Yunnan Blue-tailed Newt Forelimb And Mandibular Regeneration Organization Nag Protein Gene Expression Analysis

[Objectives] To investigate gene expression of nAG (newt anterior gradient) in mandibular and forelimb regenerative tissue with a local species -Yunnan blue tail newt (Cynops cynaurus)[Methods] Sixty wild type adult Cynops cynaurus were divided into two groups randomly. Partial mandibular amputations or left forelimb amputations were performed under general anesthesia by Tricaine. The regenerative tissues were observed on the 3,5,7,12 days later. The contralateral limb and the regenerative tissues of jaw and left forelimb were harvested respectively on the 5,12 days after amputation. The expression level of nAG was examined by RT-PCR and DNA sequencing. The expression and distribution of nAG in regenerative tissues during different period were analyzed by in Situ hybridization and statistical correlation analysis. A statistical comparison of mean option density (MOD) of nAG expression and calculate nAG protein positive expression rate was processed by software of "HPIAS-1000" and "SPSS 11-5" analysis during the epimorphosis.[Results] All experimental animals survived and jaws and limbs regenerated partially without any complications. The regenerative process in general was follow:1 hour later, the wounded stop bleeding, mouth-floor organization contraction, mandibular relatively closed, forelimb stub axial gathered; 3 days later, the wound surface smoothed, wound epidermis covered; 5 days later, apical ectodermal ridge formed; 7 days later, apical ectodermal ridge formed thickening; 12 days later, the wound surface appear transparent small bumps, regenerative blastema formed. The molecular weights of PCR products were consistented and proved with the known sequence. By in Situ hybridization, the nAG expression was shown in basal cells of epidermis and nerve fibers, which confirmed that nAG existed in regenerative tissues of Cynops cynaurus and expressed variedly in different tissues and periods. The expression of nAG was correlated with the proliferation and dedifferentiation of cells and tissues, especially in epidermis and nerve tissues. It was detected in 5 or 12 day after amputation by in Situ hybridization. On the fifth day after amputation, nAG expressed mainly in cartilage tissues and mesenchymal cells under the apical ectodermal cap as well as in the nerve tissues. On the twelfth day after amputation, nAG expressed dominatively in basal cells of epidermis. nAG expression in different time of different organizations, same time different organizations and the same organization of different time to have differences (P< 0.05).[Conclusions] 1. Cynops cynaurus has the regenerative ability of mandible and limb.2. A biological model has been created successfully on adult Cynops cynaurusr for mandibula regeneration.3. The expression of nAG was shown in regeneration tissues of jaw and limb of adult Cynops cynaurusr.4. The positive area and level of nAG expression were correlated with the regeneration of jaw and limb and the period of regeneration.5. During the regeneration process, the expression of nAG was shown in different tissues of regeneration in different times.6. nAG expressed only in cartilage tissues and mesenchymal cells, so it can indicate the proliferation and differentiation of tissue regeneration.7. nAG is a factor of proliferation and differentiation, we should study its action principle.