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目的探讨大鼠颞下颌关节髁突软骨细胞的培养方法,观察细胞的生物学特性。方法分离3只Wistar大鼠髁突软骨,采用Ⅱ型胶原酶消化法联合组织块法获取髁突软骨干细胞,采用MTT法检测细胞生长曲线,采用甲苯胺蓝染色、Ⅱ型胶原免疫组织化学法和Ⅱ型胶原免疫荧光法鉴定细胞来源。结果Ⅱ型胶原酶消化法联合组织块法消化髁突软骨组织18~20h可获得最大量细胞;体外培养软骨细胞主要呈多角形,第1~4代细胞具有稳定生物学特性,5代及5代以后逐渐出现老化细胞;MTT检测结果显示第3代细胞可最快达指数生长期;髁突软骨细胞经甲苯胺蓝染色、Ⅱ型胶原免疫组织化学染色及免疫荧光染色鉴定,结果均为阳性表达,细胞来源于软骨。结论Ⅱ型胶原酶消化法联合组织块法可作为髁突软骨细胞获取方法;4代以内细胞具有稳定的生物学特性,可用于其他实验。
Objective To investigate the culture method of condylar chondrocytes in temporomandibular joint of rats and to observe the biological characteristics of the cells. Methods Condylar cartilage of three Wistar rats was isolated and the condylar cartilage stem cells were obtained by collagenase digestion method and tissue block method. The cell growth curve was detected by MTT assay. Toluidine blue staining, type Ⅱ collagen immunohistochemistry, Type Ⅱ collagen immunofluorescence method to identify cell sources. RESULTS: The most abundant cells were obtained after digestion of condylar cartilage tissue by collagenase digestion and tissue block method. The cultured chondrocytes were mainly polygonal in shape. The first to fourth generation cells had stable biological characteristics. The fifth and fifth passages After aging, the aging cells gradually appeared. The results of MTT assay showed that the third generation cells could reach the exponential growth phase as soon as possible. The condylar chondrocytes were identified by toluidine blue staining, type Ⅱ collagen immunohistochemical staining and immunofluorescence staining. The results were positive Expression, cells derived from cartilage. Conclusions Type Ⅱ collagenase digestion combined with tissue block method can be used as a method for obtaining condylar chondrocytes. Cells within 4 passages have stable biological characteristics and can be used in other experiments.