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Cysteinyl Leukotriene Receptor 2 Modulates the Differentiation of Rat Glioma C6 Cells, and Screening for Its siRNA and New Antagonists

Author: CaiZuoLei
Tutor: WeiErQing
School: Zhejiang University
Course: Pharmacology
Keywords: Cysteinyl leukotriene receptor 2 CysLT2 receptor Rat glioma C6 cells Cell Differentiation Small interfering RNA (siRNA) Interference lentiviral expression vector Antagonist screening
CLC: R964
Type: Master's thesis
Year: 2011
Downloads: 29
Quote: 0
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Abstract


Objective: C6 rat glioma cells in the large, cysteinyl leukotriene receptor 2 (CysLT2 receptor) is the main expression CysLT receptor subtypes, but its effect is not clear characteristics. To explore the effects of this receptor subtype characteristics of this study was to investigate the following questions: (1) CysLT2 receptors on C6 cell differentiation whether the adjustment; (2) to increase research tools, screening CysLT2 receptor siRNA 'slow virus interferes expression vectors; (3) in LTD4-induced increase of intracellular calcium C6 indicators screening series of synthetic compounds, find a CysLT2 antagonistic activity by compounds. Methods: RT-PCR detection CysLT2 receptor mRNA expression, immunocytochemistry CysLT2 receptors in C6 cells was observed within the distribution. To determine LTD4 and CysLT1 receptor antagonists on cell differentiation, in order to observe the bright-field camera C6 cell morphology, immunoblotting GFAP expression was observed. To determine CysLT2 receptor siRNA, fluorescence microscopy lentiviral infection efficiency, RT-PCR and real-time PCR CysLT2 receptor mRNA expression was observed, immunoblotting CysLT2 receptor protein expression was observed by laser scanning confocal microscopy determination of calcium signaling observe agonist-induced changes in intracellular calcium. CysLT2 receptor antagonistic activity for the screening of compounds, LTD4 agonist detected in C6 cells after treatment changes in the intracellular calcium concentration as an indicator, the preliminary screening compounds with antagonist activity, and non-selective antagonist CysLT1/CysLT2 receptor and CysLT2 by Bayu9773 AP-100984-selective antagonist as a reference. Results: RT-PCR identification showed, C6 cells with high expression CysLT2 receptors, mainly distributed in the cytoplasm of C6 cells. Agonist LTD4 not induce morphological changes in C6 cells and GFAP expression; CysLT1 receptor antagonist montelukast alone without inducing differentiation of C6 glioma cells, but can be combined with LTD4 induced C6 cell processes becomes longer, the number of branches become more that changes induced differentiation. CysLT2 receptor siRNA screening received interference lentiviral expression vector; CysLT2 SH3 receptor lentiviral expression vector acting on C6 cells, inhibit their CysLT2 receptor mRNA and protein expression and LTC4-induced intracellular calcium increase. LTD41 C6 significantly increased intracellular calcium, its role is approximately 37.5% of the positive control of ATP, as a screening indicator CysLT2 receptor antagonistic activity with the compound; CysLT2 receptor antagonist AP-100984 inhibited LTD4-induced C6 cells calcium increase, CysLT1 receptor selective antagonist had no inhibitory effect; compounds DxW-1, 2,13,23,29,30 can inhibit LTD4-induced increase in intracellular calcium. Conclusions: (1) C6 cells expressed mainly CysLT2 receptors. CysLT receptor agonist alone LTD4 and CysLT1 receptor antagonist montelukast had no effect on C6 cells, and both can be combined to facilitate C6 cells, suggesting CysLT2 receptors may contribute to C6 cells. (2) successfully filter out interference CysLT2 receptor expression lentiviral vectors, at both mRNA and protein expression as well as calcium signal change them on a validation study for the future effects of CysLT2 receptors provides a useful approach. (3) agonist LTD4-induced increase of intracellular calcium C6 indicators screening a series of synthetic compounds, found DxW-1, 2,13,23,29,30 have CysLT2 receptor antagonistic activity, receptor antagonist for the study CysLT2 provide clues.

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