【作者】 刘渊；

【导师】 沈彬；

【作者基本信息】 四川大学 ， 外科学（骨科）， 2023， 博士

【摘要】 目的:骨关节炎(osteoarthritis,OA)在中老年群体中患病率高,关节疼痛、功能障碍等症状明显,严重影响正常的生活和工作,产生了很大的社会经济负担。但目前针对OA的治疗仍停留在口服止痛药物、盐酸氨基葡萄糖,关节腔注射玻璃酸钠或激素等对症治疗和终末期的关节置换手术治疗,尚无能够有效改善延缓临床OA进展的药物。主要原因在于OA病因及发病机制繁杂,随着研究深入,关节软骨退变的病理改变逐渐被公认为OA发生发展的核心变化。因此,本研究将围绕软骨退变为核心和治疗靶点展开研究。关节软骨是一种无血管且通透性较差的致密组织,正常生理条件下,软骨细胞营养供应主要自滑液及少许软骨下骨髓扩散而来。线粒体氧化磷酸化供能可能占关节软骨中总ATP产量的25%,在组织能量需求高的情况下更高。因此,保障线粒体正常工作对维护关节软骨代谢平衡非常重要。另外,线粒体功能不只为细胞代谢提供能量,还参与细胞生长、增殖、衰老和凋亡等诸多重要的病理生理调节过程。以往很多文献报道线粒体蛋白改变可能导致重要的器官和细胞功能障碍。因此,本研究将首先探究OA发展过程中软骨细胞线粒体形态及其功能变化。课题组前期研究发现miR140具有一定软骨保护作用,其表达水平随OA发展逐渐降低。通过测序及生信分析发现miR140下游靶点CAPN1与线粒体功能调节有关。CAPN1是钙依赖性半胱氨酸蛋白酶,当细胞受损致胞内ATP供能不足时,Ca2+ATP驱动泵不能调节胞内外Ca2+水平,致胞内Ca2+水平升高,激活CAPN1表达,促进线粒体自噬上游分子Parkin的清除,抑制线粒体自噬发生,加重线粒体受损,膜电位降低,活性氧(reactive oxygen species,ROS)水平升高,进一步加重细胞损伤和凋亡。因此,本研究将探究miR140/CAPN1轴对软骨细胞线粒体自噬水平的调控作用及其潜在机制。miR小分子核酸片段易降解,仍需进行甲基化修饰等加强药物稳定性,生物利用度较低,尚需药物载体优化。外泌体(exosome,Exos)作为天然细胞间通讯媒介,可通过转移蛋白和寡核苷酸来实现胞间通讯及生物学调节,具有运输稳定且不易受周围环境影响等优点。课题组前期研究发现关节软骨脱细胞基质水凝胶复合人尿源性干细胞(human urine derived stem cells,hUSCs)可有效修复大鼠膝关节软骨缺损。因此,本课题拟以hUSCs来源Exos作为递送miR140至OA软骨细胞的药物载体。综上,本研究目的如下:1.探究不同OA分期软骨组织及其细胞线粒体形态和自噬水平变化,及miR140/CAPN1 轴对OA软骨细胞线粒体形态及线粒体自噬水平的调节作用和机制;2.提取鉴定hUSC-Exos,探究不同浓度hUSC-Exos对OA软骨细胞增殖及迁移能力的影响,筛选最佳作用浓度;3.构建hUSC-140-Exos,鉴定过表达效率及其作用于软骨细胞后miR140表达水平变化,探究其对OA软骨细胞增殖能力,迁移能力,ECM合成降解平衡和线粒体自噬水平的影响;4.探究hUSC-140-Exos对大鼠OA膝关节软骨细胞线粒体自噬调节作用及OA进程的影响。材料和方法:1.申请获取伦理许可及患者知情同意后,获得正常、早期OA和中晚期OA软骨组织,并行组织学染色及OARSI评分验证分期。透射电镜观察软骨组织线粒体形态、免疫组化(immunohistochemistry,IHC)检测软骨组织/蛋白印迹(western blotting,WB)检测软骨细胞线粒体自噬分子表达水平,包括线粒体自噬经典上游调控分子Parkin,及参与自噬小体形成与降解的LC3和p62分子,以此来判断OA进展过程中软骨细胞线粒体自噬水平变化情况;2.WB检测miR140对不同分期OA软骨细胞线粒体自噬发生的调节作用。通过数据库预测、双荧光素报告实验和蛋白基因水平检测验证miR140是否可下调CAPN1表达。设置CAPN1下调组(miR140 mimics)、CAPN1上调组(CAPN1 overexpression,CAPN1 OE)、CAPN1 同时上调下调组(miR140 mimics+CAPN1 OE)、CAPN1 下调复合阳性干预组(miR140 mimics+CCCP)和 CAPN1上调复合阳性干预组(CAPN1 OE+CCCP)共5组体外细胞实验。通过检测软骨细胞线粒体形态变化、ROS水平、线粒体膜电位和线粒体自噬分子表达情况来验证miR140/CAPN1轴对软骨细胞线粒体自噬发生的调节作用;3.通过重复离心洗涤法,从青年志愿者清洁中段尿中分离、提取原代hUSCs。镜下观察细胞形态及生长活性,流式细胞分析术检测hUSCs表面特异标志物表达情况,体外诱导hUSCs成骨、成脂和成软骨分化,检测其三向分化能力。细胞增殖扩增后收集细胞上清,通过超速离心法提取hUSC-Exos。透射电镜观察Exos形态,NTA分析Exos粒径分布,WB检测Exos表面标志物。并设置一定浓度梯度检测hUSC-Exos对OA软骨细胞增殖迁移能力的调节作用;4.通过慢病毒感染构建过表达miR140的hUSCs,提取hUSC-140-Exos作用于软骨细胞,qPCR鉴定hUSCs及软骨细胞miR140表达水平。通过增殖、迁移及OA软骨细胞ECM变化检测hUSC-140-Exos对OA软骨细胞生物学改变的调节作用。并基于miR140/CAPN1轴对软骨细胞线粒体自噬发生的调节作用检测hUSC-140-Exos对OA软骨细胞线粒体形态、ROS水平、膜电位及线粒体自噬发生的调节作用;5.通过“前交叉韧带剪断+内侧半月板失稳”构建大鼠膝关节OA模型,并将大鼠分为五组:假手术组(仅切开关节囊)、阴性对照组(关节腔注射生理盐水)、hUSC-Exos 组、miR140agomir 组、以及 hUSC-140-Exos 组。于关节腔注射治疗4 w及8 w后行影像学评价,包括大鼠膝关节骨赘、关节腔游离体数量和软骨下骨微结构定量分析比较。并在伦理许可下获取大鼠膝关节标本,通过大体观察(整体病理评分)、组织学染色(OARSI评分)及IHC检测软骨细胞ECM和线粒体自噬分子变化水平,探究hUSC-140-Exos治疗大鼠膝OA的疗效及潜在机制。结果:1.相较于正常软骨,早期OA软骨表面粗糙不齐,基质着色减少,软骨细胞有水肿肥大和死亡,ECM蛋白聚糖和Ⅱ型胶原含量均减少(p<0.01,p<0.05);中晚期OA软骨层出现垂直裂缝,基质着色显著减少,细胞排列方向明显紊乱,大量软骨细胞死亡,ECM蛋白聚糖和Ⅱ型胶原含量显著减少(p<0.001,p<0.01)。OARSI评分显示,早期OA软骨评分为11.67±3.51,显著高于正常软骨组织评分(p<0.05);中晚期OA软骨评分为20.67±3.06,显著高于正常软骨组织和早期OA软骨组织评分(p<0.001,p<0.05)。TEM镜下观察软骨组织内细胞数量较少,各组标本内仅观察到少量软骨细胞。相较正常软骨组织内细胞线粒体形态,OA组软骨细胞随OA进展线粒体肿胀明显加重,且中晚期 OA 软骨细胞核固缩明显,粗面内质网轻度扩张,胞内有多个空泡。IHC检测发现,相较于正常软骨组织,早期OA软骨组织Parkin及LC3表达水平略有升高,无统计学差异,但p62表达水平显著上调(p<0.05),表现为线粒体自噬小体降解受限。而中晚期OA软骨细胞Parkin及LC3表达水平明显下调,并且LC3较早期OA软骨细胞显著下调(p<0.05),p62表达水平较正常及早期OA软骨细胞均显著上调(p<0.01,p<0.05),表现为线粒体自噬水平下降,呈受损状态。另外WB检测发现,相较于正常软骨细胞,早期OA软骨细胞Parkin及LC3表达水平显著上调(p<0.05),p62表达水平无明显变化,表现为线粒体自噬水平反应性升高;而中晚期OA软骨细胞 Parkin及LC3表达水平较早期OA软骨细胞显著下调(p<0.01),p62表达水平较正常及早期OA软骨细胞均显著上调(p<0.01),表现为线粒体自噬水平显著下降,呈受损状态。