【作者】 赵星宇；

【导师】 刘建国；

【作者基本信息】 吉林大学 ， 外科学， 2019， 博士

【摘要】 随着人类生产、生活节奏的加快,人口老龄化的加剧,骨和软骨损伤的患者呈现逐年上升的趋势。导致骨和软骨损伤的原因有很多,其中主要包括:意外事故、生产生活、骨肿瘤、先天性疾病、年龄增长等因素。在这些骨损伤的疾病中,如何治疗临界尺寸骨缺损是临床上面临的一个难题,这类骨损伤疾病很难愈合,很容易造成骨不连等后遗症。目前,临床上主要有自体骨移植、异体骨移植等治疗方法。自体骨取材来源于患者本身,虽然无免疫排斥反应,骨诱导能力强,但是来源有限,患者还要经历取骨的二次手术带来的痛苦。异体骨来源充足,可以满足临床上治疗骨缺损的大量需求,但是自体骨存在免疫排斥反应和病毒感染等风险,因此也不是骨缺损修复的最佳解决方法。为了解决上述的问题,使用骨组织工程技术制备骨缺损替代物的研究工作成为了该领域的研究的热点问题。作为骨损伤替代物应该具备以下的特点:(1)多孔结构为成骨细胞的粘附和增殖提供空间和场所,(2)具体一定的机械性能和力学强度,(3)替代物的材料应该具有良好的生物相容性和体内可吸收性,(4)具有良好的骨诱导能力,促进细胞向成骨细胞的分化。因此一个优良的临界大尺寸骨缺损替代物应该具备以上这些特性。在软骨损伤方面,本研究主要关注由软骨细胞衰老、凋亡、分化等因素导致的骨关节炎(Osteoarthritis,OA)。小分子RNA(Micro RNAs,miRNAs)作为一类内源性非编码RNA,在体内可以调控多个基因的表达,从而影响这细胞的功能。miRNAs能够和mRNA的特异性的相互作用,促进mRNA的降解并抑制mRNA的翻译,影响着相应的基因发挥作用。很多研究中已经发现在OA患者的软骨细胞中,伴随着多个miRNAs的上调和下调表达,这说明miRNAs能够参与OA的形成和病情的加剧。本研究拟从miR-495为切入点,从信号通路角度研究miR-495与OA形成的机制和关系,从而寻找一个治疗OA的新靶点。基于以上的思路,本研究的具体方案如下:(1)使用旋蝶纺丝技术,制备具有微纳米直径的骨缺损修复支架,这种技术制备的纤维支架与传统的电纺丝技术制备的纤维相比,孔隙率较高,同时具有很好的立体结构。制备纤维的材料选用的是聚乳酸-羟基乙酸共聚物(Poly(lactic-co-glycolic acid),PLGA)和羟基磷灰石(Hydroxyapatite,HA),PLGA这种材料具有良好的生物学活性和体内可降解性,HA具有良好的骨诱导能力和体内矿化作用。从材料和制备工艺角度分析,本研究中使用的PLGA和HA能够充分满足作为骨修复替代物的要求。为了进一步改善材料表面的亲水性和细胞粘附、增殖能力,利用多巴胺分子在碱性条件下聚合形成聚多巴胺(polydopamine,PDA)的性质在纤维支架表面形成一层PDA,利用PDA的粘附能力在纤维支架上粘附骨形态发生蛋白-2(Bone morphogenetic protein-2,BMP-2)生长因子。实验结果表明PLGA纤维支架在经过PDA修饰后,表面的亲水能力得到了显著的提升,BMP-2的固定能力也得到了明显的增高,经过28天缓释的检测,经过PDA固定后的BMP-2呈现出缓慢释放的特性。以上这些结果说明我们成功的制备了一种能够缓慢释放BMP-2、亲水性好、高孔隙的骨修复支架。(2)使用MC3T3-E1细胞对支架进行细胞粘附、增殖和成骨诱导能力进行评估。PLGA组的细胞增殖率是最低的,而PLGA/HA组的细胞增殖率就明显的有所提高。这主要是由于HA能够提高材料表面的亲水性、增加表面的粗糙度,这些因素都有利于细胞的粘附和增殖。当材料表面粘附有PDA后,细胞的增殖率比未涂有PDA的组有所增加。细胞增殖率最高组发生在PDA-PLGA/HA/BMP-2。在成骨基因表达方面,PLGA/HA和PDA-PLGA/HA的RUNX2基因表达量比PLGA组提升了26%和61%。PDA-PLGA/HA/BMP-2的RUNX2基因表达量是PDA-PLGA/HA的1.25倍。以上结果说明,HA和BMP-2的引入均能够有效促进成骨相关转录因子RUNX2的表达。OPN基因在PLGA中表达最低,PDA-PLGA/HA中OPN的表达量是PLGA组中OPN的表达量的4倍,PDA-PLGA/HA/BMP-2中OPN的表达量最高。(3)评估了miR-495的高表达或下调对体外软骨细胞凋亡、生长和创伤修复的影响。结果显示miR-495的高表达可显著促进软骨细胞凋亡并抑制其增殖。miR-495的转染明显阻碍了划痕创面的修复,导致软骨细胞的损伤修复能力降低。此外,我们还观察到miR-495的转染能够促进caspase-3的活化,并进一步促进软骨细胞的凋亡。miR-495可以直接下调软骨细胞AKT1的表达,而miR-495抑制剂可有效增强AKT1的表达。因此,miR-495可能通过靶向AKT1诱导软骨细胞凋亡,AKT1过表达逆转了miR-495转染对软骨细胞凋亡、生长和创伤修复的影响。此部分研究工作为治疗由于软骨细胞凋亡导致的OA疾病提供了一个药物的新靶点。本课题研究利用旋蝶纺丝制备方法和PDA固定BMP-2技术制备了一种骨缺损修复支架,这种支架可以应用于临界尺寸骨缺损修复。同时,深入的研究了miR-495在软骨细胞凋亡、衰老等方面的作用,发现了miR-495诱导软骨细胞凋亡的分子机制。更多还原

