体外条件下TGF-β3、BMP-2和DEX诱导兔滑膜间充质干细胞向软骨细胞谱系分化的研究

来源 :中国骨与关节杂志 | 被引量 : 0次 | 上传用户:chen406507025
下载到本地 , 更方便阅读
声明 : 本文档内容版权归属内容提供方 , 如果您对本文有版权争议 , 可与客服联系进行内容授权或下架
论文部分内容阅读
目的:本实验拟将分离纯化得到的滑膜间充质干细胞( synovial-derived mesenchymal stem cells,SMSCs )在体外培养条件下进行成软骨刺激诱导,并从转录和翻译两个水平寻找进入软骨细胞分化谱系的证据,进而判断转化生长因子β3( transforming growth factor-β3,TGF-β3)、骨形态发生蛋白( bone morphogenetic protein-2,BMP-2)和地塞米松( dexamethasone,DEX )诱导 SMSCs 进入软骨细胞分化谱系。方法贴壁法分离纯化得到 SMSCs,在体外培养条件下用含500 ng / ml BMP-2、10 ng / ml TGF-β3、10-7 M DEX 的高糖 DMEM 培养基进行刺激诱导,并在倒置相差显微镜下观察分化过程中其形态学的变化,以 RT-PCR检测I、II型胶原及软骨特异性Aggrecan ( AGN )的mRNA表达,细胞免疫荧光化学染色的方法检测细胞分化过程中I、II型胶原的表达,碱性甲苯胺蓝细胞化学染色检测软骨特异性GAG的表达,证实SMSCs的诱导成软骨作用。结果 SMSCs在前述诱导条件下,诱导后14天细胞逐渐由小梭形变为多角形、类软骨细胞样形态,RT-PCR可以检测到I、II型胶原及AGN基因的表达,细胞免疫荧光化学染色I、II型胶原、碱性甲苯胺蓝细胞化学染色结果呈阳性。而未经诱导的SMSCs形态基本保持梭形,基因表达和染色呈阴性,两组间差异显著。细胞免疫荧光化学染色分析SMSCs在软骨诱导培养基中诱导后14天表达I、II型胶原;未经诱导的SMSCs不表达I、II型胶原。说明SMSCs在软骨诱导培养基中诱导后14天进入软骨细胞分化谱系,可作为种子细胞在同样的诱导条件下向软骨分化。结论 SMSCs作为新的MSCs家族成员,显示出与BMSCs相似的多向分化潜能,500 ng/ml BMP-2、10 ng/ml TGF-β3、10-7 M DEX的高糖DMEM培养基中培养后14天,SMSCs已进入软骨细胞分化谱系。SMSCs可作为半月板组织工程的种子细胞。“,”Objective To explore that synovial-derived mesenchymal stem cells ( SMSCs ) differentiated into the chondrocyte lineage induced by transforming growth factor-β3 ( TGF-β3 ), bone morphogenetic protein-2 ( BMP-2 ), and dexamethasone ( DEX ) in vitro culture conditions. After isolation and purification, SMSCs were induced to chondrocytes, which was certiifed on the transcriptional and translational levels. Methods SMSCs were obtained after isolation and purification by the adherence method, which were induced in high glucose Dulbecco modiifed Eagle medium ( DMEM ) containing 500 ng/ml BMP-2, 10 ng/ml TGF-β3 and 10-7 M DEX in vitro culture conditions. The morphologic changes during the differentiation process were observed using the inverted phase contrast microscope, and collagen I, collagen II and messenger-Ribonucleic Acid ( m-RNA ) expressions of cartilage-specific Aggrecan ( AGN ) were detected by Reverse Transcription-Polymerase Chain Reaction ( RT-PCR ). The immunolfuorescence staining was used to detect the expressions of collagen I and II during the differentiation process, and the alkaline toluidine blue staining was used to detect the expressions of cartilage-speciifc group-speciifc antigen gene ( GAG ) to conifrm the capability of