2.除早期OA软骨细胞Parkin和中晚期OA软骨细胞LC3表达水平升高无统计学差异外,miR140显著提高了正常及中晚期OA软骨细胞Parkin表达水平(p<0.05),显著提高了正常及早期OA软骨细胞LC3表达水平(p<0.01,p<0.05),显著降低了正常、早期及中晚期OA软骨细胞p62表达水平(p<0.05),提示miR140对正常及OA软骨细胞均可促进其线粒体自噬发生。进一步探究miR140调控软骨细胞线粒体自噬发生的靶点发现,miR140与CAPN1存在8个碱基序列大小的结合位点,双荧光素酶报告基因检测表明miR140可靶向下调CAPN1表达(p<0.001)。另外,WB检测发现软骨细胞CAPN1表达水平随OA进展逐渐升高,与miR140表达水平正好相反,呈负相关性。而且miR140作用于软骨细胞后,其CAPN1蛋白水平和基因水平均显著降低(p<0.05,p<0.01)。因此,miR140可能通过靶向下调CAPN1表达进而调节软骨细胞线粒体自噬发生改变。通过检测上下调CAPN1表达的各组软骨细胞线粒体形态、ROS水平、线粒体膜电位和线粒体自噬分子表达水平,发现CAPN1过表达软骨细胞线粒体自噬小体数量减少、ROS堆积、线粒体膜电位降低和线粒体自噬发生停滞,而miR140显著降低CAPN1表达,缓解软骨细胞线粒体肿胀,减少ROS堆积、提高线粒体膜电位和促进线粒体自噬发生。3.本研究成功培养了米粒样形态、贴壁生长、阳性表达CD29(93.2%)、CD44(97.3%)、CD73(99.7%)、CD90(99.8%)、CD105(90.0%),不表达 CD34(0.13%)和HLA-DR(0.012%),且能够体外诱导成骨、成脂和成软骨分化的hUSCs。超速离心法分离提取 hUSC-Exos,呈球形双膜结构,粒径大小为135.5±59.5nm,表面表达 CD9、CD63 和 CD81,不表达 Calnexin。hUSC-Exos对OA软骨细胞生物调节实验表明,从低浓度(1μg/ml,5 μg/ml)到高浓度(10μg/ml)hUSC-Exos均可显著提高OA软骨细胞增殖速率和迁移能力,且呈浓度梯度效应,随浓度增大效果增强,但更高浓度(15 μg/ml,20 μg/ml)hUSC-Exos失去浓度增强效应,反而抑制OA软骨细胞增殖及迁移。4.hUSCs自身miR140表达水平很低,相比于hUSCs和阴性慢病毒感染获得的 hUSC-NC,过表达 miR140 的 hUSCs(hUSC-140)显著提高了 miR140 表达水平(p<0.001),其次,与hUSC-140-Exos共培养的软骨细胞miR140基因水平显著升高(p<0.01)。结果表明,通过hUSC-Exos递送,可以提高软骨细胞miR140的表达水平。hUSC-140-Exos对OA软骨细胞生物调节实验表明,hUSC-140-Exos不仅在增强 OA软骨细胞增殖及迁移能力方面优于等浓度的hUSC-Exos,而且显著提高了 OA软骨细胞ECMCollagen Ⅱ含量(p<0.01),显著降低了 ADAMTS5(p<0.05)和 MMP13(p<0.001)水平,改善了 OA 软骨细胞ECM合成。另外,hUSC-140-Exos缓解了 IL-1β干预引起的OA软骨细胞线粒体肿胀和结构改变,降低了胞内ROS水平(p<0.01),提高了线粒体膜电位(p<0.05),显著降低了软骨细胞CAPN1表达水平(p<0.01),提高了 Parkin(p<0.01)和LC3(p>0.05)的表达水平,显著降低了 p62的表达水平(p<0.05),提高了软骨细胞线粒体自噬水平。5.micro CT扫描大鼠膝关节发现,假手术组膝关节表面光滑,无骨赘及关节腔游离体形成,而OA组膝关节表面粗糙,有明显的骨赘及游离体形成,证明OA造模成功。关节腔注射hUSC-140-Exos治疗4周及8周后均显著减少了大鼠OA膝关节游离体及骨赘数量(p<0.05),优于hUSC-Exos和miR140 agomir。而且,hUSC-140-Exos组软骨下骨板连续,骨小梁排列整齐,未观察到骨赘形成,治疗4周和8周后有效提高大鼠OA软骨下骨BMD(p<0.01);治疗8周后显著提高骨小梁厚度(p<0.05);治疗4周和8周后显著缩小了骨小梁间隙(p<0.01),有效改善了软骨下骨微结构。观察大鼠膝关节发现,OA膝关节软骨表面粗糙,局部被破坏侵蚀,色泽异常,关节边缘有骨赘形成,而miR140 agomir治疗4周及hUSC-140-Exos治疗4周、8周后明显改善了软骨形态结构,病理评分显著低于OA组(p<0.05,p<0.05和p<0.01)。组织学染色显示,OA大鼠膝关节软骨损伤,甚至有裂缝形成,细胞排列紊乱,有水肿肥大,基质着色显著减少,潮线断裂,hUSC-Exos治疗8周(p<0.05)、miR140 agomir治疗4周和 8 周(p<0.01)和 hUSC-140-Exos 治疗 4 周和 8 周(p<0.05,p<0.001)均明显改善软骨病变,降低了 OARSI评分。IHC检测ECM变化发现,hUSC-Exos、miR140 agomir 和 hUSC-140-Exos 治疗均可提高大鼠膝软骨 ECM Collagen Ⅱ和Aggrecan含量,降低肥大变性软骨细胞标志物Collagen X表达水平,且hUSC-140-Exos 8周组效果最佳(p<0.01)。进一步探究大鼠OA膝关节软骨细胞线粒体自噬水平发现,在治疗4周后,各组软骨组织Parkin表达水平无明显变化,但大鼠OA膝关节软骨组织LC3表达水平在miR140 agomir和hUSC-140-Exos治疗4周后均显著提高(p<0.05,p<0.01),且p62的表达水平显著降低(p<0.05);在治疗8周后,miR140agomir和hUSC-140-Exos治疗明显提高了大鼠OA膝关节软骨组织Parkin(p<0.05)和LC3(p<0.01)的表达水平,显著降低了 p62(p<0.05)的表达水平,提示关节腔注射hUSC-140-Exos和miR140 agomir 均可提高大鼠OA关节软骨细胞线粒体自噬水平。结论:1.相比于正常和早期OA软骨细胞,中晚期OA软骨细胞线粒体形态肿胀,内膜嵴结构消失,且线粒体自噬水平下降。而miR140通过靶向下调CAPN1表达,提高了正常及各期OA软骨细胞线粒体自噬水平,减少胞内ROS堆积,提高线粒体膜电位,避免线粒体形态结构破坏。2.hUSC-Exos来源丰富、制备简捷,10 μg/ml的hUSC-Exos促进OA软骨细胞增殖及迁移效果最佳,可作为搭载递送miR140至OA软骨细胞的良好载体。3.本研究成功制备了 hUSC-140-Exos,显著提高了软骨细胞miR140表达水平。hUSC-140-Exos不仅提高OA软骨细胞增殖及迁移能力、增强ECM合成,而且还可下调CAPN1表达,促进软骨细胞线粒体自噬发生,改善线粒体形态及功能。4.单独使用hUSC-Exos或miR140 agomir以及两者复合的hUSC-140-Exos均缓解了大鼠OA膝关节软骨组织退变,其中hUSC-140-Exos疗效最佳。更多还原

