大鼠椎体生长板软骨细胞的改良培养及传代特征

Improved culture of chondrocytes from rat vertebral body growth plate and their passage characteristics

[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 the culture and identification of chondrocytes from rat vertebral body growth plate in vitro and observe their passage characteristics.[Method]Vertebral body growth plates were obtained from 4 Sprague-Dawley（SD） rats aged 1 week,then chondrocytes were cultured in vitro with improved trypsin-collagenase type Ⅱ digestion method.Primary chondrocytes were passaged and then identified by HE and toluidine blue staining.Cells growth curve was drawn by MTT colorimetric assay.Chondrocytes were subcultured to the sixth passage,and the changes of cell morphology were observed by an inverted phase contrast microscope,the changes of expression of collagen Ⅱ were identified with immunocytochemistry.[Result]MTT colorimetric assay showed that the growth curve of chondrocytes before the third passage presented as ＂S＂ shapes.Chondrocytes were found growing logarithmicly from the 4th day,then the growth of chondrocytes reached platform stage from the 9th day and were inhibited from the 11th day.The shape of chondrocytes would change from polygonal into long shuttle with the passages of the primary chondrocytes cultured in vitro.The collagen type Ⅱ immunocytochemical staining was extensive positive in chondrocytes before the third passage.[Conclusion]The method to isolate and culture chondrocytes in this study can acquire pure chondrocytes in short time.The cells before passage 3 maintain phenotype of chondrocyte and grow quickly.This method lay a foundation for further study on longitudinal growth of vertebral body at cell level.