【作者】 张帆；

【导师】 赵志河；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2021， 博士

【摘要】 人牙髓干细胞被认为起源于颅颌面神经棘,是一类具有分化潜能的间充质干细胞。在特定环境中,h DPSCs可以完成成骨、成脂、成软骨、神经分化等分化过程。同时,较BMSCs等较早被发现的间充质干细胞而言,h DPSCs具有较多优势。首先,h DPSCs高表达多向分化细胞因子,如OCT4、SOX2以及KLF4等。h DPSCs的获取手段无创,获取术式安全,免疫原性低使其在骨再生中的前景巨大。但目前h DPSCs调控成骨分化的作用并不明确。作为miR-17～92基因簇的重要成员之一,miR-20a-5p被证实可以参与调控多种生理/病理活动。例如,成肌细胞分化、肺泡基因表达、成血管化、免疫调节、以及成肿瘤。在骨代谢领域,目前研究提示miR-20a-5p可以促进人间充质干细胞以及人脂肪来源干细胞的成骨分化,并且可以在THP-1细胞中靶向PPARγ参与破骨细胞活动。但miR-20a-5p调控h DPSCs成骨分化的研究尚无报道。研究目的:因此,本研究拟明确miR-20a-5p在体外和体内水平h DPSCs成骨分化的调控作用,并探究其潜在的调控分子机制,为DPSCs为基础的骨再生提供新策略。研究方法:1、本研究拟鉴定h DPSCs;明确人miR-20a-5p在h DPSCs成骨分化中的变化情况;2、通过细胞转染技术过表达和抑制miR-20a-5p,分别在体外及体内水平探究其对h DPSCs成骨分化的影响。3、采用生物信息学技术预测miR-20a-5p下游潜在靶基因,以及双荧光素酶报告实验明确其交互作用;通过细胞转染技术敲减靶基因的表达量,明确miR-20a-5p/靶基因关系对在h DPSCs成骨分化中的调控作用以及作用的信号通路。研究结果:1.本实验中h DPSCs充分表达干细胞表面抗原标记物,不表达血细胞等内皮细胞表面抗原标记物,同时具有良好的成骨分化、成软骨分化、成脂分化能力;miR-20a-5p在h DPSCs成骨分化中持续高表达,可能具备调控h DPSCs成骨分化的能力。2.miR-20a-5p可在体外水平促进h DPSCs的成骨分化,同时促进裸鼠颅骨缺损处的骨修复。3.miR-20a-5p与BAMBI存在结合位点;si-BAMBI可促进h DPSCs成骨分化,并且可以部分逆转沉默miR-20a-5p对h DPSCs的抑制作用。miR-20a-5p/BAMBI关系对可激活Smad5和p38的磷酸化完成对成骨分化的调控。研究结论:miR-20a-5p可参与调控h DPSCs的成骨分化过程,miR-20a-5p可以靶向BAMBI发挥调控功能。miR-20a-5p可以激活BMPs信号通路中的Smad5和p38的磷酸化,在体内及体外水平影响h DPSCs成骨分化更多还原

【Abstract】 As members of craniomaxillofacial tissues,human dental pulp stem cells(h DPSCs)are originated from neural crest.h DPSCs have multidirectional differentiation potentials,which could differentiate into osteogenic,adipogenic,chondrogenic,and neurogenic lineage cells.Compared with mesenchymal stem cells(MSCs)derived from other tissues of human,h DPSCs possess some advantages.The increasing expression of key pluripotency factors,including OCT4,SOX2,and KLF4,are found in h DPSCs,which suggest superior property of multidirectional differentiation.It is reported that h DPSCs maintain stronger osteogenic activities when compared with the MSCs derived from bone marrow.Moreover,they could be isolated in a noninvasive and safer method without ethical controversy,and possess lower immunogenicity.Therefore,it is suggested h DPSCs could be the preferable choice of seed cells for bone regeneration,while the mechanism on osteogenic differentiation of h DPSCs remains to be clarified.As a member of miR-17～92 gene cluster,miR-20-5p has been showed to participate in a variety of cellular activities.For instance,miR-20a-5p is involved in myoblast differentiation,pulmonary surfactant gene expression,angiogenesis,immunomodulation,and tumorigenesis.Regarding the roles of miR-20a-5p in the bone metabolism,previous studies show miR-20a-5p could promote osteogenic differentiation of h MSCs and human adipose-derived stem cells(h ASCs),and miR-20a-5p could target PPARγ to regulate osteoclastogenesis of THP-1 cells.However,whether miR-20a-5p could regulate osteogenesis of h DPSCs remains unclear.Objective:This study was aimed to clarify the effect of miR-20a-5p on the osteogenesis of h DPSCs both in vitro and in vivo as well as the potential molecular mechanism.Methods:1.We will identify the h DPSCs and confirm expression of miR-20a-5p during the osteogenesis of h DPSCs.2.We will perform the cell transfection to over-express and inhibit the expression of miR-20a-5p,and investigate the effect of miR-20a-5p on osteogenesis both in vitro and vitro.3.Bioinformatics technology will be used to predict the potential target genes downstream of mir-20a-5p,and dual-luciferase reporter assay was used to clarify its interaction;The expression of target gene was knocked down by cell transfection technology to clarify the regulatory role and signal pathway of mir-20a-5p/target gene relationship in the osteogenic differentiation of h DPSCsResults:1.In this experiment,h DPSCs expressed stem cell surface antigen markers positively,and expressed endothelial cell surface antigen markers negatively.h DPSCs maintained osteogenic,chondrogenic and adipogenic differentiation ability;miR-20a-5p is continuously and highly expressed in the osteogenic differentiation of h DPSCs.2.miR-20a-5p can promote the osteogenic differentiation of h DPSCs in vitro and promote the bone repair of skull defect in nude mice.3.There are binding sites between miR-20a-5p and BAMBI;si-BAMBI can promote the osteogenic differentiation of h DPSCs and partially reverse the inhibitory effect of silencing mir-20a-5p on h DPSCs.miR-20a-5p/BAMBI interaction can activate the phosphorylation of Smad5 and p38 and complete the regulation of osteogenic differentiation.Conclusions:It is demonstrated that miR-20a-5p functioned as a regulator of BAMBI to activate the phosphorylation of Smad5 and p38 during osteogenic differentiation of hDPSCs.更多还原