【Abstract】 Objective:Osteoarthritis(OA)has a high prevalence in middle-aged and elderly people,with obvious symptoms such as pain and joint dysfunction,which seriously affects normal life and work and creates a great social and economic burden.However,at present,the treatment of OA still remains in the symptomatic treatment such as oral painkillers,glucosamine hydrochloride,intra-articular injection of hyaluronic acid,glucocorticoid,platelet-rich plasma,and end-stage arthroplasties.There is no clinical drug that can effectively delay the progression of OA.The main reason is that the etiology and pathogenesis of OA are complex,and the pathological changes of degeneration-wear-degeneration of articular cartilage are gradually recognized as the core mechanism of the occurrence and development of OA.Therefore,this study will focus on cartilage degeneration as the core and treatment target.Articular cartilage is a kind of dense tissue with no blood vessels and poor permeability.Under normal physiological conditions,the nutrition supply of chondrocytes mainly comes from synovial fluid and a little subchondral bone marrow.However,oxidative phosphorylation may account for 25%of the total ATP production in articular cartilage,even higher in the case of high tissue energy demand.Therefore,it is very important to ensure the normal work of mitochondria to maintain the metabolic balance of articular cartilage.Moreover,in addition to providing energy for cell metabolism,mitochondria also participate in many important pathophysiological regulation processes in cell growth,proliferation and aging and apoptosis.Many previous literature reports that mitochondrial protein changes may lead to the occurrence of certain diseases.These protein changes can lead to dysfunction of important organs and cells.Therefore,this study will firstly explore the morphological and functional changes of chondrocyte mitochondria during the development of OA.The previous study of the research group found that the expression level of miR140 in chondrocytes gradually decreased with the development of OA,and proved that it had a certain cartilage protection effect.Through sequencing and bioinformatics analysis,it was found that its downstream target CAPN1 was related to the regulation of mitochondrial function.CAPN1 is a calcium-dependent cysteine protease.When the intracellular ATP energy supply is insufficient due to cell damage,the Ca2+ATP drive pump cannot regulate the intracellular and extracellular Ca2+levels,resulting in an increase in the intracellular Ca2+level,activating the expression of CAPN1,aggravating mitochondrial damage,producing excessive ROS,and further aggravating cell damage and apoptosis.Therefore,this study will determine the regulatory effect of miR140 on chondrocyte mitochondria and whether it plays a regulatory role by down-regulating CAPN1.The miR140 are easy to degrade,and a series of modifications such as methylation are still needed to strengthen the stability of nucleic acid drugs.And after the injection of fluorescent labeled miR140 into the articular cavity,the frozen section detection showed that the nucleic acid drugs had poor targeting,wide distribution and low bioavailability.As a natural intercellular communication medium,exosomes(Exos)can realize intercellular communication and exert biological effects by transferring proteins and oligonucleotides.And Exos have the advantages of stable and reliable transport and transmission process and are not easily affected by the surrounding environment.The previous research of the research group found that the acellular matrix hydrogel of articular cartilage combined with hUSCs could