Study On The Effects Of 1?,25-Dihydroxyvitamin D3 On The Proliferation And Collagen Production Of Chondrocytes

Objective:1?,25?dihydroxyvitamin D₃[1,25?OH?₂D₃)]is one of the necessary fat-soluble vitamins for the human.It can regulate the growth and development of the skeletal system through direct and indirect effects.However,there are few studies on their effect of endochondral ossification?EO?.EO is a dynamic and complex process,which mainly maintains the longitudinal growth of bone.The proliferation,maturation and calcification of chondrocytes play important role in this process.Some research data have shown that the mandibular condylar cartilage is secondary cartilage,while femoral head cartilage is primary cartilage.There are different in their embryonic origin,tissue structure,growth characteristics and the response to growth factors.Therefore,in our study,we investigated the effects of 1,25?OH?2D3 on the proliferation,maturation and calcification of the femoral head chondrocytes?FCs?and the condylar chondrocytes?CCs?.Methods:The FCs and CCs were obtained from one-week old female SD rats and identified when cultured to the second-generation.1.Part I:The FCs and CCs were randomized into 6 groups in vitro:the control group,vehicle group?0.01%alcohol?,0.1nM 1,25?OH?₂D₃ group,1 nM 1,25?OH?₂D₃ group,10 nM 1,25?OH?₂D₃ group and 100nM 1,25?OH?₂D₃ group.When chondrocytes were starved,they were treated with various concentrations of 1,25?OH?2D3 for 24 h to perform CCK8 assay and detect the expression of proliferating cell nuclear antigen?PCNA?,type II collagen?COLII?,and type X collagen?COLX?in the mRNA and protein levels.2.Part II:The FCs were silenced the vitamin D receptor?VDR?by using the small interfering RNA?siRNA?and detected the interference efficiency.Then the FCs were randomized into 4 groups:the control group,1,25?OH?₂D₃ group,VDR-siRNA group,VDR-siRNA+1,25?OH?₂D₃ group,and detected the expression level of COLII by qPCR,Western blot and immunofluorescence staining.Results:Part I:1.The results of CCK8 assay showed that the OD value decreased significantly with the increase concentration of 1,25?OH?2D3,and the inhibition rate of the CCs was more obvious than that of the FCs.2.The results showed that the expression of PCNA on the mRNA and ptotein levels was gradually decreased with the increased concentration of 1,25?OH?₂D₃,and the PCNA decreased in the CCs was more obvious than that of the FCs.3.The results showed that the expression of COLII among the control group,vehicle group and the 0.1 nM group was not statistical significance?p>0.05?,the expression of COLII in the pharmacological concentrations?1-100nM?group was increased,peaked at a dose of 10 nM in the FCs,while it increased in a concentration-dependent manner in the CCs.4.The expression of COLX on the mRNA and protein levels in all group both in the FCs and CCs were not statistical significance?p>0.05?.Part II:1.In the FCs,compared the control group,the VDR silencing efficiency in the VDR-siRNA group was more than 85%.2.The results showed that compared with their in the 1,25?OH?2D3 group,the expression of COLII in the VDR-si RNA+1,25?OH?₂D₃ group was significantly decreased.3.The results of immunofluorescence staining showed that,compared with their in 1,25?OH?₂D₃ group,the fluorescence intensity of COLII in the VDR-siRNA+1,25?OH?2D3 group was significantly decreased both in the CCs and FCs.Conclusions:1.In vitro 1,25?OH?₂D₃ inhibited chondrocytes proliferation,promoted chondrocytes maturation,and had no significantly affect chondrocytes calcification.2.In vitro,compared with the FCs,1,25?OH?₂D₃ has a lower regulatory threshold and a more significant effect for the CCs.