小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

背景：研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9 的浓度呈剂量依赖正相关关系。 目的：通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原 mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。 方法：取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。 结果与结论：第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原 mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P > 0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。     

DNMT3A对骨关节炎软骨细胞SOX9基因DNA甲基化修饰的影响

目的 探讨DNMT3A对SOX9基因DNA甲基化修饰的影响。方法 培养正常软骨细胞和OA软骨细胞,显微镜下观察软骨细胞形态,甲苯胺蓝染色、Ⅱ型胶原染色鉴定软骨细胞,qRT-PCR、Western Blot检测DNMT3A、SOX9 mRNA和蛋白表达水平,亚硫酸氢盐测序检测SOX9基因DNA甲基化状态,慢病毒沉默DNMT3A观察SOX9基因DNA甲基化修饰的变化。结果 正常软骨细胞形态规则,体积较大,数量较多,OA软骨细胞形态不规则,体积较小,数量较少。与正常软骨细胞比较,OA软骨细胞中DNMT3A mRNA和蛋白表达量增高（P＜0.05）,SOX9 mRNA和蛋白表达量明显降低（P＜0.05）,SOX9基因DNA甲基化百分比增高。在OA软骨细胞中沉默DNMT3A后,SOX9基因DNA甲基化百分比明显降低。结论 DNMT3A调节SOX9基因DNA甲基化修饰参与OA软骨细胞病变。     

SOX9基因调控软骨形成的研究进展

SOX9是软骨细胞中的关键转录因子.软骨细胞在复杂的增殖分化过程中,伴有多因素、多环节的精细调控,其中SOX9参与调控并起主要软骨形成因子的作用,同时SOX9在软骨组织工程中的变化已成为软骨形成的重要检测依据,通过各种方法如SOX9转染至种子细胞、改变外界环境,最终提高SOX9表达并诱导软骨形成.文章对软骨形成中SOX...     

人类软骨糖蛋白39诱导关节软骨祖母细胞的成软骨分化

背景：有研究表明人类软骨糖蛋白39与骨关节软骨的退变与修复具有一定的关系,但其具体的作用机制并不十分明确。 目的：观察人类软骨糖蛋白39对成人膝关节软骨祖母细胞成软骨诱导分化的影响。 方法：取成人关节软骨,消化分离培养关节软骨祖母细胞;流式细胞仪检测传代细胞中能够表达CD105、CD166的细胞量并进行分离提纯。将分离的软骨祖母细胞采用单层培养法培养,传代培养至第2代后向分离培养所得的软骨祖母细胞中,分别经过含人类软骨糖蛋白39成软骨培养基及普通成软骨诱导培养基的诱导培养14 d后,通过免疫组织化学染色观察经诱导后细胞中Ⅱ型胶原的表达及通过大体组织学观察评估软骨的形成。 结果与结论：关节软骨组织中可以分离出能够表达CD105、CD166的关节软骨祖母细胞,软骨祖母细胞经过诱导分化后逐渐聚集并形成结节,经诱导后Ⅱ型胶原免疫细胞化学着色阳性,且经人类软骨糖蛋白39诱导细胞形成的结节更大,Ⅱ型胶原表达更多。结果表明,成人关节软骨中能够分离培养出具有成软骨分化能力的干细胞系细胞即软骨祖母细胞,且能够被定向诱导分化为软骨细胞,人类软骨糖蛋白39对其分化过程具有一定的促进作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