【Abstract】 With the acceleration of human production and life rhythm andaging of population,the patients with bone and cartilage injuries show an increasing trend year by year.There are many causes of bone and cartilage damage,including accidents,bone tumors,congenital diseases,aging and other factors.In these diseases of bone injury,how to treat the large size bone defect is a difficult problem in clinical practice,such bone injury diseases are difficult to heal,and it is easy to cause bone nonunion.At present,there are mainly autologous and allogeneic bone transplantation.Although there is no immune reaction and osteogenic ability,the source of autologous bone is limited,and the patient still has to experience the pain caused by the second surgery for bone extraction.Allogeneic bone has sufficient sources,which can meet the large demand of clinical treatment for bone defects.However,autogenous bone is not the best solution for bone defects due to the risk of immune rejection and viral infection.In order to solve the above problems,using bone tissue engineering technology to prepare bone defect substitutes has become an important issue in this field.As bone substitutes should possess the following characteristics:(1)the porous structure of osteoblast adhesion and proliferation,(2)the suitable mechanical properties and mechanical strength,(3)materials should have good biocompatibility,and absorbability in vivo,(4)has a good ability of osteoblast differentiation.Therefore,a good substitute for large bone defect should possess these characteristics.In the aspect of cartilage injury,this study mainly focused on OA caused by aging,apoptosis,differentiation and other factors.As a kind of endogenous non-coding RNA,miRNAs can regulate the expression of multiple genes in the body,thus affecting the function of these cell.miRNA can specifically interact with mRNA,promote the degradation of mRNA and inhibit the translation of mRNA.Many studies have found that many miRNAs are up-regulated and down-regulated in chondrocytes of patients with OA,indicating that miRNAs can be involved in the formation of OA and the progression of OA.This study intends to study the mechanism and relationship between miR-495 and the formation of OA,so as to find a new target for the treatment of OA.Based on the above ideas,the content of this study is as follows:(1)Melt-spinning method was used to prepare the bone defect repair scaffolds with micro-nano diameter.Compared with the traditional electrospinning technology,the fiber scaffolds prepared by this technology with higher porosity and better three-dimensional structure.PLGA and HA were used to prepare the fibers.PLGA has good biological activity and in vivo degradability,and HA has good osteogenic ability and mineralization.From the aspect of materials and preparation technology,PLGA and HA used in this study can fully meet the requirements of being used as bone repair substitutes.In order to further improve the hydrophilicity,cell adhesion and proliferation ability of the material surface,a layer of PDA was formed on the surface of the fiber scaffold by polymerization of DOPA under alkaline conditions,and BMP-2 growth factor was adhered on the PDA with the adhesion ability of PDA.The results showedthat the surface hydrophilic ability was significantly improved,and the absorption ability of BMP-2 was significantly increased.After 28 days of release detection,the Bmp-2 presented a slow release characteristic.These results indicate that we have successfully prepared a bone repair scaffold with slow release of BMP-2,good hydrophilicity and high porosity.(2)MC-3T3-E1 cells were used to evaluate the cell adhesion,proliferation and osteogenic induction ability of the scaffold.The PLGA group showed the lowest cell proliferation rate,while the PLGA/HA group showed significant improvement.This is mainly because HA can improve the hydrophilicity of the material surface and increase the surface roughness,all of which are conducive to cell adhesion and proliferation.When PDA was adhered to the material surface,the cell proliferation rate increased compared with the control group.The group with the highest cell proliferation rate occurred in PDA-PLGA/HA/BMP-2.In the effect of osteogenic gene expression,the expression levels of RUNX2 genes in PLGA/HA and PDA-PLGA/HA were 26% and 61% higher than those in the PLGA group.The RUNX2 gene expression of PDA-PLGA/HA/BMP-2 was 1.25 times higher than that of PDA-PLGA/HA.These results suggest that the introduction of HA and BMP-2 can effectively promote the expression of RUNX2.The expression of OPN gene was the lowest in PLGA,the expression of OPN in PDA-PLGA/HA was 4 times higher than that in PLGA group,and the expression of OPN in PDA-PLGA/HA/BMP-2 was the highest.(3)The effects of overexpression or downregulation of miR-495 on apoptosis,growth and wound repair of chondrocytes were evaluated in vitro.The results showed that the high expression of miR-495 significantly promoted the apoptosis of chondrocytes and inhibited their proliferation.The transfection of miR-495 significantly inhibited the regeneration ofscratch wounds,resulting in reduced regeneration ability of chondrocytes.In addition,we also observed that the transfection of miR-495 can promote the activation of caspase-3 and further promote the apoptosis of chondrocytes.miR-495 directly down-regulated the expression of AKT1 in chondrocytes,while miR-495 inhibitor effectively enhanced the expression of AKT1.Therefore,miR-495 may promote chondrocyte apoptosis by targeting AKT1,and AKT1 overexpression inhibited the effects of miR-495 transfection on chondrocyte apoptosis,growth.This study provides a new drug target for the treatment of OA caused by the apoptosis of chondrocytes.In this study,a kind of bone defect repair scaffold was prepared by using melt-spinning method and BMP-2 binding via PDA.This scaffold can be used for large size bone defect repair.Meanwhile,the role of miR-495 in the apoptosis of chondrocytes was further studied,and the molecular mechanism of miR-495 inducing the apoptosis of chondrocytes was discovered.更多还原