大鼠半月板纤维软骨细胞的体外培养和生物学特征鉴定

Culture and Characterization of Rat Meniscus Fibrochondrocytes in Vitro

目的:体外分离、培养和鉴定大鼠半月板纤维软骨细胞,为研究大鼠半月板纤维软骨细胞的损伤修复提供简单、可行的细胞培养方法。方法:机械分离大鼠膝关节内外侧半月板,0.1%Ⅱ型胶原酶消化配合机械吹打,10%FBS的培养基行原代和传代培养,倒置显微镜动态观察不同时间点半月板纤维软骨细胞的形态及生长情况,鉴定采用甲苯胺蓝染色法和Ⅱ型胶原免疫细胞化学染色法。CCK-8法检测大鼠纤维软骨细胞增殖。结果:在不同时间点,细胞表现出不同的生理形态,由多角形或短梭形变为梭形,最终呈现出三角形或椭圆形,并随着时间延长,细胞增殖能力逐渐增加。细胞培养48 h和72 h的OD值分别明显高于培养24 h的OD值(P<0.05),差异具有统计学意义。细胞培养48 h和72 h的OD值相比较,72 h的OD值虽有增加,但并无统计学差异(P>0.05)。经甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色为阳性。结论:该方法培养的细胞形态稳定,增殖能力强,具有体内纤维软骨细胞的生物学基本特性,可为后续实验提供简单、可靠的原代大鼠半月板纤维软骨细胞培养方法。

Objective: We want to provide a simple and feasible cell culture method for the future study of injury repair of rat meniscus by isolation, culture and identification of rat meniscus fibrochondrocytes in vitro. Methods: The lateral meniscus were mechanical separation and digestion by 0.1%Ⅱ collagenase, cultured in the 10% FBS medium. The morphology and growth situation of meniscal cells were observed by the inverted microscope dynamic at different time points, the identification of meniscus fibrochondrocytes by using toluidine blue staining and Ⅱ type collagen immune cell chemical dyeing method. The proliferation capacity of fibrochondrocytes was detected by CCK-8 method. Results: At different time points, the cells showed different physiological morphology, which was transformed from polygonal or short shuttle-shaped to fusiform, and finally appeared triangular or ellipsoidal, and the proliferation capacity of the cells increased gradually with the extension of time. The OD value at 48 h and 72 h in cell culture was significantly higher than that at24 h(P0.05), and the difference was statistically significant. The OD value of cell culture at 48 h and 72 h was increased, but there was no significant difference(P0.05). By toluidine blue staining and collagen type Ⅱ immunofluorescence stain is positive. Conclusion:Cultured cells by this method are stable in morphology, strong in proliferation, and have the basic biological characteristics of fibrochondrocytes in vivo, which can provide a simple and reliable method for rat meniscus fibrochondrocytes culture.