regulate the polarization of macrophages for the repair of cartilage defects in rats.In addition,it has also been reported that the matrix deposited by hUSCs may promote the functional cartilage repair and reconstruction mediated by tissue-specific stem cells by regulating mesenchymal epithelial transformation and inflammation-mediated macrophage polarization.Therefore,this subject intends to use Exos from hUSCs as a drug carrier to deliver miR140 to OA chondrocytes.In summary,this study intends to explore the construction of hUSCs-derived exosomes(hUSC-140-Exos)overexpressing miR140 for repairing knee OA;At the same time,the effect of hUSC-140-Exos on mitochondrial autophagy of chondrocytes was explored based on the regulatory effect of miR140/CAPN1 axis on mitochondrial function of OA chondrocytes.Therefore,the objectives of this study are listed as follows:1.To explore the changes of mitochondrial morphology and mitophagy level of cartilage tissue and its cells from different OA stages;2.To explore the regulatory effect and mechanism of miR140 on mitochondrial morphology and mitophagy level of OA chondrocytes;3.To determine the characteristics of hUSCs and hUSC-Exos,and explore its effects on the proliferation and migration ability of OA chondrocytes to select the optimal concentration;4.To prepare the hUSC-140-Exos,determine the change of miR140 expression level in hUSCs and chondrocytes,and explore the effect of hUSC-140-Exos on the proliferation ability,migration ability,extracellular matrix(ECM)remodeling,and mitophagy level of OA chondrocytes;5.To explore the effect of hUSC-140-Exos on the regulation of mitophagy in rat knee chondrocytes and the development of OA.Methods:1.After applying for ethical permission and patients’ informed consent,normal cartilage and cartilage tissue at early and medial-late stages of OA were obtained for histological staining and OARSI scoring.The mitochondrial morphology of cartilage tissue was observed by transmission electron microscope(TEM),and the expression level of mitophagy molecules in cartilage cells were detected by immunohistochemistry(IHC)and western blotting(WB),including Parkin,the classic upstream regulator of mitophagy,LC3 and p62,which are involved in the formation and degradation of autophagosomes.2.The regulatory effect of miR140 on mitophagy in OA chondrocytes at different stages was detected by WB.Whether miR140 can down-regulate the expression of CAPN1 was verified by database prediction,double fluorescein reporter experiment,qPCR and WB.Set CAPN1 down-regulation group(miR140 mimics),CAPN1 up-regulation group(CAPN1 overexpression,CAPN1 OE),CAPN1 upregulation and down-regulation group(miR140 mimics+CAPN1 OE),CAPN1 down-regulation compound positive intervention group(miR140 mimics+CCCP)and CAPN1 up-regulation compound positive intervention group(CAPN1 OE+CCCP)consisted of 5 groups,and the regulation of miR140/CAPN1 axis on chondrocyte mitophagy was verified by detecting the morphological changes of chondrocyte mitochondria,ROS level,mitochondrial membrane potential and mitophagy molecule expression.3.hUSCs were isolated from clean midstream urine of young volunteers by repeated centrifugation washes.The cell morphology and proliferation were observed under a microscope,the expression of MSCs-specific markers on the surface of hUSCs was detected by flow cytometry,the