CAJ | 학술논문

背景:兔是软骨组织工程研究中应用非常广泛的实验动物模型,关节软骨细胞的去分化现象已经被广泛认可.目的:观察体外传代培养过程中兔关节软骨细胞的去分化现象.方法:将从新西兰大白兔膝关节分离获取的原代软骨细胞进行传代培养至第7代,对细胞生长、形态、基质分泌以及基因表达等方面分别采用细胞计数,显微镜观察,F机动蛋白染色、番红O染色,糖胺聚糖定量测定以及半定量聚合链式反应进行鉴定和比较.结果与结论:光学显微镜下,兔关节软骨细胞在体外传代培养过程中细胞形态由小且圆形或多角形,逐步转变大且为成纤维细胞样的梭形形态,对F机动蛋白的染色进一步佐证了这样的形态变化.细胞计数结果表明,细胞增殖能力随代次增加显著下降,特别是第3代以后的软骨细胞基本无明显增殖;经过番红 O 染色以及定量测定糖胺聚糖的含量,发现软骨特性胞外基质分泌量从第2代细胞开始就呈现显著的降低.半定量聚合链式反应检测结果表明,随着传代次数的增加,特别是第3代以后,软骨相关特征分子(包括Ⅱ型胶原、聚集蛋白聚糖、软骨寡聚基质蛋白和SOX9等)基因表达水平下调;而与去分化相关的特征分子Ⅰ型胶原和多能蛋白聚糖基因表达水平上调,同时,细胞表面分子CD90基因表达上调,而CD14基因表达未见明显变化.结果证实,兔软骨细胞在体外传代过程中可出现快速地去分化现象,呈现出特征基因表达水平的变化,第3代以内的软骨细胞适合应用于软骨组织再生修复.
배경:토시연골조직공정연구중응용비상엄범적실험동물모형,관절연골세포적거분화현상이경피엄범인가.목적:관찰체외전대배양과정중토관절연골세포적거분화현상.방법:장종신서란대백토슬관절분리획취적원대연골세포진행전대배양지제7대,대세포생장、형태、기질분비이급기인표체등방면분별채용세포계수,현미경관찰,F궤동단백염색、번홍O염색,당알취당정량측정이급반정량취합련식반응진행감정화비교.결과여결론:광학현미경하,토관절연골세포재체외전대배양과정중세포형태유소차원형혹다각형,축보전변대차위성섬유세포양적사형형태,대F궤동단백적염색진일보좌증료저양적형태변화.세포계수결과표명,세포증식능력수대차증가현저하강,특별시제3대이후적연골세포기본무명현증식;경과번홍 O 염색이급정량측정당알취당적함량,발현연골특성포외기질분비량종제2대세포개시취정현현저적강저.반정량취합련식반응검측결과표명,수착전대차수적증가,특별시제3대이후,연골상관특정분자(포괄Ⅱ형효원、취집단백취당、연골과취기질단백화SOX9등)기인표체수평하조;이여거분화상관적특정분자Ⅰ형효원화다능단백취당기인표체수평상조,동시,세포표면분자CD90기인표체상조,이CD14기인표체미견명현변화.결과증실,토연골세포재체외전대과정중가출현쾌속지거분화현상,정현출특정기인표체수평적변화,제3대이내적연골세포괄합응용우연골조직재생수복.
@@@@BACKGROUND: Rabbits have been extensively utilized in cartilage tissue engineering as experimental models. However, research efforts remain limited concerning the dedifferentiation of rabbit articular chondrocytes. OBJECTIVE: To explore the dedifferentiation of rabbit articular chondrocytes during in vitro expansion. METHODS: Chondrocytes were isolated from articular cartilage of New Zealand white rabbits and subcultured in vitro until passage 7. Cel s were analyzed regarding cel growth, morphology, deposition of cartilaginous extracel ular matrix and gene expression using cel s counting method, microscopic examination, F-actin staining, safranine-O staining, quantitative determination of glycosaminoglycans and semiquantitative reverse transcription-polymerase chain reaction, respectively RESULTS AND CONCLUSION: Under light microscope, rabbit articular chondrocytes changed from smal round or polygonal shape into fibroblast-like morphology during the in vitro subculture, which was further confirmed with F-actin staining using phal oidin. Results of cell counting showed that, cell proliferation significantly declined with passage generations, especial y after passage 3, articular chondrocytes were not proliferating. Safranine-O staining and glycosaminoglycans quantification demonstrated that, the capacity of producing cartilaginous extracel ular matrix decreased dramatical y as early as passage 2. Semiquantitative polymerase chain reaction analysis showed that, the gene expression of col agen Ⅱ, aggrecan, cartilage oligomeric matrix protein and SOX9 was downregulated especially for cells after passage 3, while the expression of collagen I and versican was upregulated. In addition, the CD90 expression increased and CD14 expression remained unaltered. Rabbit articular chondrocytes dedifferentiate quickly upon in vitro expansion with distinct variation of gene expression profile, and cells within passage 3 are appropriate for applications in cartilage repair.