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目的:构建人源血栓素A2受体(Thromboxane A2 receptor,TXR)Label-free细胞检测体系,并采用以检测中药注射剂舒血宁注射液及其活性组分对该受体的作用。方法:构建TXRα、TXRβ表达质粒,及稳定表达TXRα或TXRβ的HEK293细胞系;通过Label-free细胞检测方法,使用TXR特异性激动剂U46619及拮抗剂SQ-29548对TXRα-HEK293和TXRβ-HEK293稳定转染细胞系进行功能鉴定,并利用该体系辨识舒血宁注射液及其总黄酮、总内酯组分对TXRα和TXRβ的作用。结果:基因测序结果表明,成功构建TXRα、TXRβ表达质粒;PCR结果表明,成功建立了TXRα-HEK293和TXRβ-HEK293稳定转染细胞系;Label-free细胞检测结果显示U46619和SQ-29548能够分别特异性激活和拮抗受体,表明TXRα和TXRβLabel-free细胞检测体系构建成功;Label-free细胞检测结果表明舒血宁注射液能够抑制TXRβ,对TXRα无影响。结论:成功建立TXRα和TXRβLabel-free细胞检测体系,并发现舒血宁注射液能够选择性作用于TXRβ,提示舒血宁注射液可通过抑制TXRβ发挥其药理活性。
OBJECTIVE: To construct a Label-free cell detection system of human Thromboxane A2 receptor (TXR) and to detect the effect of injection of Shuxuening injection and its active components on this receptor. METHODS: The TXRα, TXRβ expression plasmids and HEK293 cell lines stably expressing TXRα or TXRβ were constructed. The TXRα-HEK293 and TXRβ-HEK293 cells were stably transfected with the TXR-specific agonist U46619 and the antagonist SQ-29548 by Label-free cell assay Transfected cell lines for functional identification, and the use of the system to identify Shuxuening injection and its total flavonoids, total lactone components of TXRα and TXRβ role. Results: The gene sequencing results showed that TXRα and TXRβ expression plasmids were constructed successfully. PCR results showed that TXRα-HEK293 and TXRβ-HEK293 stably transfected cell lines were successfully established. The results of Label-free cell assay showed that U46619 and SQ-29548 were specific The results of Label-free cells showed that Shuxuening injection can inhibit TXRβ and has no effect on TXRα. Conclusion: The detection system of TXRα and TXRβLabel-free cells was successfully established. Shuxuening injection can selectively act on TXRβ, suggesting that Shuxuening injection can exert its pharmacological activity by inhibiting TXRβ.