The Effects Of Testosterone On Proliferation And Differentiation Of Sika Deer Antler Mesenchymal Cells And Chondrocytes

Deer antler is also the only organ that can completely regenerate in mammalian.Deer antlerregenerate and fall in cycle every year,involving the regeneration of various tissues,which is controlled precisely.Because of its unique physiological characteristics,deer antler become an ideal model of mammalian organs regeneration and wound healing.Testosterone is an important androgen in male mammals,which is necessary for skeletal integrity maintain.It has been reported that antler growth is closely related with the testosterone level in the female deer body,changing in cycle as the growth of the antler.Some people think that testosterone has induced proliferation and differentiation of the cells from the top of the antler proliferation and differentiation;high levels of testosterone can stimulate antler cells proliferation;at estrus the synthesis of sex hormone increases,which is this is closely related to the ossification increase at this stage.If the male is castrated at the early puberty,the antler won't grow.IHH pathway plays an important role in cartilage differentiation and development.It can bind to the receptor Ptc,which can relive the inhibition of Ptc on Smo..And Smo further activates the zinc finger transcription factor Gli,which regulates the transcription of the downstream target gene.Runx2 is a special transcriptional gene in the process of chondrocyte differentiation and chondrocyte hypertrophy,which can accelerate the maturation of chondrocytes.,However,whether the testosterone can introduce antler chondrocytes hypertrophy differentiation regulation through the IHH signaling pathway and Runx2 is still unclear.The aim of this study was to investigate the expression of AR in sika deer antler by in situ hybridization.The results of in situ hybridization showed that AR m RNA expression in the several tissues of the growing tip of Sika deer antler could be detected,indicating that androgens and its receptor play an important role in antler growth process.In the mean time,we used cellculture method in vitro,adding testosterone to cultured mesenchymal cells and chondrocytes respectively,detecting the effect of testosterone on proliferation and differentiation by MTS and real-time PCR.The MTS result showed that there was no significant increase in proliferation activity of mesenchymal cells.However,in mesenchymal cells,the cartilage marker Col ?,AGC and COMP m RNA expression is significantly higher than control group.And the proliferation rate in chondrocytes that were treated with testosterone was higher than control group.Besides,the expression of COL X was higher than that of control group.To gain a further know about how testosterone influence chondrocyte differentiation in deer antler,we used RNA interference,real-time PCR and the inhibitor of IHH to detect the relevant genes involved in IHH pathway.The results showed that after treated with testosterone,the expression of IHH and Gli1 declined,while that of Gli3 increased.After treating Cyc or IHH si RNA,it could be seen that the expression of COL X was increased,.And after interfering Gli1 in chondrocytes,the expression of COL X was also upregulated.While when Gli3 was interfered,the expression COL X was downregulated.And when IHH was knocked down,the expression of Gli1 and Gli3 decreased.In the meantime,we tested the Runx2 level in chondrocytes which were treated by testosterone,and found the level of Runx2 m RNA was increased;and when treated with Runx2 si RNA,COL X expression in chondrocytes was decreased,suggesting that Runx2 may play a positive role in antler chondrocyte differentiation,In addition,the expression of Runx2 m RNA increased when IHH and Gli1 knocked down;but when Gli3 was knocked down,Runx2 m RNA level was declined.In conclusion,testosterone plays an important role in anlter mesenchymal cells and chondrocytes proliferation and differentiation,and it can control chondrocyte differentiation via IHH pathway.