浙江医学2017，Vol.39Issue(23)：2101-2105,后插2,6.DOI:10.12056/j.issn.1006-2785.2017.39.23.2017-2027
兔关节软骨细胞的体外培养及凋亡相关基因在传代细胞中的表达
In vitro culture of rabbit articular chondrocytes and expression of apoptosis related genes in passage cells
摘要
Abstract
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 Ⅱ and 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 72h.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.关键词
软骨细胞/体外培养/烟酰胺磷酸核糖转移酶/信息沉默因子1/传代Key words
Chondrocyte/Cultured in vitro/NAMPT/Sirt1/Passage引用本文复制引用
周叶,高越,王晓楠,朱立,厉蓓..兔关节软骨细胞的体外培养及凋亡相关基因在传代细胞中的表达[J].浙江医学,2017,39(23):2101-2105,后插2,6.基金项目
浙江省中医药科技计划项目(2014ZB079) （2014ZB079）