两种可注射Pluronic F-127水凝胶复合物修复兔软骨缺损的效果差异

文题释义：Pluronic F-127：是一种聚氧化乙烯-聚氧化丙烯-聚氧化乙烯三嵌段共聚物,具有在低温下为液态、常温下为固态的特性,为温固化水凝胶,具有良好的生物相容性与生物可降解性。作为组织工程中的细胞支架,Pluronic F-127已被广泛应用于软骨与皮肤等的构建。 SOX9基因：在性别决定与分化过程中具有重要的作用,在胚胎发育过程中参与骨的形成,同时其也参与神经系统与胰腺的发育及肿瘤的发生。在骨骼形成过程中,SOX9通过与 DNA 特定区域结合促进间充质细胞的聚集：首先是在软骨前体细胞中,随后是在分化中的或成熟的前体细胞中表达,维持软骨细胞增殖,抑制其向肥大软骨细胞分化,因此SOX9在软骨形成过程中起着十分重要的作用。 背景：预实验显示,SOX9基因转染的骨髓间充质干细胞可在Pluronic F-127水凝胶内良好的生长与增殖,促进细胞外基质的分泌,增加软骨基质的表达。 目的：利用慢病毒基因诱导方式将SOX9基因转导至骨髓间充质干细胞中,将其与可注射Pluronic F-127水凝胶复合,观察Pluronic F-127水凝胶复合物修复软骨缺损的效果。 方法：利用慢病毒基因诱导方式将SOX9基因转导至骨髓间充质干细胞中,转染48 h后与Pluronic F-127水凝胶复合。取60只新西兰大白兔(武汉科技大学实验动物中心提供),建立右侧膝关节股骨髁软骨缺损模型,随机分3组处理：模型组缺损部位未植入任何材料,对照组植入未转染的骨髓间充质干细胞与Pluronic F-127水凝胶复合物,实验组植入SOX9基因转染的骨髓间充质干细胞与Pluronic F-127水凝胶复合物。术后4,12周取缺损部位组织,分别进行Micro-CT三维重建、苏木精-伊红染色、番红O染色、Ⅱ型胶原免疫组织化学染色与Wakitani软组织损伤修复组织学评分。实验获得武汉科技大学伦理委员会批准。 结果与结论：①术后12周Micro-CT三维重建显示,模型组缺损区域未见明显的修复,中央仍有较大的凹陷;对照组可见明显的修复,中央凹陷区域明显减小,可见较多的再生骨小梁结构;实验组缺损部位基本完成修复;②术后12周苏木精-伊红染色显示,模型组缺损区仍未见骨小梁结构,细胞分布紊乱,未见软骨陷窝;对照组可见较多的骨组织重建,缺损区域主要由软骨样组织与纤维组织填充;实验组骨组织重建较充分,缺损区域主要由软骨样细胞与软骨样细胞外基质填充,细胞呈柱状排列,与周围软骨相似;③术后12周番红O染色与Ⅱ型胶原免疫组织化学染色显示,模型组可见少量糖胺多糖表达,未见Ⅱ型胶原表达;对照组可见较多的糖胺多糖与Ⅱ型胶原表达,实验组糖胺多糖与Ⅱ型胶原表达最多;④实验组Wakitani软组织损伤修复组织学评分高于对照组与模型组(P < 0.05);⑤结果表明,负载SOX9基因转染骨髓间充质干细胞的Pluronic F-127水凝胶复合物可促进软骨缺损的修复。 ORCID: 0000-0002-9648-5297(樊薰勤) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

bFGF和TGF-β对原发性骨关节炎软骨间充质祖细胞增殖调控作用

目的观察bFGF和TGF-β1对原发性骨关节炎(OA)关节软骨间充质祖细胞(MPCs)的促增殖作用,为通过调控关节软骨MPCs以防治原发性OA提供理论依据。方法采用MTT法测定不同浓度bFGF和TGF-β1单独或联合作用下对原发性OA关节软骨MPCs的增殖作用。结果 10.0~50.0 ng/mL的bFGF、0.1~1.0 ng/mL的TGF-β1单独作用于传代培养原发性OA关节软骨MPCs时,其培养细胞的增殖速率显著增加(P0.05),而随着二者浓度的进一步增加,其增殖速率无显著变化(P0.05);bFGF 10.0 ng/mL+TGF-β11.0 ng/mL联合作用时,对原发性OA关节软骨MPCs具有明显促增殖作用(P0.05)。结论 bFGF、TGF-β1对原发性OA关节软骨MPCs增殖具有重要作用,不同浓度的bFGF、TGF-β1及bFGF+TGF-β1促增殖作用不同,提示通过适宜浓度的bFGF、TGF-β1对原发性OA关节软骨MPCs的作用,可能具有促进原发性OA关节软骨损伤修复的效果。     