osteogenic,adipogenic and chondrogenic differentiation of hUSCs were induced in vitro,and their three-way differentiation ability was detected.After cell proliferation and expansion,the Exos-free cell culture supernatant was collected,and hUSC-Exos were extracted by ultracentrifugation.The shape of Exos was observed by TEM,the particle size distribution of Exos was analyzed by nanoparticle tracking analysis(NTA),and the surface markers of Exos were detected by WB.And set a certain concentration gradient to detect its regulating effect on the biological changes of OA chondrocytes.4.hUSCs overexpressing miR140 were constructed by lentivirus infection,hUSC140-Exos were extracted and acted on chondrocytes.The expression levels of miR140 in hUSCs and chondrocytes were determined by qPCR.The regulatory effect of hUSC-140-Exos on the biological changes of OA chondrocytes was detected by proliferation test,migration test,and ECM detection of OA chondrocytes.Based on the regulation of miR140/CAPN1 axis on the occurrence of mitophagy in chondrocytes,the regulatory effects of hUSC-140-Exos on ROS level,mitochondrial morphology,membrane potential and mitophagy in OA chondrocytes were detected.5.The rats were divided into five groups:normal control group(only the j oint capsule was cut),OA group(injection of saline into the joint cavity),hUSC-Exos group,miR140 agomir group,and hUSC-140-Exos group.Imaging evaluations were performed at 4 and 8 weeks after the intra-articular injection treatment,including the comparison of the number of osteophytes in the rat knee joint,the number of free bodies in the joint cavity,and the quantitative analysis and comparison of the microstructure of the subchondral bone.Rat knee joint specimens were obtained under ethical permission,using gross observation(overall pathological score),histological staining(OARSI score)and the levels of chondrocyte extracellular matrix and mitophagy molecules detected by IHC to explore the efficacy and underlying mechanism of hUSC-140-Exos on repairing rat knee OA.Results:1.Compared with normal cartilage,the surface of cartilage at early stage of OA is rough and uneven,and the matrix coloring is reduced.Chondrocytes showed edema,hypertrophy and death,and the content of ECM proteoglycan and type II collagen decreased(p<0.01,p<0.05);vertical cracks appeared in the cartilage at medial-late stage of OA,the matrix coloring was significantly reduced,and the cell arrangement direction was obviously disordered.Chondrocytes died,and ECM proteoglycan and type II collagen contents were significantly reduced(p<0.001,p<0.01).The OARSI score of cartilage at early stage of OA was 11.67±3.51,which were significantly higher than that of normal cartilage(p<0.05);And the score of cartilage at medial-late stage of OA was 20.67±3.06,which were significantly higher than that of normal cartilage and cartilage at early stage of OA(p<0.001,p<0.05).Under the TEM microscope,the number of cells in the cartilage tissue was small,and only a few chondrocytes were observed in the specimens of each group.Compared with the mitochondrial morphology in normal cartilage tissue,the mitochondrial swelling of chondrocytes in the OA group increased significantly with the progression of OA,and the chondrocytes in the middle and late stages of OA had obvious pyknosis,mild expansion of the rough endoplasmic