Expression Of Apoptosis And Proliferation Genes In Injured Rabbit Articular Chondrocytes

0bjective: To establish the injury model of rabbit articular chondrocyte in vitro andanalyse the expression change of caspase-3and PCNA in different time-point of injurymodel.Methods:1. Articular cartilage was aseptically dissected from the bilateral knee jointsof4weeks old healthy new Zealand white rabbit. Articular chondrocytes were harvestedby the mechanical separation combined with enzyme digestion method, and which werecultured and passaged in vitro. Type â…¡collagen immunofluorescence assay wereemployed to identify the articular chondrocytes.2. According to the recent study, theinjury model was established.3. The gene expression of caspase-3, PCNA and collagenâ…¡of injuried articular chondrocytes at1,3,7days and the normal group were studiedby real-time quantitative polymerase chain reaction(qPCR).4. The protein expression ofcaspase-3and PCNA of injuried articular chondrocytes at1,3,5,7days and the normalgroup were studied by Western Blot.Results:1. The type â…¡collagen immunofluorescence assay demonstrated: The articularchondrocytes separated from the bilateral knee joints were successfully cultured.2.According to the present study about cell injury, the scratch wound model wasestablished, and then the morphology change at different time-points showed: the areawhich the pressure directly act on was empty at1minute after scratching; thechondrocytes at the edge of scratch had some biologically changes at1day afterscratching; the chondrocytes at the edge of scratch had proliferation activities at day3;at day7, the empty area was filled by proliferation chondrocytes.3. The result ofrealtime PCR showed: Compared with the normal cell group, the levels of caspase-3mRNA increased at day1after injury, and which at day3and7were obviously lowerthan that at day1and normal group(P<0.05). Compared with the normal cell group, the levels of PCNA mRNA decreased at day1after injury, and which at day3changed notobviously than that at day1, began to increase at day7. Compared with the normal cellgroup, the levels of collagen â…¡ mRNA decreased obviously at day1afterinjury(P<0.05), began to increase at day3and reached normal at day7after injury.4.Western Blot showed: Compared with the genes expression, the expression of proteinhave the similar pattern. Compared with the normal cell group, the levels of caspase-3protein increased at day1after injury, and then reach the peak at day3; at day5thefalling trend appeared; the level of caspase-3protein at day7was similar to normalgroup. The day3group was obviously higher than that at day7and normalgroup(P<0.05). the level of PCNA protein showed the falling down trend appeared atday1and day3,and then began to arise at day5and day7. The day5group wasobviously higher than day3group(P<0.05).Conclusions: The study of the chondrocytes injury model shows: With the mechanicalinjury stimulating, chondrocyte apoptosis peaks in few days after injury, following byincreases in cellular proliferation. These findings indicate chondrocytes cultured in vitrohave the anti-apoptotic ability, which can provide the experimental and theory basis intreating the articular cartilage injury.