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目的:探讨行之有效的人胚髁状突软骨细胞体外分离培养方法及研究其生物学特性。方法:用0.25%胰蛋白酶和0.2%Ⅱ型胶原酶分阶段联合消化法分离髁状突软骨细胞,将分离的软骨细胞与未消化完的小片软骨共同置入预涂多聚赖氨酸的培养瓶中培养,通过倒置显微镜,免疫组织化学染色、电镜观察等方法与传统培养方法对比,检测软骨细胞的分离后存活率、贴壁生长速度及传代7次内髁状突软骨细胞表型改变相伴随的形态学及生物学特征。结果:本方法培养的人髁突软骨细胞存活率可达98%以上,细胞贴壁能力强,与传统培养方法形成细胞单层的速度相比具有统计学意义(p<0.05)。7代以内培养的髁状突软骨细胞免疫组织化学染色阳性,透射电镜可见细胞内有丰富的粗面内质网及线粒体。而传统培养方法所获得的人髁状突软骨细胞存活率为90%左右,培养3代以后免疫组织化学染色显示其软骨细胞表型逐渐减弱,培养至第7代其免疫组织化学染色为阴性。结论:本方法培养的髁状突软骨细胞存活率高,且能维持软骨细胞的特有表型,至少能保持软骨细胞7代稳定,是一种简便、有效的培养方法。
OBJECTIVE: To explore the method of isolation and culture of human embryonic condylar chondrocytes in vitro and its biological characteristics. METHODS: Condylar chondrocytes were separated by phase-by-digestion with 0.25% trypsin and 0.2% collagenase Ⅱ, and the isolated chondrocytes were incubated with undigested small pieces of cartilage in precoated polylysine The cells were cultured, and the survival rate, adherent growth rate and the phenotypic change of the condylar cartilage cells were detected by inverted microscope, immunohistochemical staining and electron microscope observation compared with the traditional culture methods Accompanying morphological and biological characteristics. Results: The survival rate of human condylar chondrocytes cultured in this method can reach more than 98%, and the cell adhesion ability is strong, which is statistically significant (p <0.05) compared with the rate of monolayer formation by traditional culture method. Condylar chondrocytes cultured for 7 generations were positive for immunohistochemistry staining. Transmission electron microscopy showed abundant rough endoplasmic reticulum and mitochondria in the cells. However, the survival rate of human condylar chondrocytes obtained by the traditional culturing method was about 90%. Immunohistochemical staining showed that the chondrocyte phenotype gradually weakened after 3 generations of culture, and the seventh passage was negative for immunohistochemical staining. CONCLUSION: The method has the advantages of high survival rate of condylar chondrocytes, maintaining the unique phenotype of chondrocytes, and keeping the stability of chondrocytes for at least 7 generations, which is a simple and effective culture method.