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Chinese Journal of Biotechnology ; (12): 636-643, 2014.
Article in Chinese | WPRIM | ID: wpr-279476


PAC1 is the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) preferring receptor, which belongs to class B G protein-coupled receptors (GPCR) family. PAC1 mediates the most effects of PACAP as neurotransmitter, neuroregulator and neuroprotectant, while its high expression has close relationship with some physiological and pathological processes such as nerve-injury and tumor. To further understand the function of PAC1, a cell line that expressed inducible PAC1 was constructed to achieve Doxycycline (Dox) dependent expression of PAC1 in CHO (Chinese hamster ovary) cell using the improved Tet (tetracycline)-on Advanced System. First, the PAC1-EYFP fusion gene composed of PAC1 gene and gene encoding EYFP (enhanced yellow fluorescent protein) was sub-cloned to the tetracycline response element pTRE-Tight vector to construct the recombinant vector pEYFP-PAC1-EYFP by double enzyme digestion. Second, the tetracycline regulation components pTet-On advanced vector and the response element pTRE-PAC1-EYFP vector were both introduced into CHO cells successively and the positive clones were screened with G418 and hygromycin respectively. Third, the controlled expression of PAC1-EYFP in CHO was induced by tetracycline analogues Dox in different concentrations and the different levels of receptor PAC1-EYFP were detected. The results of fluorescence analysis and western blotting show that the cell strain with Dox dependent expression of PAC1-EYFP named PAC1-Tet-CHO was obtained. Moreover, in PAC1-Tet-CHO cells the expression of PAC1-EYFP was induced by Dox in a dose-dependent manner. The inducible expression of PAC1 still was stable after sub-culturing for more than 10 passages. It was also found by MTT assay that the higher expression level of PAC1 endowed the cells with higher proliferative viabilities. The construction of controlled expression system of PAC1 will lay a foundation for the further research on PAC1 profiles.

Animals , Blotting, Western , CHO Cells , Cloning, Molecular , Cricetinae , Cricetulus , Genetic Vectors , Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide
Article in Chinese | WPRIM | ID: wpr-592153


Objective To explore the key factors which affect maintenance cost of large-scale medical equipment. Methods The basic factors which affect maintenance cost of medical imagining equipment were summarized. The relationships of them are analyzed and then an interpretative structure mode of each factor was set. Additionally, the multiple relationship and controllability between each factor were also analyzed and the judging standard of key factors was set. Results The maintenance policy and mode were the key factors which affect maintenance cost of medical imagining equipment. Conclusion High quality maintenance for equipment condition and scientific optimization of the maintenance policy are necessary to fundamentally reduce the maintenance cost of large-scale medical equipment.