大鼠软骨细胞与兔软骨细胞的培养与比较  

Culture and comparison of rat and rabbit chondrocytes

作　　者：张振 罗辉宇 曾炼 ZHANG Zhen;LUO Hui-yu;ZENG Lian(Department of Anesthesiology,Xiangyang First People􀆳s Hospital,Hubei Medical University,Xiangyang 441100,China)

机构地区：[1]湖北医药学院附属襄阳市第一人民医院麻醉科,湖北襄阳441000

出　　处：《骨科》2020年第4期323-328,共6页ORTHOPAEDICS

摘　　要：目的体外分离培养大鼠及兔膝关节软骨细胞,观察并比较两种软骨细胞的培养特点。方法采用机械-酶消化法处理SD大鼠幼鼠和新西兰兔的软骨组织,传代培养,倒置显微镜观察细胞形态,细胞计数法测定生长曲线,实时定量PCR测定不同代数的软骨细胞Ⅱ型胶原与含血小板结合蛋白基序的解聚蛋白样金属蛋白酶5(a disintegrin and metalloproteinase with thrombospondin motifs 5, ADAMTS5)表达水平,甲苯胺蓝染色及Ⅱ型胶原酶免疫荧光染色对细胞进行鉴定。结果原代培养大鼠软骨细胞12 h,内贴壁成多角形或不规则型,排列成"铺路石"状,培养3~5 d后进入快速增殖期,1周后进行细胞传代培养。兔软骨细胞相对贴壁缓慢,生长滞后。两类软骨细胞随着培养代数的增加,Ⅱ型胶原蛋白的mRNA表达量逐渐减少,ADAMTS5的表达逐代升高,表明随着培养代数的增加,软骨细胞活力逐渐下降。相同代数的大鼠软骨细胞活性较兔软骨细胞更高。甲苯胺蓝染色可见大鼠软骨细胞内成蓝色异染的糖胺多糖成分。不同波段的荧光激发下,Ⅱ型胶原酶免疫荧光染色可见大鼠原代培养的软骨细胞胞浆和胞膜呈清晰的绿色荧光,兔软骨细胞呈红色荧光,细胞核为明亮的蓝色荧光。结论本实验通过比较大鼠及新西兰兔软骨细胞的培养与特点,证实随培养代数增加,软骨细胞活性逐渐降低,且相同代数大鼠软骨细胞较兔软骨细胞具备更高活性。Objective To isolate and culture rat and rabbit knee chondrocytes in vitro, observe and compare the culture characteristics of the chondrocytes in rats and rabbits. Methods Chondrocytes were obtained by the mechanical separation combined with type Ⅱ collagenase enzymatic digestion from the cartilage of young SD rats and New Zealand white rabbits, and subcultured. The cell morphology was observed by an inverted microscope, growth curve was measured by cell counting, the expression levels of collagen Ⅱand a disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS5) in different generations of chondrocytes were detected by real-time PCR, and the cells were identified by toluidine blue staining and typeⅡ collagenase immunofluorescence staining. Results Primary cultured rat chondrocytes were presented with polygonal or irregular shape after 12 h, and arranged as"paving stones". After culture for 3-5 d, chondrocytes entered a period of rapid proliferation and cell subculture was carried out after 1 week. The chondrocytes of rabbits were adhered and grew slower than the rat chondrocytes. By comparing the expression of Collagen Ⅱand ADAMTS5 in two types of different generation chondrocytes, it was found that the expression of Collagen Ⅱin both chondrocytes decreased from generation to generation and that of ADAMTS5 increased, indicating that the activity of chondrocytes gradually decreased with the increase of chondrocytes generation. And the rat chondrocytes of the same generation had higher activity than rabbit chondrocytes. The toluidine blue staining revealed that there were some blue-stained glycosaminoglycan components in chondrocytes. Under the fluorescence excitation of different wave bands, collagenase type Ⅱ immunofluorescence staining showed clear green fluorescence in the cytoplasm and membrane of primary cultured rat chondrocytes, and red fluorescence of rabbit chondrocytes, the nucleus was bright blue fluorescence. Conclusion The study compared the culture and characteristics of cho

关 键 词：关节软骨 软骨细胞 细胞分离培养 

分 类 号：R684.3[医药卫生—骨科学]