摘要
目的验证体外Ⅱ型胶原酶消化法培养兔软骨细胞的可行性,探讨凋亡及抗凋亡基因在软骨细胞传代中的变化,寻找关节软骨退变老化研究的最适宜靶细胞。方法在无菌条件下取6周龄新西兰大白兔双侧膝关节软骨,采用Ⅱ型胶原酶消化并机械吹打的方法分离关节软骨细胞并进行原代、传代培养;形态学观察时采用甲苯胺蓝染色法对关节软骨细胞进行鉴定;RT-PCR法检测P0~P4代软骨细胞烟酰胺磷酸核糖转移酶(NAMPT)、信息沉默因子1(Sirt1)、p53、Bax基因的mRNA相对表达量,四甲基偶氮唑盐(MTT)法检测各代关节软骨细胞增殖情况。结果显微镜下观察兔原代软骨细胞大多呈透亮椭圆形、短梭形、多角形特征,72h全部贴壁生长。甲苯胺蓝染色显示培养的软骨细胞细胞质染成浅蓝色,细胞核染成深蓝色;前3代软骨细胞表型稳定,增殖力良好;连续培养至P4代后,绝大部分细胞变为长梭形和不规则形状。随着软骨细胞的传代,NAMPT的表达量逐渐下调。与P0代软骨细胞相比,Sirt1的表达量在P1代细胞中明显上调,在P2代细胞急剧下降至P0代以下,在P3~P4代细胞中Sirt1的表达量逐渐下调。凋亡基因p 53和Ba x的表达量随着软骨细胞的传代而上调。前3代软骨细胞增殖差异无统计学意义(均P>0.05);在培养到第4~7天时,P1~P3代与P4代软骨细胞增殖差异有统计学意义(P<0.05)。结论采用体外Ⅱ型胶原酶消化法培养兔关节软骨细胞是切实可行的。随着软骨细胞的传代,抗凋亡基因NAMPT、Sirt1的表达逐渐下调,凋亡基因p53、Bax的表达逐渐上调。P1~P3代关节软骨细胞作为研究关节软骨凋亡的细胞体系是合适的。
Objective To verify the feasibility of in vitro culture of rabbit chondrocytes with collagenase Ⅱ digestion method and to investigate the changes of apoptosis and anti apoptotic genes in chondrocyte passages. Methods The knee joint cartilage was obtained under aseptic condition from 6 week-New Zealand white rabbits. The chondrocytes were isolated and digested with type collagenase Ⅱand primary culture and subculture were carried out. The articular chondrocytes were identified by morphological observation with toluidine blue staining. RT-PCR method was used to detect the relative expression of NAMPT, Sirt1, P53 and Bax genes in chondrocytes of various generations, and the proliferation of chondrocytes in articular cartilage was detected by MTT method. Results Under the microscope observation of rabbit articular chondrocytes were mostly in oval translucent, short spindle, polygonal appearance, and the adherent growth was almost completely in 72 h. Toluidine blue staining showed light blue cytoplasm and deep blue nuclei in cultured chondrocytes. The first 3 generations of chondrocytes showed stable phenotype and good proliferation. In the fourth generation, most of the cells changed to long spindle and irregular-shaped. With the passage of chondrocytes, the expression of NAMPT decreased gradually. Compared with the P0 generation of cartilage cells, the expression of Sirt1 in P1 cells was significantly up-regulate, then decreased rapidly in P2 cells,and down-regulated in P3-P4 cells. The expression levels of apoptotic genes p53 and Bax were up-regulated with the passage of chondrocytes. MTT showed that there was no significant difference in the proliferation of chondrocytes among the first 3 generations(all P >0.05). When cultured on day 4-7, the proliferation of chondrocytes was significantly different between P1-P3 and P4 generations(P<0.05). Conclusion It is feasible to culture rabbit articular chondrocytes by collagenase digestion method in vitro. With the passage of chondrocytes, the expression of anti apoptotic genes NAMPT and Sirt1 decreased gradually, and the expression of apoptotic genes P53 and Bax increased gradually. P1-P3 joint chondrocytes are suitable for the study of chondrocyte apoptosis.
出处
《浙江医学》
CAS
2017年第23期2101-2105,2191,共6页
Zhejiang Medical Journal
基金
浙江省中医药科技计划项目(2014ZB079)