软骨细胞培养液诱导肌源性干细胞转化为软骨样细胞

目的:体外定向诱导大鼠肌源性干细胞(MDSCs)向软骨细胞转化,并对诱导细胞进行鉴定.方法:用差速贴壁的方法对SD大鼠MDSCs进行分离和培养.同时将软骨细胞进行体外培养,收集软骨细胞培养液作为MDSCs诱导液.将MDSCs进行诱导分化,9d后取出标本,行甲苯胺蓝染色检测软骨基质的分泌,免疫组织化学检测软骨特异性Ⅱ型胶原表达.RT-PCR检测Ⅱ型胶原和aggrecan mRNA表达.结果:5d后镜下见细胞形态由梭形向多边多角形转化.9d后诱导组甲苯胺蓝染色阳性,Ⅱ型胶原免疫组织化学检测阳性,RT-PCR检测Ⅱ型胶原和aggrecan mRNA呈阳性表达.结论:软骨细胞培养液可诱导MDSC向软骨细胞转化.     

血小板裂解液对Wistar大鼠创伤性膝软骨损伤中Runx2、SOX9表达的影响

目的 探讨血小板裂解液(PL)在修复Wistar大鼠创伤性膝软骨损伤中对Runx2、SOX9表达的影响。方法 将30只Wistar大鼠随机分成对照组、生理盐水组和血小板裂解液组,每组10只。HE染色和番红O-固绿染色观察各组大鼠膝关节软骨情况,并进行Mankin评分;免疫组织化学染色检测大鼠膝关节软骨Runx2、SOX9的表达;Western blot检测大鼠膝关节软骨Runx2、SOX9的表达。结果 经过PL注射后,创伤性膝软骨损伤大鼠软骨组织损伤明显减轻,软骨Mankin评分明显降低,膝关节软骨组织Runx2、SOX9阳性表达细胞数明显降低,膝关节软骨组织Runx2、SOX9的蛋白表达明显降低(P<0.01)。结论 血小板裂解液可促进Wistar大鼠软骨组织结构性恢复,降低软骨损伤后Runx2、SOX9表达水平。     

鹿茸多肽诱导骨髓间充质干细胞体外向软骨表型的分化

背景：实验证实鹿茸多肽可以促进体外培养软骨细胞的增殖和细胞外基质糖胺多糖、Ⅱ型胶原、Aggrecan蛋白的表达。 目的：通过对体外培养的兔骨髓间充质干细胞在特定培养液作用下向软骨细胞表型分化的研究,探讨鹿茸多肽对其软骨分化的影响。 方法：将第3代兔骨髓间充质干细胞随机分为空白对照组、诱导组、鹿茸多肽组,分别采用普通培养液、诱导培养液、含10 mg/L鹿茸多肽的诱导培养液于离心管内进行培养;并取兔的关节软骨细胞作为关节软骨组。分别于1,2,3周后取材,通过组织学、生物化学和RT-PCR技术,对离心管内构建的软骨组织进行形态学和细胞功能状态的观察。 结果与结论：空白对照组培养2周后,细胞团块逐渐崩解,无法进行苏木精-伊红染色。诱导组、鹿茸多肽组细胞团块除有轻度收缩外,呈白色半透明状;苏木精-伊红染色发现部分细胞为圆形或卵圆形,表层细胞密度大;诱导组、鹿茸多肽组糖胺多糖含量及Ⅱ型胶原mRNA表达随培养时间延长而增多,各时间点诱导组、鹿茸多肽组含量均高于空白对照组(P < 0.05);各时间点鹿茸多肽组糖胺多糖含量及Ⅱ型胶原mRNA表达均高于诱导组,但低于关节软骨组 (P< 0.05)。提示骨髓间充质干细胞在特定培养条件下能向软骨细胞表型分化,且鹿茸多肽对其定向软骨分化有明显促进作用。虽然在体外可以构建出软骨组织,但其与关节软骨质量相比仍有很大差距。     