reticulum,and multiple vacuoles in the cells.IHC detection found that compared with normal cartilage,the expression levels of Parkin and LC3 in cartilage at early stage of OA were slightly increased,with no significant difference,but the expression level of p62 was significantly up-regulated(p<0.05),showing that the degradation of mitophagosomes was limited.The expression levels of Parkin andLC3 in chondrocytes at medial-late stages of OA were significantly downregulated,and LC3 was significantly down-regulated compared with chondrocytes at early stage of OA(p<0.05),and the expression level of p62 was significantly up-regulated compared with normal and chondrocytes at early stage of OA(p<0.01,p<0.05),manifested as a decrease in the level of mitophagy.In addition,WB detection found that compared with normal chondrocytes,the expression levels of Parkin and LC3 in chondrocytes at early stage of OA were significantly upregulated(p<0.05,p<0.05),and there was no obvious difference in the expression level of p62,suggesting the increased level of mitophagy at early stage of OA.However,the expression levels of Parkin and LC3 in chondrocytes at medial-late stages of OA were significantly down-regulated than that in chondrocytes at early stage of OA(p<0.01,p<0.01),and the expression level of p62 was significantly up-regulated than normal chondrocytes and chondrocytes at early stage of OA(p<0.01,p<0.01),which showed that the level of mitophagy decreased at mediallate stage of OA.2.Except that there was no significant difference in the expression level of Parkin in chondrocytes at early stage of OA and LC3 in chondrocytes at medial-late stage of OA,miR140 significantly increased the expression level of Parkin in normal chondrocytes and chondrocytes at medial-late stage of OA(p<0.05,p<0.05),significantly increased the expression level of LC3 in normal chondrocytes and chondrocytes at early stage of OA(p<0.01,p<0.05),and significantly decreased the expression level of p62 in normal chondrocytes and chondrocytes at early,medial-late stage of OA(p<0.05,p<0.05,p<0.05),suggesting that miR140 promotes mitophagy in both normal and OA chondrocytes.Further exploration of the target of miR140 regulating the level of mitophagy in chondrocytes found that miR140 and CAPN1 have a binding site with a size of 8 base sequences,and dualluciferase reporter gene detection showed that miR140 down-regulated the expression of CAPN1(p<0.001).In addition,WB detection found that the expression level of CAPN1 in chondrocytes gradually increased with the progression of OA,which was inversely correlated with the expression level of miR140.Moreover,miR140 significantly decreased both the protein and gene level of CAPN1 in chondrocytes(p<0.05,p<0.01).Therefore,miR140 may regulate the changes of mitophagy in chondrocytes by targeting and downregulating the expression of CAPN1.By detecting the mitochondrial morphology,ROS level,mitochondrial membrane potential and expression level of mitophagy molecules in each group of chondrocytes found that the number of mitophagosomes decreased,ROS accumulated,mitochondrial membrane potential decreased and mitophagy level reduced when the CAPN1 was over-expressed in chondrocytes,while miR140 significantly decreased the expression of CAPN1 in chondrocytes,alleviated mitochondrial swelling,reduced ROS accumulation,increased