Localization Of α_1B-adrenoceptor Using PEG-Prazosin-QDs And Developing Of A Drug Screeing Model

In chaptor one, the commonly used fluorescence techniques in drug screening on cellular and molecular level were firstly reviewed. The fluorescence techniques were the major technique in high throughput screening and high content screening as their high sensitivity and simple operation. The emphasis in this part was on total internal reflection fluorescence microscopy and flow cytometry involved in the experiment. Secondly, the adrenergic receptor and its coupling with ligands were introduced. Then, we described the modification and bioconjugation of quantum dot in detail and the importance of polyethylene glycol in the modification in especial. At last, we make a summary of the applications of modified QDs in drug screening.In chapter two, we began with the structure of adrenergic receptor and analyzed the coupling of antagonist Prazosin-PEG-biotin with it by the computer-aided drug design software. We done the work to obtain the proper PEG length which could make the Prazosin-PEG-biotin stretch the receptor cavity and decrease the steric hindrance from the QDs in theory. From the other hand, when the molecular weight of PEG was more than 2000, it could decrease the nonspecific adsorption of QDs apparently. Taken together, we fixed 2000 as a proper molecular weight of PEG and got the Prazosin-PEG2ooo-biotin. Then, we used QDs-streptavidin labeled Prazosin-PEG2ooo-biotin positioning theα1B-AR on HEK293α1B cell membrane by TIRFM. In the experiment, we performed our study on many factors which effect the location ofα1B-AR, including concentration and incubation time and then obtained the optimum condition ofα1B-AR location at last. The results demonstrated Prazosin-PEG2ooo-QDs could combine withα1B-AR more quickly and the HEK293 cell made a weaker nonspecific adsorption in comparative with Prazosin-QDs. In this case, the membrane receptor and the cell could maintain a good condition. These two apparent improvements indicated that the steric hindrance and nonspecific adsorption of QDs could be solved in some extent by PEG.In chapter three, we determined the mean fluorescence intensity of the two kinds cells in different concentrations and with different incubation time of Prazosin-PEG2ooo-QDs at first and then verified the optimum condition by TRIFM:30 nM,15 min. The results proved the MFI of HEK293α1B with Prazosin-PEG2ooo-QDs was higher than with Prazosin-QDs and HEK293 made a weaker nonspecific adsorption to Prazosin-PEG2ooo-QDs than Prazosin-QDs. In the part two, we screened three new antagonists of al-AR using TRIFM and FCM. By analysis the result, we concluded the affinity sequence of three antagonist withα1B-AR was Carvedilol> labetalol hydrochloride> alfuzosin hydrochloride. By modification Prazosin-QDs with PEG, the incubation time between Prazosin andα1B-AR was greatly decreased and membrane receptor could be maintained in a good condition. Moreover, the increasment of MFI by FCM and reduction of nonspecific adsorption to QDs made the screening model basing on ligand-receptor more accurate.