【作者】 张敏；

【导师】 陈文川；

【作者基本信息】 四川大学 ， 口腔医学（专业学位）， 2023， 硕士

【摘要】 研究背景:颅颌面位置突出,因故导致的骨缺损常损害患者的身心健康,此难题困扰临床多年,自体骨不再满足日益增长的需求,骨组织工程(BTE)近几十年来快速的发展成为一个可替代方案。BTE倾向于结合生物和工程技术结合的策略,使用生物材料为组织提供生物活性的仿生平台,移植或招募具有分化功能的祖细胞,利用供体或移植物中的生长因子诱导新骨形成,进而形成骨整合和骨传导,其中骨髓间充质干细胞(BMSCs)是用于BTE的“金标准”。然而,BMSCs的收集手段具有侵入性,体外培养增殖能力有限,且BMSCs的自我更新和增殖潜能随着个体年龄增长而减弱。因此,挖掘更优渥廉价、分化潜能高、供应量大的替代者为大势所趋。人脐带间充质干细胞(h UCMSCs)来源于胎盘脐带的沃顿胶质(Wharton jelly),具有多谱系分化潜能、低免疫原性,且供应量大,具有与BMSCs相似的成骨分化机制。然而,h UCMSCs在体内的分化效能仍不足,临床应用受限。环状RNA(circ RNAs)是一类小的非编码RNA分子,首尾3’端和5’端共价连接形成闭环结构。环状结构使它们免于降解,从而具有稳定的表达、时间和组织特异性。众多研究揭示circ RNAs可以通过mi RNA海绵吸附、蛋白海绵吸附、m RNA陷阱等来调节成骨相关基因的表达,表明circ RNAs具有调节成骨的潜力。本课题组前期利用高通量测序手段和生物信息学的方法分析了成骨诱导与标准培养的h UCMSCs中circ RNAs的表达差异。发现circ RNAs,特别是circ-CTTN(has＿circ＿0003376)高水平表达,提示它们可能在h UCMSCs的成骨分化中发挥重要作用。研究目的:本实验旨在探究circ-CTTN对h UCMSCs成骨分化的影响。通改造h UCMSCs的circ-CTTN的表达水平,检测成骨分化的效果,随后对其进行体内功能验证。研究方法:利用腺病毒载体转染技术,在h UCMSCs中过表达circ-CTTN,以空载体及未转染的细胞作为对照。构建h UCMSCs工程化细胞后,在体外进行成骨诱导,检测成骨相关m RNA的表达水平及ALP和ARS的染色等细胞表型。利用骨组织工程技术,将过表达circ-CTTN的h UCMSCs、空转染的h UCMSCs、未转染h UCMSCs分别与明胶-甲基丙烯酸酯(Gel MA)水凝胶支架材料混合用于修复大鼠颅骨的临界骨缺损。术后分别于2周、4周和8周收取样本,多聚甲醛固定后进行micro-CT扫描,分析各样本颅骨缺损区的骨小梁密度、分离度等参数。分别对样本进行石蜡切片、包埋、组织学切片、染色、免疫组化染色观察缺损区的修复效果及表达情况。研究结果:在本研究中,体外成骨诱导显示过表达的circ-CTTN表现出更显著的成骨相关表型,同时成骨相关基因高表达。在大鼠颅骨缺损中,Gel MA水凝胶用于负载h UCMSCs,过表达的circ-CTTN组表现出比其他组更大的骨愈合面积和更密集分布的骨小梁。组织学切片和免疫组化染色追踪h UCMSCs细胞的转归,切片显示h UCMSCs在宿主组织缺损区能持续发挥生物活性。结论:circ-CTTN的过表达可以促进h UCMSCs的成骨分化,加速骨重建。本研究为促进h UCMSCs在BTE的临床应用提供了新的策略及依据。更多还原

