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目的:在Sf9昆虫细胞中表达D_1受体,并研究左旋氯代斯阔任对重组D_1受体的激动作用。方法:构建含D_1受体cDNA的重组杆状病毒,以其感染Sf9昆虫细胞得到D_1受体表达。[~3H]SCH23390受体结合检测重组D_1受体的药理特性。[~3H]SCH23390受体结合和cAMP测定实验检测左旋氯代斯阔任对重组D_1受体的激动作用。结果:在Sf9昆虫细胞中成功表达D_1受体,[~3H]SCH23390与重组D_1受体最大结合量(B_(max))为(0.94±0.06)nmol/g蛋白,[~3H]SCH23390与重组D_1受体的结合解离常数(K_d)为(1.9±0.3)nmol/L,其药理特性与小牛纹状体脑匀浆所得结果一致。左旋氯代斯阔任对重组D_1受体有高亲和力,解离常数K_i为(6.3±1.4)nmol/L;并剂量依赖地引起胞内cAMP增加,EC_(50)为0.72μmol/L(95％可信限为0.67-0.77μmol/L),表现出D_1激动作用。结论:在杆状病毒/昆虫细胞Sf9中,成功建立了D_1受体异源表达系统。在细胞分子水平,直接证实了左旋氯代斯阔任对D_1受体的激动作用。
OBJECTIVE: To express D_1 receptor in Sf9 insect cells and to study the agonism of L-chloroceracecin on recombinant D_1 receptor. Methods: Recombinant baculovirus containing D_1 receptor cDNA was constructed and infected with Sf9 insect cells to obtain D_1 receptor expression. [~ 3H] SCH23390 Receptor Binding to Detect the Pharmacological Characteristics of Recombinant D_1 Receptors. [~ 3H] SCH23390 receptor binding and cAMP assay was used to test the agitation effect of L-SST on recombinant D_1 receptor. Results: The D_1 receptor was successfully expressed in Sf9 insect cells. The maximal binding capacity of [~ 3H] SCH23390 to recombinant D_1 receptor was (0.94 ± 0.06) nmol / g protein. [~ 3H] SCH23390 and recombinant The binding dissociation constant (K_d) of D_1 receptor was (1.9 ± 0.3) nmol / L, and its pharmacological properties were consistent with those obtained from brains of equine striatum. L-Chlorothiazolone has a high affinity to recombinant D_1 receptor with a K_i dissociation constant of (6.3 ± 1.4) nmol / L and a dose-dependent increase of intracellular cAMP with EC50 of 0.72 μmol / L % Confidence limits 0.67-0.77μmol / L), showing D_1 agonism. Conclusion: The D_1 receptor heterologous expression system was successfully established in baculovirus / insect cell Sf9. At the cellular and molecular level, L-chlorothyrotropin directly confirms the agonism of D_1 receptor.