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Effects And Regulatory Mechanisms Of IGF-1 On The Regeneration Of Sika Deer Antlers

Posted on:2018-10-02Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z Q YangFull Text:PDF
GTID:1313330515480348Subject:Basic veterinary science
Abstract/Summary:PDF Full Text Request
Deer antler is the male characteristics of the vast majority of deer,which regenerates and sheds annually.Antler is the tender angle which has not yet been ossification before the growth of the first stage,At later growth stage,velvet sheds and completely ossifies with formation of hard bone,called hard antlers.Antler develops as an extension from pedicle,annually periodic regenetate,and it is the only organ that can fully regenerate,so it is an ideal animal model for studying mammalian organ regeneration and wound healing.Antler is a bony organ,and its growth pattern is similar to long bone growth in mammals.After regeneration,mesenchymal cells,chondrocytes,osteoblasts and osteoclasts differentiate and proliferate,which is in a dynamic balance,finally form hard antlers after ossification.Antler regeneration process is regulated by a variety of cytokines and growth factors.Insulin-like growth factors 1(IGF-1)are essential for the proliferation and differentiation of chondrocytes,but their underlying molecular mechanism is still unknown.The influence of IGF-1on antler chondrocytes proliferation and differentiation has not been reported.In this study,the expression of IGF-1 in sika deer antler and its effect on the proliferation and differentiation of antler chondrocytes and the signal mechanism were studied by in situ hybridization,real-time PCR,gene overexpression,RNA interference and flow cytometry.In situ hybridization results showed that IGF-1 was highe expressed in the deer antler cartilage layer,suggesting that IGF-1 may play an important role in regulating the regeneration of antler.In order to study the effect of IGF-1 on the proliferation of chondrocytes in antler,the antler chondrocytes were isolated and cultured in vitro.After treatment with recombinant human IGF-1 protein,the cell proliferation was detected by MTS colorimetric assay,and the effect of IGF-1 on cell cycle of antler chondrocytes was detected by flow cytometry.The results exhibited that IGF-1 couldsignificantly promote antler chondrocytes proliferation,decrease G0/G1 ratio and increase the proportion of S phase.However,these effects were abrogated by a pretreatment with IGF-1 receptor inhibitor(PQ401).To analyze the effect of IGF-1 on the differentiation of antler chondrocytes,recombinant human IGF-1 protein was added to chondrocytes,use real-time PCR to detect chondrocyte marker type II collagen(Col II),Aggrencan(AGC)and cartilage oligomieric matrix protein(COMP),prehypertrophic chondrocyte marker indian hedgehog(IHH)and Osterix(OSX).The results showed that the expression levels of Col II,AGC and COMP were significantly decreased in chondrocytes treated with recombinant human IGF-1 protein,while the expressions of IHH and OSX in chondrocytes were significantly increased.These effects of IGF-1 on the expression of Col II,AGC,COMP,IHH and OSX were inhibited by PQ401.Overexpression of IGF-1 inhibited Col II,AGC,COMP expression in antler chondrocytes and promoted IHH and OSX expression.The transfection with IGF-1 si RNA enhanced Col II,AGC and COMP m RNA levels,and decreased the expressions of IHH and OSX.These suggest that IGF-1 has an effect on promoting the differentiation of antler chondrocytes into hypertrophic chondrocytes.During the development of chondrocytes,IGF-1 can affect chondrocytes differentiation by down-regulating insulin receptor substrate 1(IRS1),and IRS2 levels.After transfection with IRS1 or IRS2 si RNA,Col II,AGC,COMP expressions were inhibited,while IHH and OSX expressions were enhanced.IRS1 or IRS2 si RNA was transfected into antler chondrocytes and then added recombinant human IGF-1 protein,the expression of Col II,AGC,COMP,IHH and OSX was detected by real-time PCR.It was found that transfection with IRS1 and IRS2 si RNA significantly enhanced the inhibitory effect of IGF-1 on Col II,AGC and COMP and promoting effect on expression of IHH and OSX.IGF-1 can regulate the differentiation of chondrocytes by modulating expression of RUNX1 which could inhibit chondrocytes to differentiate into hypertrophic chondrocytes.The expressions of Col II,AGC and COMP increased significantly after treament with recombinant human RUNX1 protein,while IHH and OSX expressions significantly decreased.Inhibition of RUNX1 expression hasthe opposite effect.Further studies showed that IGF-1 could inhibit the expression of RUNX1.RUNX1 si RNA was transfected into antler chondrocytes and then added recombinant human RUNX1 protein,the results showed that IGF-1 could significantly decreased Col II,AGC and COMP expressions in RUNX1 si RNA transfected chondrocytes,while IHH and OSX expression were significantly enhanced.IGF-1 si RNA was transfected into antler chondrocytes and then added recombinant human RUNX1 protein,the results showed that RUNX1 could significantly enhance Co L II,AGC and COMP expressions in IGF-1 si RNA transfected chondrocytes,while decreased IHH and OSX levels.Meanwhile recombinant human RUNX1 protein could up-regulate the expressions of Col II,AGC and COMP in antler chondrocytes pretreated with PQ401,and attenuated IHH and OSX expressions.RUNX1 could mediate IRS1 and IRS2 on the differentiation of antler chondrocytes,and RUNX1 expression in chondrocytes was significantly decreased after IRS1 or IRS2 was inhibited,while the expressions of IRS1 and IRS2 did not change after treatment with recombinant human RUNX1 protein or transfected with RUNX1 si RNA.IRS1 or IRS2 was transfected into antler chondrocytes and then treated with recombinant human RUNX1 protein.The results showed that RUNX1 could increase Col II,AGC and COMP expressions in chondrocytes transfected with IRS1 or IRS2 si RNA,and attenuated the effect of IRS1 or IRS2 si RNA on the IHH and OSX expressions.IRS1/2 mediates the regulation of IGF-1 on the expression of RUNX1 in antler chondrocytes,and the inhibition of IRS1 or IRS2 enhances the inhibitory effect of IGF-1 on RUNX1 expression.In summary,IGF-1 promotes the proliferation and differentiation of antler chondrocytes,IRS1/2 may act downstream of IGF-1 and regulate differentiation from chondrocyte to prehypertrophic chondrocytes by RUNX1.
Keywords/Search Tags:IGF-1, antler, chondrocyte, differentiation, sika deer
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