优化基于细胞的测定法，以在 96 孔板格式中定量测试物质的抗炎/抗过敏潜力。
Optimization of cell-based assays to quantify the anti-inflammatory/allergic potential of test substances in 96-well format.
机构信息
Department of Cellular Assay, R&D Centre, Natural Remedies Pvt. Ltd, Veerasandra Industrial Area, Bangalore, India.
出版信息
Inflammopharmacology. 2011 Jun;19(3):169-81. doi: 10.1007/s10787-010-0065-1. Epub 2010 Oct 31.
OBJECTIVE
There is an insistent need for robust, reliable, and optimized assays for screening novel drugs targeting the inflammatory/allergic markers. The present study describes about the optimization of eight cell-based assays utilizing mammalian cell lines in 96-well format for quantifying anti-inflammatory/allergic drug candidates.
MATERIALS AND METHODS
We estimated the inhibitory response of reference compounds: 1400 W dihydrochloride on LPS-induced NO release, celecoxib on LPS-induced PGE(2) production and dexamethasone on LPS-induced pro-inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha production by J774A.1 murine macrophages. Response of acetylsalicylic acid and celecoxib was studied on A23187-induced TXB(2) production; captopril on A23187-stimulated LTB(4) production by HL-60 cells. Effect of ketotifen fumarate was evaluated on A23187-elicited histamine release by RBL-2H3 cells. Each experiment was repeated twice to assess the reproducibility and suitability of the assays by determining appropriate statistical tools viz. %CV, S/B and Z' factor.
RESULTS
1400 W dihydrochloride was capable of inhibiting LPS-induced NO levels (IC(50) = 10.7 μM). Dexamethasone attenuated LPS-induced IL-1 beta (IC(50) = 70 nM), IL-6 (IC(50) = 58 nM) and TNF-alpha (IC(50) = 44 nM) release, whereas celecoxib, a specific COX-2 inhibitor showed marked reduction in LPS-induced PGE(2) (IC(50) = 23 nM) production. Captopril (IC(50) = 48 μM) and ketotifen fumarate (IC(50) = 36.4 μM) demonstrated potent inhibitory effect against A23187-stimulated LTB(4) and histamine levels, respectively. Both acetylsalicylic acid (IC(50) = 5.5 μM) and celecoxib (IC(50) = 7.9 nM) exhibited concentration-dependent decrease in TXB(2) production. Results for all the cell assays from two experiments showed a Z' factor varying from 0.30 to 0.99; the S/B ratio ranged from 2.39 to 24.92; %CV ranged between 1.52 and 20.14.
CONCLUSION
The results proclaim that these cell-based assays can act as ideal tools for screening new anti-inflammatory/anti-allergic compounds.
目的
迫切需要用于筛选针对炎症/过敏标志物的新型药物的稳健、可靠和优化的检测方法。本研究描述了利用哺乳动物细胞系在 96 孔板中优化八种基于细胞的测定法，以定量测定抗炎/抗过敏候选药物。
材料和方法
我们估计了参考化合物的抑制反应：1400W 二盐酸盐对 LPS 诱导的 NO 释放，塞来昔布对 LPS 诱导的 PGE（2）产生以及地塞米松对 LPS 诱导的促炎细胞因子 IL-1β、IL-6 和 TNF-α产生的抑制作用。J774A.1 鼠巨噬细胞。阿司匹林和塞来昔布的乙酰水杨酸对 A23187 诱导的 TXB（2）产生的反应；卡托普利对 HL-60 细胞中 A23187 刺激的 LTB（4）产生的反应。富马酸酮替芬的作用通过 RBL-2H3 细胞评估 A23187 引发的组胺释放。通过确定适当的统计工具，即%CV，S / B 和 Z'因子，重复进行了两次实验，以评估实验的可重复性和适用性。
结果
1400W 二盐酸盐能够抑制 LPS 诱导的 NO 水平（IC（50）= 10.7μM）。地塞米松减弱了 LPS 诱导的 IL-1β（IC（50）= 70 nM），IL-6（IC（50）= 58 nM）和 TNF-α（IC（50）= 44 nM）释放，而特异性 COX-2抑制剂塞来昔布则显著降低了 LPS 诱导的 PGE（2）（IC（50）= 23 nM）的产生。卡托普利（IC（50）= 48μM）和富马酸酮替芬（IC（50）= 36.4μM）分别对 A23187 刺激的 LTB（4）和组胺水平表现出有效的抑制作用。阿司匹林（IC（50）= 5.5μM）和塞来昔布（IC（50）= 7.9 nM）均表现出浓度依赖性的 TXB（2）产生减少。两次实验中所有细胞测定的结果均显示 Z'因子变化范围为 0.30 至 0.99；S / B 比值范围为 2.39 至 24.92；%CV 范围为 1.52 至 20.14。
结论
结果表明，这些基于细胞的测定法可以作为筛选新型抗炎/抗过敏化合物的理想工具。
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