide new ideas and methods for the treatment of the corresponding diseases.Objective: Exploring the causes of low bone density in WD;Preliminary study on the mechanism of low osteogenic activity in WD;Screening small molecules to promote osteogenic differentiation of WD.Methods: Osteogenesis induction from both normal and WD iPSCs was performed by the embryoid bodies(EBs)formation method and addition of osteogenic medium.And we compared whether there is a difference in osteogenesis between the normal controls and WD from both genotype and phenotype by real-time quantification and Alzizarin Red staining.The gene expression profile of WD and normal iPSCs-induced osteoblasts were analyzed using RNA-Seq technology.And we compared the differential genes between normal controls and WD,and then analyzed the sequencing results to obtain that the low osteogenic activity in the WD may be associated with the β-catenin pathway.Further,it is confirmed whether the β-catenin pathway is involved in regulating the low osteogenic activity in the WD by western blot and immunofluorescence.Finally,we found that SKL2001,a small molecule compound that can stabilize intracellular β-catenin by disrupting its interaction with axin.Further,we verified whether SKL2001 can promote osteogenic differentiation in the WD by real-time quantification,western blot and immunofluorescence.Results: Osteogenesis induction from both normal and WD iPSCs was performed by the embryoid bodies(EBs)formation method and addition of osteogenic medium.In all stages of osteogenic differentiation,the expression of the osteogenic marker gene in all stages of osteogenesis induction were significantly lower in the WD osteoblasts compared to the normal controls,and the mineralization level was significantly lower in the WD osteoblasts than the normal controls.Transcriptome sequencing suggested that there was a significant difference in β-catenin pathway between osteoblasts from WD and healthy control.Then we found that the total protein expression of β-catenin is lower in the WD osteoblasts compared to the normal controls by western blot,and immunofluorescence assays and western blot also revealed that the β-catenin nuclear protein level of the WD on days 7 and 21 during osteogenic differentiation was significantly lower compared to the normal controls.Finally,we found that SKL2001,a small molecule compound,stabilizes intracellular β-catenin by disrupting the interaction between β-catenin and axin.The result showed that the expression of the osteogenic marker gene in the WD osteoblasts treated with SKL2001 was significantly higher than that WD controls by real-time quantification.And the nucleus of β-catenin in the WD osteoblasts treated with SKL2001 was significantly higher than WD controls by western blot and immunofluorescence.But these did not show a dose-dependent.Conclusion: In this study,osteogenesis induction from both normal and WD iPSCs was performed by the embryoid bodies(EBs)formation method and addition of osteogenic medium,and for the first time,we found that WD-iPSCs derived osteoblasts demonstrated a decreased osteogenesis activity than their normal controls.Through gene expression profiling,we identified and validated that there was a significant difference in β-catenin pathway between osteoblasts from WD and healthy control.Most meaningfully,when treating with SKL2001,a small molecule compound that can stabilize intracellular β-catenin by disrupting its interaction with axin.,can reverse decreased osteogenesis of WD.Therefore,our above findings indicated that low bone density and osteoporosis of WD patients might be due to the decreased osteogenesis caused by aberrant β-catenin signaling pathway,and these patients might benefit from β-catenin targeting strategies to improve the imbalance on bone remodeling.更多还原