[Observation of replicative senescence of rat chondrocytes in vitro].
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 beta-galactosidase (beta-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 II, 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 beta-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, PI decreased (16.2%), while in P1, 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 II diminished,all detections above were significantly different (P<0.01) when compared the 4th passage with the preceding passages.


CONCLUSIONS
Chondrocytes show the onset of senescence in the 4th passage.
Lin
Jianhua
J
Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University, Fuzhou Fujian, 350005, PR China. jianhual@126.com
Chen
Xiaodong
X
Deng
Lingxiao
L
D000818:Animals; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D024043:Collagen Type II / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005434:Flow Cytometry; D006025:Glycosaminoglycans / Q000378:metabolism; D007150:Immunohistochemistry; D008297:Male; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D001616:beta-Galactosidase / Q000378:metabolism
D024043:Collagen Type II; D006025:Glycosaminoglycans; D018809:Proliferating Cell Nuclear Antigen; D001616:beta-Galactosidase