【Abstract】 Background: Bone defect caused by prominent craniofacial position often damages patients’ physical and mental health,a clinical problem for many years.In addition,autologous bone transplantation no longer meets the growing demand.Bone tissue engineering(BTE)has developed rapidly in recent decades as an alternative.BTE tends to combine the strategy of biological and engineering technology and uses biological materials to provide a bionic platform for tissues with biological activity.Transplantation or recruitment of progenitor cells with differentiation function utilizes growth factors in donors or grafts to induce new bone formation,followed by the formation of bone integration and bone conduction.Bone marrow mesenchymal stem cells(BMSCs)are the "gold standard" for BTE.However,the collection method of BMSCs is invasive,and its ability to proliferate in vitro is limited.Moreover,the self-renewal and proliferation potential of BMSCs decreases with the age of individuals.Therefore,the general trend is to explore substitutes with better advantages,lower cost,high differentiation potential,and ample supply.Human umbilical cord mesenchymal stem cells(h UCMSCs)are derived from the Wharton jelly of the placental umbilical cord and have multiple lineage differentiation potential,low immunogenicity,and ample supply,with a similar osteogenic differentiation mechanism to BMSCs.However,the differentiation efficiency of h UCMSCs in vivo is still insufficient,and their clinical application is limited.Cyclic RNA(circ RNAs)is a class of small non-coding RNA molecules,with the end 3’ and the end 5’covalently linked to form a closed-loop structure.The cyclic structures protect them from degradation and thus provide stable expression,time,and tissue specificity.Many studies have revealed that circ RNAs could regulate the expression of genes related to osteogenesis through mi RNA sponge adsorption,protein sponge adsorption,m RNA trap,etc.,indicating that circ RNAs had the potential to regulate osteogenesis.In the previous study,we analyzed the difference in the expression of circ RNAs between osteogenic induction and traditional culture of h UCMSCs based on highthroughput sequencing technology and bioinformatics methods.The high level of expression of circ RNAs,especially circ-CTTN(has-circ-0003376),was found,suggesting that they might play an essential role in the osteogenic differentiation of h UCMSCs.Objectives: This experiment aimed to investigate the effects of circ-CTTN on the osteogenic differentiation of h UCMSCs.The osteogenic differentiation was detected by regulating the expression of circ-CTTN followed by in vivo functional verification.Methods: The adenovirus vector transfection technique was used to overexpress circ-CTTN in h UCMSCs,with the empty vector and non-transfected cells as the control.After the h UCMSCs engineered cells were constructed,they were subjected to osteogenic induction in vitro and detected for the expression of osteogenic-related m RNA and the cell phenotype,such as ALP and ARS staining.HUCMSCs overexpressing circ-CTTN,empty-transfected h UCMSCs,and non-transfected h UCMSCs were respectively mixed with gelatin-methacrylate(Gel MA)hydrogel for the repair of critical bone defects in rat cranial using bone tissue engineering technology.The samples were collected at 2 weeks,4 weeks,and 8 weeks after surgery.After being fixed with paraformaldehyde,micro-CT scanning was performed to analyze each sample’s trabecular density,resolution,and other parameters of the skull defect area.Paraffin section,embedding,histological section,staining,and immunohistochemical staining were performed on the samples to observe the repair effect and expression of the defective area.Results: In this study,in vitro osteogenesis induction showed that the overexpressed circ-CTTN exhibited a more prominent osteogenic-related phenotype,with high expression of osteogenic-related genes.In the rat skull defect,where Gel MA hydrogel was loaded with h UCMSCs,the overexpressed circ-CTTN group exhibited a larger bone healing area and more densely distributed trabeculae than the other groups.Histological sections and immunohistochemical staining were used to trace the fate of h UCMSCs cells,which showed significant expression in the host tissue defect area.Conclusions: The overexpression of circ-CTTN can promote the osteogenic differentiation of h UCMSCs and accelerate bone reconstruction.This study has provided a new strategy and laid a solid theoretical basis for promoting the clinical application of h UCMSCs in BTE.更多还原