CAJ | 학술논문

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。
목적：본실험의장분리순화득도적활막간충질간세포( synovial-derived mesenchymal stem cells，SMSCs )재체외배양조건하진행성연골자격유도，병종전록화번역량개수평심조진입연골세포분화보계적증거，진이판단전화생장인자β3( transforming growth factor-β3，TGF-β3)、골형태발생단백( bone morphogenetic protein-2，BMP-2)화지새미송( dexamethasone，DEX )유도 SMSCs 진입연골세포분화보계。방법첩벽법분리순화득도 SMSCs，재체외배양조건하용함500 ng / ml BMP-2、10 ng / ml TGF-β3、10-7 M DEX 적고당 DMEM 배양기진행자격유도，병재도치상차현미경하관찰분화과정중기형태학적변화，이 RT-PCR검측I、II형효원급연골특이성Aggrecan ( AGN )적mRNA표체，세포면역형광화학염색적방법검측세포분화과정중I、II형효원적표체，감성갑분알람세포화학염색검측연골특이성GAG적표체，증실SMSCs적유도성연골작용。결과 SMSCs재전술유도조건하，유도후14천세포축점유소사형변위다각형、류연골세포양형태，RT-PCR가이검측도I、II형효원급AGN기인적표체，세포면역형광화학염색I、II형효원、감성갑분알람세포화학염색결과정양성。이미경유도적SMSCs형태기본보지사형，기인표체화염색정음성，량조간차이현저。세포면역형광화학염색분석SMSCs재연골유도배양기중유도후14천표체I、II형효원；미경유도적SMSCs불표체I、II형효원。설명SMSCs재연골유도배양기중유도후14천진입연골세포분화보계，가작위충자세포재동양적유도조건하향연골분화。결론 SMSCs작위신적MSCs가족성원，현시출여BMSCs상사적다향분화잠능，500 ng/ml BMP-2、10 ng/ml TGF-β3、10-7 M DEX적고당DMEM배양기중배양후14천，SMSCs이진입연골세포분화보계。SMSCs가작위반월판조직공정적충자세포。
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.