【作者】 韩晓峰；

【导师】 刘祖德；

【作者基本信息】 上海交通大学 ， 外科学（骨外）（专业学位）， 2019， 博士

【摘要】 骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)在机体内具有潜在的多方向分化能力,可作为种子细胞参与生物体的组织构建与再生,然而怎样调控骨髓间充质干细胞向特定的功能细胞分化仍然存在许多不确定因素,只有充分阐明间骨髓充质干细胞诱导分化明确的分子调节机制,才能对其的诱导分化方向进行特定性地干预。研究表明转录调节因子是现阶段传统的调控手段,其中表观遗传修饰在骨髓间充质干细胞诱导分化调控的进程中起着至关重要的作用,研究中常用的表观遗传方式的调控主要是蛋白DNA的甲基化修饰。蓬松蛋白(Dishevelled)是涉及典型和非典型Wnt信号通路的蛋白质家族。蓬松蛋白是直接作用于卷曲受体下游的细胞质磷蛋白,其在胚胎和成年人之间都发挥重要作用,影响着从细胞分化和细胞极性到社会行为的整个过程。研究目的:本论文通过构建人骨髓间充质干细胞成骨诱导分化模型,利用特异性的DNA甲基化抑制剂(5-氮杂-2’-脱氧胞嘧啶)来研究蓬松蛋白中DNA启动子区CpG岛的甲基化修饰对骨髓间充质干细胞成骨诱导分化的进程影响和调控作用,并探讨甲基化修饰对差异表达性基因表达水平的影响。研究方法:1、骨髓间充质干细胞在成骨培养基中诱导分化,利用细胞染色剂茜素红鉴定其成骨分化程度,AKP试剂盒检测成骨分化过程中碱性磷酸酶的活性和表达量。利用RT-PCR和蛋白免疫法检测诱导分化过程中蓬松蛋白的表达水平。2、应用甲基化特异性PCR(MSP)方法检测骨髓间充质干细胞成骨诱导分化前后蓬松蛋白DNA启动子区CpG岛甲基化程度。3、应用RT-PCR和蛋白免疫印迹法检测甲基转移酶抑制剂(5-氮杂-2’-脱氧胞嘧啶)处理前后Wnt、GSK3、Axin、Dishevelled、β-catenin的表达水平。4、利用甲基化转移酶对蓬松蛋白启动子CpG岛进行甲基化后,对转录了甲基化启动子的骨髓间充质干细胞进行高通量测序分析差异性表达基因的表达水平。5、加入甲基转移酶抑制剂(5-氮杂-2’-脱氧胞嘧啶)处理骨髓间充质干细胞,RT-PCR和蛋白免疫印迹法检测差异基因随分化天数变化的表达情况。6、设计合成差异基因的增强子(mimics)和抑制子(inhibitors),并将其转染至骨髓间充质干细胞中,利用RT-PCR和蛋白免疫印迹法检测差异基因的表达水平,利用茜素红染色鉴定转染后的干细胞成骨分化程度。研究结果:1、随着诱导分化骨髓间充质干细胞的时间增长,其成骨分化的程度越来越大,同时碱性磷酸酶和蓬松蛋白的表达量也呈上升的趋势。2、在诱导分化7、14、21天后,骨髓间充质干细胞中蓬松蛋白启动子区域CpG岛甲基化程度随着诱导分化的进行而降低。3、在加入甲基化酶抑制剂(5-氮杂-2’-脱氧胞嘧啶)共同作用于成骨分化的骨髓间充质干细胞后,相对于DMSO实验对照组,随着诱导分化时间的增加,Wnt、GSK3、Axin、Dishevelled、β-catenin的表达水平都呈上升趋势。4、通过对甲基化启动子转染的骨髓间充质干细胞进行高通量分析后,发现PP2A在分化的细胞中表达异常,通过调控表达实验进一步证实,过表达差异基因PP2A能够促进骨髓间充质干细胞的分化进程。结论:本论文充分证明了蓬松蛋白中DNA启动子区CpG岛的甲基化修饰程度对骨髓间充质干细胞的分化具有调节和控制的作用,我们借助调节干细胞内的差异化基因PP2A的表达水平,来调节骨髓干细胞分化的进程,这可为骨髓间充质干细胞在成骨诱导分化的研究和应用提供了重要依据和实验基础。更多还原

