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Localization of α_1B-adrenoceptor Using PEG-Prazosin-QDs and Developing of a Drug Screeing Model

Author: WangLiPing
Tutor: WangLei
School: Shandong University
Course: Pharmaceutical Analysis
Keywords: PEG- quantum dot complexes of prazosin α1B- adrenergic receptor Total internal reflection fluorescence microscopy Flow cytometry Drug screening
CLC: R96
Type: Master's thesis
Year: 2011
Downloads: 35
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Abstract

The first chapter on the fluorescence technique commonly used in drug screening technology in the cell and molecular level. Fluorescence techniques because of its high sensitivity, the method is simple and commonly used in high-throughput and high-content screening of drug technology, which focuses on two technologies involved in the experiment - total internal reflection fluorescence microscopy and flow cytometry . Secondly, the adrenergic receptor involved in the experiment, and analysis from the spatial structure of the receptor ligand coupling. Subsequently, the fluorescent dyes are used in the experiment - quantum dots modified and biological conjugate aspects of the situation to conduct a detailed description, especially polymer-PEG in the modification of the quantum dots play an important role, a quantum dot has been modified after the fluorescent characteristics get better improvement. Finally, based on the modified quantum dot probes in drug screening applications are reviewed. Chapter II adrenergic receptor structure, the use of computer simulation technology, and analysis from the spatial structure of the adrenergic receptor antagonist Prazosin-PEG-biotin molecules coupled theory to determine the length of the PEG molecule, length connected to the ligand on the quantum dot can protrude outside the receptor hole, Prazosin-PEG-QDs probe thus obtained is reduced due to steric hindrance caused by too much of the volume of the quantum dot can be membrane α1B -AR is the maximum amount of binding. On the other hand, has been reported, the PEG molecular weight exceeds 2000, it is possible to significantly reduce the non-specific adsorption of the QDs, comprehensive consideration to determine the length of the PEG 2000 to obtain PEG functionalized Prazosin-PEG2ooo-biotin molecule. Coupled with streptavidin and biotinylated quantum dots (streptavidin-QDs) Prazosin-PEG2ooo-biotin, the formation of Prazosin-PEG2ooo-QDs, and then locate the cell surface HEK293aiB-AR α1B-AR. Prazosin-PEG2ooo-QDs with concentration and incubation time of the cells of these two effects receptor labeled factors experiment we conducted a study to determine Prazosin-PEG2ooo-QDs positioning HEK293 membrane best α1B-AR conditions, and compare the rate of Prazosin-QDs and Prazosin-PEG2ooo-QDs at the same concentration, α1B-AR, confirmed the Prazosin-PEG2ooo-QDs significantly faster binding to the cell membrane, the experimental results indicate prazosin and QDs inserted between PEG2000, can reduce the steric hindrance of the quantum dots, greatly shorten its membrane receptor binding, and largely maintaining the membrane receptor and the activity of the cells; the same time, the surface is not α1B-expression the AR-negative cells in the same concentration as Prazosin-PEG2000-QDs Prazosin-QDs, significantly weaker adsorption on the former. By two significant changes PEG2000 modified quantum dots steric hindrance and non-specific adsorption of problems have been greatly improved. Chapter III of this paper, the first verified by flow cytometry Prazosin-PEG2ooo-QDs through TRIFM derived membrane receptors. Verified after the combination of PEG modification of of Prazosin-PEG2ooo-QDs with the cell membrane receptor cells much higher than the average fluorescence intensity under the same conditions Prazosin-QDs treated cells, and non-specific adsorption of Prazosin-PEG2ooo-QDs Prazosin-QDs compared to relatively small. Combination of three kinds of α1-AR antagonists were screened on the basis of the above experiments, the two methods by total internal reflection fluorescence microscopy and flow cytometry, and by comparing the experimental results, it is determined to be screened antagonist α1B-AR affinity activity order is: Carvedilol gt; hydrochloride labetalol gt; hydrochloric A furosemide prazosin. After PEG modification drugs - quantum dot complexes can greatly shorten the incubation time of the drug with cell membrane receptors and cells maintained better activity, improve work efficiency; And HEK293α1B modified quantum dot binding more apparent from the average fluorescence intensity after the improvement of the cell to its non-specific adsorption is greatly reduced, so that the FCM statistical mean fluorescent intensity, the yin and yang of experimental comparison, so that the receptor - ligand screening model screening effect is more better.

Localization of α1B-adrenoceptor Using PEG-Prazosin-QDs and Developing of a Drug Screeing Model

Abstract

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Localization of α_1B-adrenoceptor Using PEG-Prazosin-QDs and Developing of a Drug Screeing Model