大鼠软骨细胞复制性老化的体外观察

OBSERVATION OF REPLICATIVE SENESCENCE OF RAT CHONDROCYTES IN VITRO

目的对体外培养大鼠软骨细胞复制性老化行为进行观察,为进一步研究组织工程软骨退变机制,以及用药物干预并逆转其退变提供实验参考。方法4周龄SD大鼠,体重185～200g,断颈处死,分离培养软骨细胞,采用组织化学法检测传代培养的P1、P2、P3及P4代软骨细胞老化相关β-半乳糖苷酶,透射电镜观察细胞结构,免疫细胞化学法检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达,阿力新蓝染色检测胞外基质硫酸糖胺多糖(sulfat-glycosaminoglycan,GAG)含量和分子结构,RT-PCR方法检测软骨细胞型胶原表达,流式细胞仪分析细胞周期及增殖指数(proliferative index,PI)。结果β-半乳糖苷酶检测:P1、P2代软骨细胞偶见阳性染色;P3代可见少数细胞阳性染色,与P1、P2代比较差异有统计学意义(P<0.01);P4代与P1～P3代比较,阳性染色显著增加,差异有统计学意义(P<0.01)。透射电静镜观察:P1、P2代细胞胞浆内细胞器较多,胞核及核仁清晰,无凋亡软骨细胞;P3代细胞核浆比较大,胞浆内细胞器减少,胶原网络结构模糊不清;P4代细胞核浆比例增大,核固缩。P4代细胞G0/G1期含量增加(83.8%),而P1、P2及P3代分别为79.1%,79.2%,80.8%。P4代细胞PI为16.2%,P1、P2、P3代PI分别为20.9%、20.8%、19.2%。PCNA表达、GAG含量、长链分子百分比、型胶原表达减少等指标P4代与前代比较差异均有统计学意义(P<0.01)。结论体外培养的大鼠软骨细胞P4代出现老化。

Objective To observe the replicative senescence of rat articular chondrocyte cultured in vitro so as to provide reference for the succeeding experiment of using medicine interfere and reverse the cataplasia of tissue engineering cartilage or probing cataplasia mechanism. Methods Different generations（P1, P2, P3 and P4） of the chondrocytes were detected with the methods of histochemistry for β-galactosidase （β-gal）, electron-microscope for ultromicrostructure, immunocytochemistry for proliferating cell nuclear antigen （PCNA）,alcian blue stain for content and structure of sulfat-glycosaminoglycan （GAG） of extracellular matrix （ECM） ,reverse transcription-polymerase chain reaction （RT-PCR） for content of collagen I ,flow cytometry for cell life cycle and proliferative index（PI） to observe senescence of chondrocytes. Results In the 4th passage,the chondrocytes emerging quantitively positive express of β-gal, cyto-architecture cataplasia such as caryoplasm ratio increasing and karyopycnosis emerging under electron- microscope ,cell life cycle being detented on G1 phase （83. 8%）,while in P1, P2, P3 the content of G1 phase was 79.1%, 79.2%, 80.8% respectively. In the 4th passage, PIdecreased（16.2%）,while inP1, P2, P3, it was 20.9%, 20.8%, 19. 2%. The positive percentage of PCNA, the content of GAG （long chain molecule） and the positive expression of collagen I diminished,all detections above were significantly different （P〈0. 01） when compared the 4th passage with the preceding passages. Conclusion Chondrocytes show the onset of senescence in the 4th passage.