SMSCs to differentiate into the chondrocyte lineage. Results After 14 days’ induction, the small spindle SMSCs were gradually changed into the polygonal morphology, just like the chondrocytes.Collagen I, collagen II and expressions of AGN could be detected by RT-PCR. The results of immunolfuorescence staining of collagen I and II and alkaline toluidine blue staining were positive. Without the induction, SMSCs basically maintained the spindle-shape morphology, and the gene expressions and staining results were negative. The differences between them were statistically signiifcant. Based on the results of immunolfuorescence staining, SMSCs expressed collagen I and II after 14 days’ induction in DMEM. Without the induction, SMSCs did not express collagen I or II. It was illustrated that SMSCs differentiated into the chondrocyte lineage after 14 days’ induction in DMEM, which could be used as the seed cells to differentiate into chondrocytes under the same condition. Conclusions As a new member in the family of mesenchymal stem cells ( MSCs ), SMSCs show the multi-differentiation potential which is similar to that of bone marrow-derived mesenchymal stem cells ( BMSCs ). After induced in the high glucose DMEM containing 500 ng/ml BMP-2, 10 ng/ml TGF-β3 and 10-7 M DEX, SMSCs enter into the chondrocyte lineage at the 14th day, which can be used as the seed cells for the meniscus tissue engineering.
其他文献
目的了解维吾尔族糖尿病及糖调节受损患者膳食摄入状况,为制定相关干预措施提供科学依据。方法采用整群抽样的方法,横断面调查乌鲁木齐市6个社区维吾尔族40岁~常住居民中选择
期刊
为观察侧方内括约肌切断术(侧切术)治疗肛裂的临床疗效,采用该术式治疗肛裂146例(试验组),与传统的切扩术治疗肛裂98例(对照组)进行对比观察.结果显示,试验组在治愈率与术后
由中国康复医学会脊柱脊髓损伤专业委员会微创脊柱外科学组、《中国骨与关节杂志》编辑部、卫计委内镜与微创医师定期考核培训学校与中国人民解放军总医院第一附属医院全军骨
近年来,我们采用CO2激光切除配合干扰素局部注射和中药坐浴治疗肛周尖锐湿疣65例,效果满意,现报告如下.rn临床资料:65例肛周尖锐湿疣中,男36例,女29例;年龄19~62岁,平均(35±1
为比较两种手术方式治疗陈旧性肛裂的临床效果,采用侧位内括约肌切断术治疗陈旧性肛裂42例,后正中内括约肌切断术治疗陈旧性肛裂44例.结果显示,侧位内括约肌切断术有效率95.2
目的 探讨外源性蛋氨酸(nethionine,Met)对饮水砷暴露小鼠脑中砷形态分布及NO代谢影响.方法 将小鼠随机分为对照组、染砷组及低、中、高剂量Met干预组.饮水染砷4周,最后1周在
国际临床骨密度测量学会(ISCD)发布了髋部定量CT(QCT)临床应用的最新共识.ISCD致力于汇总归纳定量CT的临床应用证据并在2015年ISCD指南修订会议上发布共识指南报告,讨论并一
目的探讨腹腔镜手术和开腹手术治疗老年良性卵巢肿瘤患者对T细胞亚群及神经内分泌功能的影响。方法选取老年良性卵巢肿瘤患者86例随机分为对照组和观察组各43例,给予对照组患
期刊
@@
采用复方亚甲蓝肛周局部皮下注射治疗肛门湿疹78例,取得满意疗效,现报告如下.rn临床资料:本组男56例,女22例;年龄18~52岁,平均33.5岁;病程15d至3年,平均8个月.肛周局部均有不
目的分析中晚期妊娠妇女的心电图改变情况。方法选取2013年9月1日-2015年1月1日在该院就医的124例妇女为研究对象,其中中晚期妊娠妇女62例为干预组,健康体检妇女62例为对照组
期刊
@@