【Abstract】 Bone marrow mesenchymal stem cells(BMSCs)are considered to be one of the most promising therapeutic cell sources as they encompass a plasticity of multiple cell lineages.The challenge in using these cells lies in developing well-defined protocols for directing cellular differentiation to generate a desired lineage,thus orienting its differentiation direction.In addition to the traditional transcription factor regulation,epigenetic modification also plays an important role in the regulation of bone marrow mesenchymal stem cells differentiation.DNA methylation is one of the main ways of epigenetic regulation.Dishevelled(Dsh)is a family of proteins that deals with typical and atypical Wnt signaling pathways.Dsh is a cytoplasmic phosphoprotein that acts directly on the downstream of the crimp receptor,which plays an important role in both embryos and adults,and affects the whole process from cell differentiation to social behavior.Research purposesIn this paper,by using osteoblast differentiation of BMSCs a research model,the effect of DNA methylation of Dishevelled protein on osteogenic differentiation of BMSCs was studied by DNA methylation inhibitor(5-azacytidine),and explored the potential effect of methylation modification on differential gene expression.Research methods(1)Human bone marrow mesenchymal stem cells induced differentiation in osteogenic medium,and the degree of differentiation of stem cells was identified by alizarin red staining.AKP kit was used to detect the activity and expression of alkaline phosphatase during osteogenic differentiation.The expression level of Dishevelled protein was seen by the way of RT-PCR and Western blot.(2)The methylation of Cp G island in the promoter region of Dishevelled protein gene was detected by methylation specific PCR(MSP).(3)The expression levels of Wnt,GSK3,Axin,Dishevelled and β-catenin were detected by RT-PCR and Western blot before and after treatment with methyltransferase inhibitor 5-aza-2’-deoxycytidine.(4)The Cp G island methylation of Dishevelled promoter was using specific methylatedtransferase,and carried out by high-throughput sequencing analysis of transcriptional cells to analyze the expression of differentially expressed genes.(5)Human bone marrow mesenchymal stem cells were treated with 5-Aza-2’-deoxycytidine,a methyltransferase inhibitor,and the expression of PP2 A was detected by RT-PCR and Western blot.(6)The mimics and inhibitors of PP2 A were synthesized and transfected into humanbone marrow mesenchymal stem cells.The expression of PP2 A gene was detected by RT-PCR and Western blot.The differentiation of stem cells was identified by alizarin red staining.Research results(1)With the bone marrow mesenchymal stem cells in osteogenic medium induced differentiation time,the greater the degree of osteogenic differentiation,while the alkaline phosphatase and Dishevelled protein expression also showed a rising trend.(2)The degree of methylation of Cp G island in the promoter region of bone marrow mesenchymal stem cells decreased with the induction of differentiation after 7,14,and 21 days after induction.(3)With the addition of methylase inhibitor-5-aza-2’-deoxycytosine to act on osteogenic differentiation of mesenchymal stem cells,compared with the DMSO control group,with the induction of differentiation time increased,Wnt,GSK3,Axin,Dishevelled and β-catenin showed an increasing trend.(4)After high-throughput analysis of human bone marrow mesenchymal stem cells transfected with methylated promoter,PP2 A was found to be abnormally expressed in differentiated cells.It was further confirmed by regulation of expression experiments that overexpression of PP2 A gene can promote proliferation of mesenchymal stem cells Differentiation.ConclusionThis article irrefutablely demonstrated that the regulation of Cp G island methylation in the promoter region of Dishevelled protein can regulate the differentiation of mesenchymal stem cells by regulating the expression level of regulatory factor PP2 A in the regulation of BMSCs differentiation.Stem cells in osteogenic differentiation and application of the study provided an important basis and experimental basis.更多还原