Ⅱ型胶原酶消化法可短时间获得大量纯化大鼠关节软骨细胞

Purified rat chondrocytes can be gained by using typeⅡ collagenase enzyme digestion method in short time

背景:建立一种经济、快捷、切实可行的软骨细胞分离培养体系对于软骨体外实验研究有着重要的意义。目的:探讨与改进大鼠关节软骨细胞的培养方法。方法:无菌条件下取新生1周龄SD雄性大鼠双侧髋及膝关节软骨,采用Ⅱ型胶原酶消化法分离软骨细胞并进行原代、传代培养及鉴定。结果与结论:倒置相差显微镜下见原代培养的软骨细胞12h后开始贴壁,3d左右可形成单层,4d左右即可传代。传至第6代后,部分细胞变为梭形;第7代后,绝大部分细胞变为长梭形和不规则形状,增殖能力减弱。甲苯胺蓝染色显示培养的软骨细胞核呈异染性,免疫荧光染色显示培养的软骨细胞Ⅱ型胶原呈阳性表达。说明采用此方法可在短时间内获得大量纯化的大鼠软骨细胞。

BACKGROUND： To establish an economic, efficient, practical isolation and culture system of chondrocytes has important significance to cartilage in vitro experiments. OBJECTIVE： To investigate and improve the culture method of rat articular chondrocytes. METHODS： Rat cartilage of bilateral hip and knee was resected from one week old male rats under aseptic condition. Chondrocytes were isolated by type ]I collagenase enzyme digestion method. The cells were cultured and passaged, then identified. RESULTS AND CONCLUSION： Inverted phase contrast microscope observation showed that the primary cultured chondrocyt adherented at the 12th hour after cultivation. The monolayer formation occurred on the 3rd day after cultivation. And cells were ready to be passaged on the 4~ day after cultivation. After passaged for six generations, some cells represented a spindle-like appearance. In the 7th generation, most cells turned into a long and irregular appearance, and cell proliferation capacity diminished. Toluidine blue staining showed that the nucleis of cultured chondrocytes were metachromatic. Immunofiuorescent staining showed that cultured chondrocytes had a positive expression of collagen type ]1. These findings illustrate that a large number of purified rat chondrocytes can be gained by this method in a short time.