Study Of The Cytoskeleton Of Primary And Passage Rabbit Knee Articular Chondrocytes

Objectives: Used laser confocal scanning microscope(LCSM) to observe the morphology of cytoskeleton(CSK) and estimated the fluorescence intensity of CSK proteins of primary and passaged chondrocytes respectively, aimed to elucidate the changes of CSK during chondrocytes passage.Methods: 3 male New Zealand White rabbits were sacrificed by air embolism. The chondrocytes of their knee joints were taken out under aseptic conditions and cultured in the plates. The primary(P0) and passaged 1,2(P1,P2) chondrocytes were inoculated into 24 well plates which round cover slips were put at the bottoms. Cell climbing slices were fixed when chondrocytes were spreaded to the slices nuder the inverted microscope. The CSK proteins: actin, vimentin, tubulin and vinculin were stained by immunofluorescence antibody on P0, P1 and P2 cell climbing slices respectively. The morphology of CSK were observed by LCSM and the fluorescence intensity of CSK proteins were detected quantitatively by the fluorescence intensity software.Results: The microfilaments of chondrocytes were filamentous, distributed at the periphery of the cytoplasm and approached to the membrane, spreaded along the cell peripheria, and no microfilaments surrounded the nuclear. The microfilaments became thicker during cell passage and thicker actin fibers were observed in P2 chondrocytes. The vinculins were punctiform, distributed at the terminal of the microfilaments where approach to membrane. The amounts of the punctiform vinculins were decreased. The intermediate filaments of chondrocytes were also filamentous, spreaded though whole cytoplasm and interlaced to be networks; Dense distribution of intermediate filaments were found surrounding the nuclear in a part of chondrocytes; The intermediate filaments networks became loosen and dense distribution surrounding the nuclear decreased in P1 and P2 chondrocytes. The fluorescence intensity of intermediate filaments were not so uniform in the cytoplasm. The microtubules were radial through whole cytoplasm and interlaced to be loosen networks; The P1 and P2 chondrocytes formed more microtubule processes than P0 at the cell periphery. The fluorescence intensity of microtubules were more uniform in the cytoplasm. The results of fluorescence intensity of P0, P1 and P2 chondrocytes CSK proteins showed as follow: The fluorescence intensity of actin of P1 and P2 chondrocytes were significantly increased than P0(P00.05). The fluorescence intensity of vimentin and tubulin were significantly decreased during passage(P0>P1>P2, comparison between any two means P<0.05).Conclusions: The CSK morphology of passaged chondrocytes changed comparison with primary chondrocytes, manifested that: the microfilaments became thicker; the amounts of the punctiform vinculins were decreased; the intermediate filaments networks became loosen and dense distribution surrounding the nuclear decreased; more microtubule processes were formed at the cell periphery during passage. Comparison with primary chondrocytes, The quantity of actin was significantly increased while the quantity of vimentin and tubulin were significantly decreased in passaged chondrocytes. These results suggested that the morphology and proteins quantity of chondrocyte CSK changed during chondrocytes passage.