mitochondrial membrane potential,and promotes mitophagy.3.In this study,rice grain-like morphology,adherent growth,positive expression of CD29(93.2%),CD44(97.3%),CD73(99.7%),CD90(99.8%),CD105(90.0%),no expression of CD34(0.13%)and HLA-DR(0.012%)were successfully cultured,and hUSCs that can induce osteogenic,adipogenic and chondrogenic differentiation in vitro meet the definition criteria for MSCs proposed by the International Society for Cell Therapy Committee on Mesenchymal and Tissue Stem Cells(ISCT).The hUSC-Exos were isolated and extracted by ultracentrifugation.They had a spherical double-membrane structure with a particle size of 135.5±59.5nm.They expressed CD9,CD63 and CD81 on the surface,but did not express Calnexin,which met the definition of Exos.The bioregulation experiment of hUSC-Exos on OA chondrocytes showed that 1,5,10 μg/ml hUSC-Exos could significantly improve the proliferation rate and migration ability,and showed a concentration gradient effect,the effect was enhanced with the increase of the concentration,but the higher concentration(15μg/ml,20 μg/ml)hUSC-Exos lost the concentration enhancement effect,showed a negative effect of inhibiting the proliferation and migration of OA chondrocytes.Therefore,10 μg/ml hUSC-Exos were chosen for the subsequent experiments.4.The expression level of miR140 in hUSCs is very low.Compared with hUSCs and hUSC-NC obtained by negative lentivirus infection,hUSCs overexpressing miR140(hUSC-140)significantly increased the expression level of miR140(p<0.001).And the gene level of miR140 in chondrocytes co-culture with hUSC140-Exos was significantly increased(p<0.01).It is proved that the expected goal of increasing the expression level of miR140 in chondrocytes can be realized through hUSC-Exos delivery.The bioregulation experiment of hUSC-140-Exos on OA chondrocytes showed that hUSC-140-Exos not only was superior to hUSCExos at the same concentration in terms of enhancing the proliferation rate and migration ability of OA chondrocytes,but also significantly improved the expression level of Collagen Ⅱ(p<0.01),decreased the expression level of ADAMTS5(p<0.05)and MMP13(p<0.001).In addition,hUSC-140-Exos alleviated the mitochondrial swelling and structural changes of OA chondrocytes induced by IL-1β intervention,reduced intracellular ROS level(p<0.01),increased mitochondrial membrane potential(p<0.05),significantly down-regulated the expression level of CAPN1(p<0.01),and increased the expression level of Parkin(p<0.01)and LC3(p>0.05),and decreased the expression level of p62(p<0.05),promoted the mitophagy of chondrocytes.5.Three-dimensional reconstruction of rat knee joints scanned by micro-CT revealed that intra-articular injection of hUSC-140-Exos significantly reduced the number of free bodies and osteophytes in knees at 4 and 8 weeks(p<0.05,p<0.05),was superior to hUSC-Exos and miR140 agomir.Moreover,in the hUSC-140-Exos group,the subchondral bone plate was continuous and the bone trabeculae were arranged neatly,no osteophyte formation was observed.After 4 weeks and 8 weeks of treatment,hUSC-140-Exos effectively increased the BMD(p<0.01,p<0.01);after 8 weeks of treatment,it significantly increased the Tb.Th(p<0.05);after 4 weeks and 8 weeks of treatment,it significantly improved Tb.Sp(p<0.01),effectively improved subchondral bone microstructure.The overall observation of