GDF-5和Dex通过Wnt/β-catenin信号通路促进rBMSCs体外成软骨分化

目的利用生长分化因子(GDF-5)联合地塞米松(Dex)诱导大鼠BMSCs成软骨分化,并探讨Wnt/β-catenin信号通路在其中的作用。方法用全骨髓贴壁法分离培养SD大鼠BMSCs,取P3代细胞随机分成6组:BMSCs组、BMSCs+Dex组、BMSCs+GDF-5组、BMSCs+GDF-5+Dex组、BMSCs+GDF-5+Dex+SB216763组和BMSCs+GDF-5+Dex+XAV-939组。连续诱导培养14 d,倒置相差显微镜观察细胞形态,Alcian blue染色检测细胞的蛋白聚糖,RT-PCR检测成软骨分化相关基因aggrecan、Col2、Sox9、Col1,Wnt/β-catenin信号通路相关基因Dvl1、Gsk3β、β-catenin mRNA表达,Western blot检测Ⅱ型胶原蛋白和β-catenin蛋白表达。结果与BMSCs组相比,BMSCs+GDF-5+Dex组能够使诱导的细胞蛋白聚糖分泌明显增加;Aggrecan、Col2、Sox9基因表达明显增加(P0.05);Ⅱ型胶原蛋白表达增加(P0.05);β-catenin mRNA及蛋白表达水平也增加(P0.05)。与BMSCs+GDF-5+Dex组相比,添加SB216763组蛋白聚糖,Ⅱ型胶原基因和蛋白表达增加(P0.05);相反,添加XAV-939组相应的成软骨分化基因及蛋白表达下降(P0.05)。结论 GDF-5联合地塞米松(Dex)可诱导大鼠BMSCs成软骨分化,Wnt/β-catenin信号通路可能在其中发挥促进作用。     

SOX Trio Decrease in the Articular Cartilage with the Advancement of Osteoarthritis

SOX trio (SOX-5, SOX-6, and SOX-9) maintain the chondrocytic phenotypes and are vital for chondrogenesis in embryonic development. The purpose of this study is to investigate the change in the expression of SOX trio with the advancement of osteoarthritis (OA) in human articular cartilage (AC). Human OA samples from eight patients were obtained from the distal femoral condyles during total knee arthroplasty. Minimally OA cartilage taken from areas with no obvious surface defects on lateral condyles was compared with advanced OA cartilage obtained from areas within 1 cm of overt lesion located on medial condyle surface. SOX-5, SOX-6, and SOX-9 gene expressions significantly decreased by 41% (p = 0.047), 46% (p = 0.047), and 56% (p = 0.029) in advanced OA area compared with the minimally OA area. There was a significant decrease in aggrecan and type II collagen (COL2A1) gene expressions by 73% (p = 0.029) and 65% (p = 0.029), respectively, in advanced OA area compared with the minimally OA area. From Western blotting and immunohistochemistry, SOX-5, SOX-6, SOX-9, type II collagen, and aggrecan protein expressions also significantly decreased in advanced OA cartilage compared with minimally OA cartilage. DNA methylation study of SOX-9 promoter regions revealed no difference in the epigenetic status between the two areas. It is concluded that SOX trio gene and protein decreased with advancement of OA in human articular cartilage.     

SOX trio decrease in the articular cartilage with the advancement of osteoarthritis

SOX trio (SOX-5, SOX-6, and SOX-9) maintain the chondrocytic phenotypes and are vital for chondrogenesis in embryonic development. The purpose of this study is to investigate the change in the expression of SOX trio with the advancement of osteoarthritis (OA) in human articular cartilage (AC). Human OA samples from eight patients were obtained from the distal femoral condyles during total knee arthroplasty. Minimally OA cartilage taken from areas with no obvious surface defects on lateral condyles was compared with advanced OA cartilage obtained from areas within 1 cm of overt lesion located on medial condyle surface. SOX-5, SOX-6, and SOX-9 gene expressions significantly decreased by 41% (p = 0.047), 46% (p = 0.047), and 56% (p = 0.029) in advanced OA area compared with the minimally OA area. There was a significant decrease in aggrecan and type II collagen (COL2A1) gene expressions by 73% (p = 0.029) and 65% (p = 0.029), respectively, in advanced OA area compared with the minimally OA area. From Western blotting and immunohistochemistry, SOX-5, SOX-6, SOX-9, type II collagen, and aggrecan protein expressions also significantly decreased in advanced OA cartilage compared with minimally OA cartilage. DNA methylation study of SOX-9 promoter regions revealed no difference in the epigenetic status between the two areas. It is concluded that SOX trio gene and protein decreased with advancement of OA in human articular cartilage.     

Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.     

