| Background objective: With the development of modern molecular biology, wound healing and tissue repair have gradually been one of the most potential and indispensable part of trauma research. The first barrier to the stimuli and harmful factors is the skin, therefore, dermal healing is the indispensable part of wound healing and tissue repair. The cutis is the result of three kinds of embryo-cells differentiation, consists of epidermis and dermis which have cells of multi-types. When the skin is wounded, the course of healing includes all kinds of the dermal cells and the extracellular matrix, which involve about hundreds of genes in regulation. Although a lot of research work has been done, it is far away from the understanding dermal healing course, genes expression and regulation. Our research's objective is to clone, to screen, to identify the cDNA segment, and to discuss the genes expression of the dermal tissue repair course by the means of constructing a gene subtractive cDNA library between the tissues of dermal healing phase and normal dermal ones.Method: (1) We subscribed the dermal tissues of trauma healing phase (5 days after the trauma) as the Tester, while the normal ones is the Driver. By the techniques of Suppression Subtractive Hybridization (SSH), first we extracted the mRNA from the tissues of healing phase and the normal ones, then reverse-transcripted them into the first and the second cDNA segment. We divided the Tester cDNA deposed by Rsa I into halves and different primers are connected. After 2 subsequences of cDNA subtractive and PCR, at last constructed the cDNA library between the tissues of healing phase and normal ones by T/A clone of the selective amplified PCR products and T-carrier; (2) Select positive clones by roughly screening the Blue-White colony which based on the a-complementation principal and more precisely identifying them using PCR; (3)Thepositive clones were differently screened and the different expressed segments were identified; (4)The sequence of different expressed segments were analyzed, and searched and compared by using NCBI BLASTN, the data base includes Non-redundant GenBank + EMBL + DDBJ + PDB Sequences and Non-redundant Database of GenBank EST Division; (5)Our research was proved be reliable by Northern Blot, which also helped to compare the screened genes expression of the normal and Ih after trauma and 5 days after trauma.Results: (1) We successfully constructed the cDNA library between the dermal healing phase(5 days after trauma) and the normal tissue by the means of Suppression Subtractive Hybridization(SSH); (2)The truly positive clones which had 128 positive clone roughly screened by Blue-White colony were 70 by PCR-selecting; (3)The positive clones were difference screened and the identified segments were 37; (4) 38 segments sequence analysis confirmed 37 segment similar to the human genes that are already known(97%~100%); but only 1 segment of 665bp which partly coincides with the latter is supposed to be EST segment compared to the unknown human genes. We have already made an application to the dbEST data base of Genbank and registered (the logging number is BM499225); (5) The Keratin 6e and P -2-microglobulin of known genes were not appeared in the relative references of tissue healing and repair after trauma; (6)4 segments confirmed by the Northern blot were expressed significantly differently in the dermal tissues after Ih and 5 days.Conclusion: SSH proved to be efficient in the screening course, we successfully constructed a subtractive cDNA library between dermal tissues of the healing phase of trauma and the normal ones; (2)By combining the Blue-White colony with the PCR, the positive clones can be selected rapidly and accurately; (3)Sequence analysis confirmed that among the 38 difference expression segment, 37 segment similar to the human genes that are already known(97%~100%); but only 1 segment of 665bp which partly coincides with the latter is supposed to be EST segment compared to the unknown human genes. We have already...
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