| AimsScreen tolerance related differential fragments from gene level, and investigate the molecular mechanism of tolerance from the area of cytosolic calcium, mitochondrial membrane potential and cytosolic pH.MethodsConstruct HEK293 cell lines stably expressed μ-/κ-opioid receptor gene respectively by transfection and dilution. Identify receptor number on each cell line by receptor binding assay. Check opioid receptor signal transduction integration by cAMP assay.Investigate activity changes of adenylate cyclase on μ-CHO cells after treatment of 10-6 M DAMGO for 0120 min. μ-CHO cells were exposed to μ-receptor selective agonist DAMGO for 72 hours to develop addiction and tolerance. The specific expressed genes were screened out by differential display PCR, cloning and Northern blot and were sequenced. The sequences were compared with the database of NIH NCBI. Analyze the relationship between homologous proteins and tolerance, and find out the points for further research.Observe the changes of cytosolic calcium, mitochondrial membrane potential and cytosolic pH on μ-, δ- and κ-CHO cells, μ-, κ-HEK.293 cells and PC12 cells after both short-term and long-term treatment of opiates by confocal laser scanning microscopy and flowcytometry. Analyze the relationship among the changes and their effects on cell desensitization and tolerance; analyze the differences among three kinds of subtype receptors.Observe apoptosis signals on PC12 cell after morphine treatment by confocal laser scanning microscopy and flowcytometry, and analyze cell cycle changes after administration.Results1. We got 7 stably expressed μ-opioid receptor cell lines and 3 stably expressed κ-opioid receptor cell lines all together. According to the results of receptor binding assay, Bmax of μ10-HEK.293 is 0.02308 pmol /105 cells, Kd to 3H-diprenorphine is 1.9455 nM; Bmax of κ19-HEK293 is 0.01607 pmol /105 cells, Kd to 3H-diprenorphine is 1.080 nM. Activity of AC both on μ10-HEK293 cells after treatment of 10-910-5 M morphine for 10 min and on μ19-HEK293 cells after treatment of 10-1010-10 M U50,488 for 10 min decreased and was concentration-dependent.2. In the process of treatment of 10-6 M DAMGO on μ-CHO cells for 0120 min, the inhibition rate of AC was most significant at 2 min, the concentration of cAMP in cells was 5.21% compared to control group; then the inhibition was gradually decreased as the time went on, the concentration of...
|
PDF Full Text Request