Effects Of Shh Signaling Pathway On Apoptosis Activity Of The Condylar Hyperplasia Chondrocytes

Condylar hyperplasia(CH)is an excessive growing disease of unilateral condyle that occurred in teenagers,and it always leads to facial asymmetry,which causes facial deformity and occlusal disturbance.It is self-limited and there is no gender difference in prevalence rate.However,the etiology of CH still remains unknown.Removing the involved condyle by surgery is the main therapy.Hence,carrying out the basic research is helpful to explore new treatment way which would relieve or avoid the pain of surgery.Recent studies have demonstrated that condyle overgrowth is the main cause.Condylar cartilage is the growth center of condyle,and the growth of condylar cartilage includes proliferation,differentiation,hypertrophy and apotosis of chondrocytes.In our previous study,we have found that chondrocytes of CH have a higher rate of proliferation,but there is still few reports about the effect of the change of apotosis to CH.Sonic hedgehog(Shh)signaling pathway plays a crucial role in the growth and development of normal cartilage.The main components of Shh signaling pathwayinclude Shh,membrane receptor Ptc and Smo,transcription factor Gli.Tavella S et al had found that overexpressed Shh could inhibit the chondrocytes' apotosis of articular cartilage.But there is still few report about the effect of Shh on the chondrocytes' apoptosis of condylar hyperplasia.Therefore,we aim to text the expression level of Shh signaling pathway in CH,explore the influence of Shh on the chondrocytes' apoptosis of condylar hyperplasia,and the potential mechanism.1,Collection of specimens from normal condylar cartilage and CH cartilageMethods: 1,Sources of our specimens: We got the normal condylar cartilage from patients with condylar fracture(the fragment can't be fixed)in the department of Oral and Maxillofacial Surgery,School of Stomatology,Fourth Military Medical University;and the pathological specimens were from the CH patients confirmed by SPECT in the department of Oral and Maxillofacial Surgery,School of Stomatology,Wu Han University.2,Management of tissue specimens: 3 normal specimens and 3 pathological specimens with intact structure were selected.Fixed by paraformaldehyde,and then decalcify by EDTA.3,Seperation and culturing of chondrocytes: Chongrocytes were seperated by enzyme digestion.Collected chondrocytes of the senond passage and then cultured them in alginate beads to protect chondrocytes from dedifferentiation.Results: Most of the chondrocytes are polygon and triangle,a few proportion are a fusiform or roundness shape.Chondrocytes aggregated into clusters which looked like“paving stone”.Morphology of alginate beads is water-drop shape or roundness,and the encapsulated chondrocytes presented as round lightspots.Conclusion:Using mechanical and enzymatic digestion method could obtain condylar cartilage cells with high cell activity.2,Dection of the expression level of Shh pathway in CH chondrocytesMethods: 1,Immunohistochemistry was used to test the expression of Shh and Smo in cartilages from both groups.2,Chondrocytes from both groups were collected on the 21 st day,and then Westernblotting and Real-time PCR were chosen to detect the expression of Shh and Smo.Results: Immunohistochemical staining showed that there was a abnormally high expression level of Shh and Smo in CH group,especially in the undifferentiated mesenchymal layer and hypertrophy layer.And compared with normal chondrocytes,the protein and gene expression levels of CH chondrocytes were significantly increased(P<0.05).Conclusion: Shh signaling pathway had abnormally high expression in CH cartilage.3,The effect of Shh pathway on the apotosis of CH chondrocytesMethods: Chondrocytes were randomly divided into 3 groups: normal condylar chondrocytes(NC),chondrocytes from condylar hyperplasia(CH),CH+Cyclopamine.Cyclopamine(a specific repressor of Shh signaling pathway)was added in the medium at day 5,12 and 19.Chondrocytes were collected on the 21 st day,and then Western blotting and Real-time PCR were chosen to detect the expression of Cleaved-caspase3.Results: Compared with NC,the protein and gene expression levels of Cleaved-caspase3 in CH were significantly decreased(P<0.05),after blocked the Shh signaling pathway by Cyclopamine,the protein and gene expression levels of Cleaved-caspase3 was significantly increased(P<0.05).Conclusion: Shh signaling pathway could inhibit the apotosis of CH chondrocytes.4,The potential mechanism of Shh signaling pathway inhibit the apotosis of CH chondrocytesMethods: Chondrocytes were randomly divided into 4 groups: CH,CH+Cyclopamine,CH+ LY294002,CH+ U0126.Cyclopamine,LY294002(a specific repressor of PI3K/AKT)and U0126(a specific repressor of MAPK/ERK)were added in the medium at day 5,12 and 19,and chondrocytes were collected on the 21 st day.The expression level of Cleaved-caspase3,AKT,ERK,P-AKT and P-ERK was detected by Western blotting and the gene expression level of Cleaved-caspase3 was texted by Real-time PCR.Results: Compared with CH,the expression levels of AKT and ERK had no obvious changes in CH+Cyclopamine group(P>0.05),but the expression levels ofP-AKT and P-ERK were significantly increased(P<0.05).By comparing with CH,the protein and gene expression levels of Cleaved-caspase3 were significantly decreased in both CH+ LY294002 and CH+ U0126 groups(P<0.05).Conclusion: Shh could activate the PI3K/AKT and MAPK/ERK pathway,and inhibit the apotosis of CH chondrocytes though the PI3K/AKT and MAPK/ERK pathway.