C57BL/6小鼠关节软骨细胞的提取及凋亡模型的建立

Extraction of C57BL/6 mouse articular chondrocytes and establishment of apoptosis model

目的体外培养C57BL/6小鼠关节软骨细胞,建立符合骨关节炎实验需求的凋亡模型。方法采用两步酶消化配合机械吹打法分离C57BL/6小鼠关节软骨细胞,倒置相差显微镜分别观察5代软骨细胞的形态及生长情况,甲苯胺蓝、Ⅱ型胶原免疫荧光染色鉴定软骨细胞,CCK-8法分别检测5代软骨细胞的活力情况,实时荧光定量(RT-PCR)法分别检测5代软骨细胞中Ⅱ型胶原mRNA的变化。在培养基中加入不同浓度(0,5,10,20μmol/L)的星形孢菌素及200μmol/L H_(2)O_(2)作用24 h,通过检测细胞凋亡率,选择合适的浓度,并利用AOEB法进一步验证10μmol/L星形孢菌素的促凋亡作用。结果体外培养的C57BL/6小鼠关节软骨细胞,第1代类圆形,第2代三角形,第3代三角形及短梭形,3代后软骨细胞主要呈长梭形且形态不规则,胞核增大,胞质边缘模糊化。经甲苯胺蓝和Ⅱ型胶原免疫荧光染色证实为软骨细胞。与第1代软骨细胞相比,第4代和第5代软骨细胞的细胞活力显著降低(均P<0.05)。与第1代软骨细胞相比,第4代和第5代软骨细胞的Ⅱ型胶原mRNA相对表达量显著降低(均P<0.001)。与H_(2)O_(2)阳性对照相比,10μmol/L星形孢菌素作用软骨细胞24 h凋亡率为(63.97±1.67)%,差异无统计学意义。AOEB结果显示,与正常细胞相比,10μmol/L星形孢菌素作用软骨细胞后,凋亡早期细胞核质体呈绿色,细胞形状不规则;凋亡晚期细胞,核质体呈橙色,细胞核碎裂成点状,大小不一。结论本研究成功培养了C57BL/6小鼠关节软骨细胞,10μmol/L星形孢菌素作用3代内软骨细胞24 h建立凋亡模型最适宜。

Objective To culture C57 BL/6 mouse articular chondrocytes in vitro and establish an apoptosis model for the osteoarthritis experiment. Methods Two-step enzyme digestion combined with mechanical pipetting were used to isolate C57 BL/6 mouse articular chondrocytes. The morphology and the growth of five generations were observed under inverted phase contrast microscope. Toluidine blue and type Ⅱ collagen immunofluorescence staining were used to identify chondrocytes. CCK-8 method was used to detect the viabi-lity of five generations, and real-time PCR(RT-PCR) was used to detect the changes of type Ⅱ collagen mRNA in five generations. Different concentrations(0, 5, 10, 20 μmol/L) of staurosporine and 200 μmol/L H_(2)O_(2)were added in the medium for 24 h to select the appropriate concentration by detecting the rate of apoptosis, respectively. The pro-apoptotic effect of staurosporine(10 μmol/L) was further verified by AOEB. Results The first generation C57 BL/6 mouse articular chondrocytes were suborbicular, the second generation cells showed triangular, the third generation chondrocytes were triangular or short spindled, and the fourth and fifth generation chondrocytes were mainly long spindled or irregular, with enlarged cellular nuclei and vague cytoplasmic edges. They were confirmed as chondrocytes by toluidine blue and type Ⅱ collagen immunofluorescence staining. Compared with the first generation chondrocytes, the cell viabilities of the fourth and fifth generations were significantly reduced(both P<0.05). And compared with the first generation chondrocytes, the relative expression of type Ⅱ collagen mRNA in the fourth and fifth generations were significantly reduced(both P<0.001). The apoptosis rate of chondrocytes was(63.97±1.67)% after treated with 10 μmol/L staurosporine for 24 h, and there was no statistically significant difference when compared with H_(2)O_(2)positive control. The AOEB results showed that, after the treatment of staurosporine(10 μmol/L), the chondrocytes became morphological irregular and their nucleoplasts were green during the early phase of apoptosis, while their nucleoplasts were orange and nuclei were fragmented into punctiform with different sizes during the late phase of apoptosis. Conclusion The C57 BL/6 mouse articular chondrocytes were successfully cultured. It is the most suitable to establish an apoptosis model by treating chondrocytes within three generations with 10 μmol/L staurosporine for 24 h.