Expression Of P70 S6 Kinase In Rabbit Periodontal Tissues Remodeling During Orthodontic Tooth Movement

ObjectiveTo build animal models and detect the expression and the change of p70 S6K which is the substrate of mTOR by immunohistochemistry,RT-PCR and Western-blot test techniques.The aim is to study the role of p70 S6K in periodontal tissues remodeling during orthodontic tooth movement.Methods1.Building animal modelsThirty-six rabbits(weight 2.0±0.2 kg)were chosen to establish rabbit models for the study.The rabbits were divided into 4 groups(9 rabbits per group):3,5,7 and 14d group,The right maxillary teeth of each animal treated by orthodontic were the test groups,a coil spring was located from the upper first molar to the upper incisor and the 80g mesial force was applied to pull the upper first molar,whereas the left teeth were untreated(control).The animals were sacrificed at different time intervals(3,5,7,14d) respectively.2.Immunohistochemistry assayThe paraffin wax speciments of periodontal tissues in control and test group were made by immunohistochemistry staining to observe the expression of p70 S6K and analyse the significance.3.RT-PCRAccording to the instruction of reverse transcription kit,the first strand of cDNA was synthesized with oligo dT primer.The expression of p70 S6K mRNA was detected by RT-PCR in periodontal tissues remodeling during orthodontic tooth movement.The result of p70 S6K expression was analysed with agarose gel electrophoresis.4.Western BlottingTissues were washed with PBS and lysed at 4℃with 25mM Tris.HCI,Ph 7.4/50Mm NaCl/0.5%sodium deoxycholate/2%Nonidet P-40(NP40)/0.2%SDS/1μM phenylmethylsulfonyl fluoride(PMSF)/50μg/ml aprotinin/ 50μM leupeptin. Lysates were resolved by SDS/10%(for p70 S6K)polyacrylamide gels and transferred to nitrocellulose filters.After blocking of filters with a solution containing 5%BSA, the filters were incubated with a rabbit polyclonal antibody raised against the common carboxyl-terminal sequence of the p70 S6K isoforms,Specific reactive proteins were detected by an enhanced chemiluminescence(ECL)method,employing a sheep anti-rabbit IgG antibody linked to horseradish peroxidase.5.Statistical analysisTtest was used for statistical analysis between the fixed appliance group and the unfixed group with SPSS 11.5 sofeware For Windows.P＜0.05 was considered statistically significant.Results1.Histological studyIn the group of test,the derangement of tranecula of bone and cells arrayed disorderly,resorption of alveolar bone were observed in histological study,there were many holes in the alveolarbone,occasionally occurred osteoclasts.2.The location of p70 S6K in periodontal tissuesp70 S6K immunohistochemistry results indicated that the expression of p70 S6K observed and increased in periodontal tissues,the expression of p70 S6K is mainly located in fibroblast,osteoblast,vascular endothelial cell and esteocycte appeared positive. 3.RT-PCRRT-PCR showed:Compared with the control,the expression of p70 S6K mRNA was observed to change at 3d,the expression of p70 S6K mRNA in treatment periodontal tissues was higher than control,and then decreased.There was significant difference from 3d to 14d in statistical analysis,especially at 7d(P＜0.01).4.Western BlottingThe result of Western-blot is the same as the RT-PCR.The expression of p70 S6K protein increased during orthodontic tooth movement,and then decreased.There was significant difference from 3d to 14d in statistical analysis,especially at 7d(P＜0.01).Conclusions1.The expression of p70 S6K increased and it was observed in periodontal tissues, the expression of p70 S6K was mainly located in fibroblast,osteoblast,vascular endothelial cell and esteocycte appeared positive.2.Higher expression of p70 S6K was detected during the orthodontic tooth movement.3.The expression of p70 S6K was the highest at 7d followed by periodontal remodeling.4.p70 S6K participated in periodontal tissues remodeling during orthodontic tooth movement.