SOX trio-co-transduced adipose stem cells in fibrin gel to enhance cartilage repair and delay the progression of osteoarthritis in the rat

The aim of this study was to test the hypotheses that retroviral gene transfer of SOX trio enhances the in vitro chondrogenic differentiation of ASCs, and that SOX trio-co-transduced ASCs in fibrin gel promote the healing of osteochondral defects, and arrest the progression of surgically-induced osteoarthritis in a rat model. ASCs isolated from inguinal fat in rats were transduced with SOX trio genes using retrovirus, and further cultured in vitro in pellets for 21 days, then analyzed for gene and protein expression of SOX trio and chondrogenic markers. SOX trio-co-transduced ASCs in fibrin gel were implanted on the osteochondral defect created in the patellar groove of the distal femur, and also injected into the knee joints of rats with surgically-induced osteoarthritis. Rats were sacrificed after 8 weeks, and analyzed grossly and microscopically. After 21 days, ASCs transduced with SOX-5, -6, or -9 had hundreds-fold greater gene expression of each gene compared with the control with the SOX protein expression matching gene expression. SOX trio-co-transduction significantly increased GAG contents as well as type II collagen gene and protein expression. ASCs co-transduced with SOX trio significantly promoted the in vivo cartilage healing in osteochondral defect model, and prevented the progression of degenerative changes in surgically-induced osteoarthritis.     

Downregulation of MicroRNA-495 Alleviates IL-1β Responses among Chondrocytes by Preventing SOX9 Reduction

PurposeOur previous work demonstrated that miRNA-495 targets SOX9 to inhibit chondrogenesis of mesenchymal stem cells. In this study, we aimed to investigate whether miRNA-495-mediated SOX9 regulation could be a novel therapeutic target for osteoarthritis (OA) using an in vitro cell culture model.Materials and MethodsAn in vitro model mimicking the OA environment was established using TC28a2 normal human chondrocyte cells. Interleukin-1β (IL-1β, 10 ng/mL) was utilized to induce inflammation-related changes in TC28a2 cells. Safranin O staining and glycosaminoglycan assay were used to detect changes in proteoglycans among TC28a2 cells. Expression levels of COX-2, ADAMTS5, MMP13, SOX9, CCL4, and COL2A1 were examined by qRT-PCR and/or Western blotting. Immunohistochemistry was performed to detect SOX9 and CCL4 proteins in human cartilage tissues obtained from patients with OA.ResultsmiRNA-495 was upregulated in IL-1β-treated TC28a2 cells and chondrocytes from damaged cartilage tissues of patients with OA. Anti-miR-495 abolished the effect of IL-1β in TC28a2 cells and rescued the protein levels of SOX9 and COL2A1, which were reduced by IL-1β. SOX9 was downregulated in the damaged cartilage tissues of patients with OA, and knockdown of SOX9 abolished the effect of anti-miR-495 on IL-1β-treated TC28a2 cells.ConclusionWe demonstrated that inhibition of miRNA-495 alleviates IL-1β-induced inflammatory responses in chondrocytes by rescuing SOX9 expression. Accordingly, miRNA-495 could be a potential novel target for OA therapy, and the application of anti-miR-495 to chondrocytes could be a therapeutic strategy for treating OA.     

Heterozygous SOX9 Mutations Allowing for Residual DNA‐binding and Transcriptional Activation Lead to the Acampomelic Variant of Campomelic Dysplasia

Campomelic dysplasia is a malformation syndrome with multiple symptoms including characteristic shortness and bowing of the long bones (campomelia). CD, often lethal due to airway malformations, is caused by heterozygous mutations in SOX9, an SRY‐related gene regulating testis and chondrocyte development including expression of many cartilage genes such as type II collagen. Male to female sex reversal occurs in the majority of affected individuals with an XY karyotype. A mild form without campomelia exists, in which sex‐reversal may be also absent. We report here two novel SOX9 missense mutations in a male (c.495C>G; p.His165Gln) and a female (c.337A>G; p.Met113Val) within the DNA‐binding domain leading to non‐lethal acampomelic CD. Functional analyses of mutant proteins demonstrate residual DNA‐binding and transactivation of SOX9‐regulated genes. Combining our data and reports from the literature we postulate a genotype‐phenotype correlation: SOX9 mutations allowing for residual function lead to a mild form of CD in which campomelia and sex reversal may be absent. © 2010 Wiley‐Liss, Inc.