the knee joints of rats showed that the cartilage surface of the OA knee joints was rough,partially destroyed and eroded,with abnormal color,and osteophyte was formed.After 4 weeks of miR140 agomir treatment and 4 weeks and 8 weeks of hUSC-140-Exos treatment,the cartilage morphology was improved,and the pathological score was significantly lower than that of the OA group(p<0.05,p<0.05 and p<0.01).Histological staining showed that the articular cartilage of OA rats was damaged,even cracks were formed,the cells were arranged in disorder,there was edema and hypertrophy,the coloring of the matrix was significantly reduced,the tide line was broken,and the subchondral marrow cavity tissue invaded into the cartilage.After 8 weeks of hUSC-Exos treatment(p<0.05),and 4 weeks and 8 weeks of miR140 agomir treatment for(p<0.01,p<0.01),and 4 weeks and 8 weeks of hUSC-140-Exos treatment(p<0.05,p<0.001),significantly improved cartilage lesions and decreased OARSI scores.In addition,hUSC-Exos,miR140 agomir and hUSC-140-Exos increased the expression level of CollagenⅡ and aggrecan of chondrocytes,reduced the content of hypertrophic chondrocyte marker Collagen X,and hUSC-140-Exos group at 8-weeks had the best efficacy(p<0.01,p<0.01,and p<0.01).Further exploring the level of mitophagy in rat OA knee chondrocytes found that,after 4 weeks of treatment,there was no significant difference in the expression level of Parkin in chondrocytes,but the expression level of LC3 was significantly increased after miR140 agomir or hUSC-140-Exos treated(p<0.05,p<0.01),and expression level of p62 was significantly decreased(p<0.05,p<0.05);after 8 weeks of treatment,miR140 agomir and hUSC-140-Exos significantly increased the expression level of Parkin(p<0.05,p<0.05)and LC3(p<0.01,p<0.01),decreased the expression level of p62(p<0.05,p<0.05),suggesting that intra-articular injection of hUSC140-Exos and miR140 agomir can promote mitophagy in rat OA articular chondrocytes.Conclusion:1.Compared with normal and chondrocytes at early stage of OA,the mitochondrial morphology of chondrocytes at medial-late stages of OA was swollen,the intimal cristae structure disappeared,and mitophagy was impaired and stagnated.2.miR140 improved the level of mitophagy in normal and OA chondrocytes at different stages,and reduced intracellular ROS accumulation,increased mitochondrial membrane potential,and avoided damage to mitochondrial morphology and structure through decreasing the expression level of CAPN1.3.hUSC-Exos are rich in sources and easy to prepare.10 μg/ml of hUSC-Exos has the best effect on promoting the proliferation and migration of OA chondrocytes.Therefore,hUSC-Exos could be used as a good carrier to deliver miR140 to OA chondrocytes.4.hUSC-140-Exos were successfully prepared,which significantly increased the expression level of miR140 in chondrocytes.Moreover,hUSC-140-Exos is not only superior to hUSC-Exos at the same concentration in improving the proliferation and migration ability of OA chondrocytes,and enhancing the synthesis of ECM,but also down-regulates the expression of CAPN1,promotes the occurrence of mitophagy in chondrocytes,and improves the function of mitochondria.5.The use of hUSC-Exos or miR140 agomir alone and the combined hUSC-140Exos both promoted the cartilage repair in rat OA knee j oints,among which hUSC140-Exos showed the best efficacy.更多还原