Correlation Of FOXO4 Transcription Factor In Rat Chondrocytes Degeneration

Objective:To detect the expression of transcription factor FOXO4 in rat chondrocytes,establishing an effective model of chondrocyte degeneration,to investigate the correlation of FOXO4 in rat chondrocytes degeneration.Methods:(1)The femoral head cartilage was obtained from 4 weeks old and 16 weeks old SD rats respectively,and the cells were isolated by collagen digestion method using collagen type Ⅱ,obtain normal and natural degeneration of the primary chondrocytes.(2)Chondrocytes at passage 1 were identified by Toluidine blue staining,Alcian blue staining and Immunocytochemical staining of collagen type Ⅱ.(3)The experiment was divided into three groups.Control group: chondrocytes at passage 2 from 4 weeks old rats;Natural degeneration group: chondrocytes at passage 2 from 16 weeks old rats;Induced degeneration group: chondrocytes from 4weeks old rats were induced with 1l-1β(10ng/mL)from the first generation to the second generation,and the induction of the second generation chondrocytes were used for follow-up experiments.(4)The morphology of cells was observed under a microscope,and the degree of cells senescence was tested by using the beta-galactosidase staining method;(5)The expression level of mRNA of FOXO4 and col2α were detected by RT-PCR.the protein expression levels of FOXO4 and col2α were detected by Western Blot.Results:(1)The joint of femoral head cartilage and subchondral bone of 4-week old rats is more relaxed and easy to be taken down.The femoral head cartilage are translucent,thick and thick in the middle,with a tough and brittle texture and easy to be cut.In 16 weeks old rats,the joint of femoral head cartilage and subchondral bone are dense and not easy to be removed completely.The cartilage is obviously thinner and solider,hard to be cut.(2)The primitive cartilage cells from 4 weeks old rats could grow largely by adhering to the wall after 48 h,homogeneous distribution.after 1 week,the cells proliferate and were filled with cell bottoms;there are very few the primitive cartilage cells from 16 weeks old rats could grow by adhering to the wall after 48 h,then form accumulation clusters and gradually proliferate,filled with cell bottoms about 2weeks.(3)There aren’t obvious differences at morphology about chondrocytes at passage 1 between 4 weeks and 16 weeks old rats,and both are triangular or polygonal.The cytoplasm is blue and the nucleus is dark blue in oluidine blue staining,The cytoplasm is pale blue in Alcian blue staining,and Immunocytochemical staining of collagen type Ⅱshow an tan of cytoplasm,nucleus of dark brown.All three methods of identification of chondrocytes were positive.(4)We observe the chondrocytes at passage 2.the control group cells are fusiform,and with abundant cytoplasm.The cells of natural degeneration and induced degeneration are in long spindle shape or irregular shape.For the beta-galactosidase staining,the results show that the cells from control group showed no obvious staining,and the cells in the natural degeneration group and the induced degeneration group show a large number of cells with a dark blue(5)RT-PCR results show that the mRNA expression levels of col2α in the natural degeneration group and the induced degeneration group are significantly reduced compared with the control group [(0.07±0.02)vs(0.53±0.06),p<0.001;(0.15±0.03)vs(0.53±0.06),p<0.001].The mRNA expression level of FOXO4 was significantly increased [(0.58±0.03)vs(0.08±0.02),p<0.001;(0.83±0.03)vs(0.08±0.02),p<0.001].(6)Western Blot results shows that the protein expression levels of collagen typeⅡ in the natural degeneration and induce degeneration group are significantly reduced compared with control group[(0.14±0.01)vs(0.51± 0.02),p < 0.001;(0.11±0.03)vs(0.51±0.02),p < 0.001];FOXO4 protein expression significantly increase[(0.16±0.02)vs(0.08 ±0.01),p < 0.05;(0.21±0.02)vs(0.08±0.01),p < 0.01).Conclusion:(1)Spontaneous degeneration and 1l-1β are two kinds of effective chondrocyte degeneration models,which could be used for the experimental study of cartilage degeneration or osteoarthritis.(2)The transcription factor FOXO4 can be expressed in rat chondrocytes;(3)FOXO4 was involved in rat chondrocytes degeneration,and its expression was positively correlated with the degree of cartilage degeneration.