Preliminary Screening And Analysis Of Proteins Associated With Scarless Healing Of Fetal Rabbit Skin

Background and Objective: Scarless healing is the main mode of trauma repair. On one hand, scars can overlay the trauma surface and enhance the strength of repaired tissue. On the other hand, scars destroy outlook, impairs some functions and worsens patients'psychological health. Studies have found that trauma healing can be achieved through scarless healing in many fetal animals. The mechanism may involve with diferentially expression of genes or some differentially expressed proteins. The present study aims to establish a scarless healing model of fetal rabbits. Total protein of fetal rabbit skin tissues were separated by two-dimensional electrophoresis technique. After comparison and analysis to determine the proteins associated with scarless healing of skin in fetal rabbits, the role of the associated proteins in scarless healing can be revealed and experimental basis can be further complemented in the intervention and treatment of scar formation after injury. Methods 1. Scarless healing model of fetal rabbits Twenty-five 20-day fetal rabbits were subjected to incision injury on the back. Among them, 5 were born after 10-day. Skin tissues were collected from surplus injured fetal rabbits, intact fetal rabbits and adult rabbits 24 hours after injury as samples of fetal trauma(FT), fetal control (FC)and adult trauma control(ATC), respectively. ProteoExtractTM Complete Mammalian Proteome Extraction Kit was used to extract total protein of these skin tissues respectively. Concentration is determined by Bradford method to further determine samples volume of total protein. 2. Two-dimensional gel electrophoresis of total protein of the skin and mass spectrographic of differentially expressed protein spots Immobilized pH gradient (IPG) strip (pH:4-7, length:11cm) were used in two-dimensional gel electrophoresis with triple volume of protein. Total focusing was 60000Vh. Separation gel was 10%Acrylamide gel. The gels were stained with Silver Stain Plus Kit. 2-DE map of skin total protein in the three groups were studied with ImageMaster 2D Platinum to determine the differentially expressed protein gel points. Differentially expressed skin protein spots were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrogram (MALDI-TOF-MS) to obtain the peptide finger prints of these protein spots. Then, the finger prints were searched in peptide library and the functions were studied. Results 1. Scarless healing model of fetal rabbits No evident scar formation was noted in 5 rabbits born after operation. In the rest fetal rabbits were randomly assigned to Group FT and Group FC. Pregnant rabbits were in Group ATC. In this way, with elimination of individual different to some extent, consistency in genetic background of samples can be warranted. Fetal rabbits were wounded with small incisions. Survival rates of adult rabbits and wounded fetal rabbits were 100% and 80%, respectively, 24h after operation. This wound mode is appropriate for fetal rabbits wounds. In the present study, total protein was extracted by ProteoExtractTM Complete Mammalian Proteome Extraction Kit. Results of protein concentrations revealed that protein were efficiently and stably extracted with this kit. 2. Mass spectrographic analysis of differentially expressed protein spots Only expressed protein spots in Group FT were analyzed by MALDI-TOF-MS, (owing to economic reasons). The results showed that seven protein spots displayed similar with some identified protein. The score of 10 protein spots were lower than 76, or species difference was too significant and they might be unidentified rabbit proteins. The rest 3 protein spots may contain just a little protein, or protein separation was not complete. The searching results revealed nothing significant. After homology analysis and similarity retrieval, seven protein fragments were identified and they were : chaperonin or mitochondrial protein P1 precursor, Vimentin(Vim), tubulin, beta polypeptide , heterogeneous nuclear ribonucleoprotein H (hnRNP H), α-enolase. Identified proteins screened were associated with cell signal transduction,proliferation, differentiation, development and other functions, consistent with the results in the study with improved SSH. We postulate that Vim, microtubule protein βpolypeptide, etc. can promote cell proliferation, differentiation and development after skin wound in fetal rabbits, and can maintain normal morphology and function of cells. hnRNP H regulates gene transcription and translation,accelerate the synthesis of collagen protein. Meanwhile, chaperonin assists polypeptide in folding,assemblage,transportation,prevents anormal folding and accelerate refolding of unfolded polypeptide and normal assemblage. Moreover, α-enolase can function as an enzyme and regulate the process. Thus, collagen fibers and other components may maintain normal alignment, suppress excessive scar formation and complete the scarless healing in feta rabbits. Conclusions 1. This study further consummates the scarless healing model of fetal rabbit skin and establishes the technique of two-dimensional electrophoresis of total protein in rabbit skin tissues. 2. Twenty, thirteen and five expressed protein spots were identified in Groups FT, FC and ATC, respectively. In group FT, identified protein associated with healing after incision may promote synthesis of collagen protein, normal folding and assemblage, accelerate cell proliferation, differentiation and development, participate and enhance scarless wound healing in rabbits, through regulation of gene transcription and translation. Other unknown proteins will be determined by sequence analysis of amino acids in the future.