Résumé
Objective: To reconstruct a substractive complementary deoxyribornucleic acid (cDNA) library of genes sensitive to mechanical stretch in human osteoblast like cells.Methods: Mechanical stretch at 12 cycles per minute was applied to human osteoblast like cells Saos-2 and the deformation of the stretched cells was 12%. Twelve hours after loading, mRNAs were isolated from both stretched and unstretched cells. Substractive cDNA library of the genes sensitive to stretch was constructed with the technique based on polymerase chain reaction (PCR) and substractive hybridization. Primary sequencing of clones in the library was carried out. Results: A substractive cDNA library of genes sensitive to stretch was constructed with a capacity of about 200 clones. According to the results of sequencing, most genes in the library were related to the mechanical stimulation. One novel gene fragment was obtained. Conclusion: The method used in the experiment is effective in cloning genes sensitive to mechanical stretch.
Résumé
Objective:To identify the function of the 5′-flank upstream regulation region of human CD226 gene.Methods:The upstream regulation region of CD226 was cloned by PCR and ligated into pGL3 vector. Then the vector was transfected into Jurkat cell and luciferase activity was detected after 48 h culture.Results:CD226 gene may have two promoters, P1 and P2,which were located at the region of -843--319 bp and +1-+181 bp respectively, and PMA can up-regulate P1 while down-regulate P2. Both P1 and P2 can be up-regulated by A23187, especially P2.Conclusion:CD226 gene may have two promoters, and PMA and A23187 can regulate CD226 promoter activity in the similar pattern of protein level.
Résumé
Objective:To clone mouse CD226(platelet and T cell activation antigen1,PTA1).Methods:Specific primers were designed and sythesized according to the EST sequence from GenBank,which had 51% homology to human CD226 on the amino acid level.And then,the cDNA of mouse CD226 was cloned from the thymus of 4 week old BALB/c mouse by using RACE technique.Results:The length of mouse CD226 cDNA is 2 223 bp,with the open reading frame(ORF) of 1 002 bp,encoding 333 amino acids,which is 3 amino acids shorter than its counterpart in human.The mouse CD226 belongs to IgSF,and shares 53% homology with human PTA1 on amino acid level.Besides,three isoforms of mouse PTA1 were also cloned at the mean time.Conclusion:The molecular cloning of mouse PTA1 lays the foundation for the in vivo studies on the biological function of this molecule,as well as the studies of this molecule in gene knockout mouse and transgenic mouse.
Résumé
Objective:To investigate the expression and function of CD226 on NK cell clone Methods:NK cell clone was established by limited dilution, and identified by FCM The function of CD226 on the cytotoxicity of NK cell clone was detected by RCA and the NK cell clone secreted cytokines in the supernatants during the killing phase were measured by ELISA Results:One NK cell clone was obtained by limited dilution The cytotoxicity of this clone was upregulated markedly by CD226 mAb, and the secretion levels of IFN ? and GM CSF by NK cell clone increased obviously in RCA Conclusion:CD226 is a novel NK cell activation receptor, and the elevated IFN ? and GM CSF levels may be related to CD226 mAb enhanced NK function