α_(1D)受体cDNA转染细胞、大鼠主动脉组织及其培养细胞的细胞膜色谱研究

Comparative Studies on Cell Membrane Chromatography with Rat Aorta Tissue,Cultured Rat Aorta Smooth Muscle Cell and α_(1D) Subtypes Over Expressed Cell

目的为了增加细胞膜色谱法的选择性和特异性,用细胞膜色谱法分别研究不同浓度药物BMY7378(α1D-AR亚型高选择性药物)与大鼠主动脉组织、原代培养大鼠主动脉平滑肌细胞和α1D-AR转染细胞膜色谱柱的生物亲和作用。方法根据大鼠主动脉细胞表达以α1D-AR占优势,分别构建大鼠主动脉组织匀浆细胞膜色谱柱、培养大鼠主动脉平滑肌细胞膜色谱柱和受体质粒转染所得受体高表达HEK293α1D细胞膜色谱柱。用细胞膜色谱法研究6种浓度的BMY7378与受体的亲和色谱特性,分别测定其容量因子并进行相关性分析。结果在大鼠主动脉组织制备的细胞膜固定相(cell membranestationary phase,CMSP)上,最小检测到的BMY7378浓度为1.00 mmol·L-1;在大鼠主动脉平滑肌细胞CMSP上,最小检测到的BMY7378浓度为0.03 mmol·L-1;在α1D-AR细胞CMSP上,最小检测到的BMY7378浓度为0.10 mmol·L-1。结论与组织器官制备的CMSP的传统细胞膜色谱法比较,培养的细胞或细胞株色谱系统具有更高的灵敏度。可扩大细胞膜色谱法的应用范围,试用于受体亚型选择性药物的高效筛选。

OBJECTIVE To invetigate the chromatography affinities of subtype-selective α1D- adrenergic receptor（AR） antagonist （BMY7378） to rat aorta tissue cells, primary cultured rat aorta smooth muscle cell and α1D- AR subtype for improving the selectivity and specificity of CMC. METHODS According to predominant α1D-AR express in rat aorta, the homogenized rat aorta tissue cells chromatographic column, primary, cultured rat aorta smooth muscle cell chromatographic column and α1D-AR subtypes over-expressed in the human embryonic kidney （HEK293） cell lines chromatographic column were designed and constructed. Six different concentrations of the BMY7378 were available in CMC experiments. Capacity factors （k＇）of different concentrations of BMY7378 , as a chromatographic parameter, were investigated and calculated. RESULTS With the preparation of CMSP of rat aorta tissue, the lowest concentration of BMY7378 was 1.00 mmol· L ^-1. With CMSP preparing of primary cultured rat aorta smooth muscle cell, the lowest concentration of BMY7378 was 0. 03 mmol· L ^-1 The lowest concentration of BMY7378 with α1D-AR subtype obtained from CMC was 0. 10 mmol · L ^-1. CONCLUSION Compared with traditional CMC method, the new method will further improve sensitivity and specificity of CMC and can be used to screen receptor-subtype drugs.