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[目的]建立体外培养及鉴定大鼠椎体生长板软骨细胞的方法,观察软骨细胞的传代特征。[方法]采用改良胰酶和Ⅱ型胶原酶序贯消化法对4只1周龄SD大鼠椎体生长板软骨细胞进行体外分离、培养。原代细胞传代后采用细胞HE染色和甲苯胺蓝染色对软骨细胞进行鉴定,采用MTT比色法绘制细胞生长曲线。将细胞传代至第6代,采用倒置相差显微镜观察各代软骨细胞的形态,采用免疫细胞化学染色鉴定各代软骨细胞Ⅱ型胶原的表达。[结果]MTT比色法显示,传3代以前的软骨细胞的生长曲线近似“S”形,从第4 d开始细胞呈对数生长,第9 d达平台期,至第11 d开始出现生长抑制。体外培养的软骨细胞随着传代次数的增加,细胞形态由原代的多角形逐渐变为长梭形。传3代以前的软骨细胞Ⅱ型胶原免疫细胞化学呈强阳性。[结论]本研究所采用的软骨细胞分离和培养方法,能在短时间内获得高纯度的软骨细胞,传3代及以前的细胞具有软骨细胞的特征性表型,增殖较快,这一方法为更深入地从细胞水平研究椎体的纵向生长奠定了基础。
[Objective] To establish a method for culturing and identifying chondrocytes in rat vertebral growth plate in vitro and observe the passage characteristics of chondrocytes. [Method] Chondrocytes of 4-week-old SD rat vertebral growth plate were isolated and cultured in vitro by modified trypsin and collagenase Ⅱ sequential digestion. Chondrocytes were identified by HE staining and toluidine blue staining after passage of primary cells, and cell growth curve was drawn by MTT colorimetric method. The cells were passaged to passage 6, and the morphology of chondrocytes was observed by inverted phase contrast microscope. The expression of type Ⅱ collagen in chondrocytes was identified by immunocytochemical staining. [Result] The MTT colorimetric method showed that the growth curve of chondrocytes before passage 3 was similar to “S” shape. The cells grew logarithmically from day 4, and reached the plateau on the 9th day. From the 11th day, Growth inhibition occurred. Chondrocytes cultured in vitro with the passage of times increased, the cell morphology from the original polygon gradually fusiform. Chondrogenesis type Ⅱ collagen immunocytochemistry before passage 3 was strongly positive. [Conclusion] The chondrocytes isolation and culture method used in this study can obtain high-purity chondrocytes in a short time. The passage 3 and previous cells have the characteristic phenotype of chondrocytes and proliferate rapidly. This method Which lays the foundation for further studies on the longitudinal growth of vertebral body at the cellular level.