Study On The Relationship Between PI3K/AKt Signaling Pathway And Orthodontic Tooth Movement

ObjectiveTo build animal models and detect the expression and the change of PI3K,AKt by RQ-PCR and Western-blot test techniques. The aim is to study the role of PI3K/AKt signaling pathway in periodontal tissues remodeling during orthodontic tooth movement.Methods1. Building animal modelsTwenty-four rabbits (weight 2.0±0.2 kg) were chosen to establish rabbit models for the study. The rabbits were divided into 4 groups (six 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. RQ-PCRAccording to the instruction of reverse transcription kit, the first strand of cDNA was synthesized with oligo dT primer.The expression of PI3K and AKt mRNA was detected by RQ-PCR in periodontal tissues remodeling during orthodontic tooth movement. All RT product, sequenc examination system moves in ABI Prism 7500HT. After 3 independent samples undergo 3 independent experiments, obtains the data carries on the analysis with the formula RQ=2-ΔΔCt. 3. 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/8%(for PI3K,AKt) 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 PI3KandAKt isoforms, Specific reactive proteins were detected by an enhanced chemiluminescence (ECL) method,employing a sheep anti-rabbit IgG antibody linked to horseradish peroxidase.4. Statistical analysisT test was used for statistical analysis between the fixed appliance group and the unfixed group with SPSS 12.0 sofeware For Windows. P<0.05 was considered statistically significant.Results1.RQ-PCRRQ-PCR showed:Compared with the control, the expression of PI3KandAKt mRNA was observed to change at 3d, the expression of PI3K and AKt mRNA in treatment periodontal tissues was higher than control, and then decreased. There was significant difference from 3d to 14d in statistical analysis(P<0.05), especially at 7d (P＜0.01).2. Western BlottingThe result of Western-blot is the same as the RQ-PCR. The expression of PI3KandAKt protein increased during orthodontic tooth movement, and then decreased. There was significant difference from 3d to 14d in statistical analysis(P<0.05), especially at 7d(P<0.01). Conclusions1. Higher expression of PI3K and AKt was detected during the orthodontic tooth movement.2. PI3K/AKt signalling participated in periodontal tissues remodeling during orthodontic tooth movement.