Analysis Of Genetic Regulation And Cytokines Expressions Of Distraction Osteogenesis Reconstruction For Cleft Palate On Rhesus

ObjectiveTo investigate the osteogenesis mechanism by analysis of the expression of insulin-like growth factor-Ⅰ(IGF-Ⅰ), alkaline phosphatase (ALP), osteopotin(OPN) and osteocalcin (OC) in the reconstruction of cleft palate (CP) with distraction osteogenesis (DO) in rhesus.Methods1 The CP animal models (n=23) were established surgically.21 rhesus in experimental group underwent DO to close the soft and bony defect, followed by consolidations. Every 3 animals were killed and the specimen were taken out after consolidation of 1,2,4,6,8,12,24 weeks. IGF-Ⅰ, ALP, OPN and OC mRNA were detected by Real-Time RT-PCR.Further more,the effects of IGF-Ⅰ, ALP, OPN and OC were detected by ELISA. The results are compared with those of the experimental control and healthy control groups (each of 2 animals).2 For an In-depth understanding of the interactive mechanism of these four osteoblast-relative factors, correlative analysis of the mRNA and protein expression with Pearson Statistics is used. The correlation coefficient(r) was calculated and statistical "t" test were applied.Results1 Since consolidation, the mRNA of IGF-Ⅰand ALP increased significantly at one week and reached the peak at two weeks, but decrease to control level after 12 weeks of consolidation. The expression of IGF-Ⅰalso increased to peak level aftert two weeks of consolidation. The expression of ALT increased significantly since consolidation and reach the peak value after six weeks. They all decreased to nearly control level after 8-12 weeks.2 The mRNA expression of OPN were observed highly up-regulated to since the early phase of new bone formation in distraction gap since the 2ed week and reached the apex of 7.59±0.37 in 4th week, while that of OC started increasing in 4th week and reached the top level in 6th week, in which from 4.98±0.21 to 7.94±0.31. Subsequently, these expression switched to decreasing tendency gradually, but still significantly higher than the experimental and healthy control groups until 24th week. ELISA test results indicated that OPN and OC protein expressions were 4.75±0.15 ng/mg,4.86±0.09 ng/mg and 3.18±0.16 ng/mg,3.63±0.33 ng/mg, respectively, demonstrated the highest level during 4th to 6th weeks. The protein expressions showed the changing curves accordant with those of mRNA expression respectively.3 The mRNA expression levels of fore pairs, which included IGF-I and ALP (r=0.88546), IGF-I and OPN (r=0.65549), OPN and ALP (r=0.82380), OPN and OC (r=0.74945), suggested that there was a significant positive correlation between each pair. There was a significant positive correlation between 6 groups:IGF-Iand ALP (r=0.68922), IGF-Iand OPN (r=0.68541), OPN and ALP (r=0.99468), OPN and OC (r=0.0.88352), ALP and OC (r=0.88352). Meanwhile, our study displayed that there might be a positive regulatory mechanism between them.Conclusion1 The palate cleft can be successfully closed with new formed bone after DO. The mechanism of bone consolidation is intramembranous bone formation.2 The correlative study on the mRNA and protein expression levels of IGF-I, ALP, OPN, OC suggested that there was a significant positive correlation between the mRNA and protein expressions levels of IGF-I and ALP, IGF-I and OPN, OPN and ALP, which indicated that there might be a positive regulatory mechanism between each pairs of them.3 Throughout this study of different experimental stages, the curve of mRNA expression and that of protein synthesis expression of ALP, IGF-I, OPN, OC had a consistent trend. In our study, osteoblast-related genes was investigated from m-RNA level to protein level, so as to understand the regulation mechanism of new bone formation in DO preferably in different levels.