新型α-黑素细胞刺激素类似物亲和力和生物学效应的测定

Detection of binding activity and biologic effect of a novel α-melanocyte-stimulating hormone analogue

检测新型α-黑素细胞刺激素(α-MSH)类似物与受体的亲和力和生物学效应。构建黑皮质激素受体表达质粒,经测序鉴定后以磷酸钙-DNA共沉淀法转染HEK-293细胞,48h后加入含900μg.mL-1G418的细胞维持液,稳定表达后以流式细胞仪检测。采用放射性配体结合分析测定新型α-MSH类似物对受体的亲和力,并以[3H]环磷酸腺苷(cAMP)测定盒检测新型α-MSH类似物作用细胞后的cAMP水平。结果显示,新型α-MSH类似物对MC1R,MC3R,MC4R及MC5R的抑制常数Ki分别为(0.159±0.040),(35.430±6.743),(19.293±2.780)和(2.230±0.670)nmol.L-1。其对MC1R,MC3R,MC4R及MC5R的EC50值分别为(0.45±0.07),(7.80±0.65),(2.55±0.23)和(0.33±0.09)nmol.L-1。新型α-MSH类似物是MC1R和MC5R高选择性的激动剂。

Binding activity and biologic effect of a novel α-melanocyte-stimulating hormone analogue were tested on cells transiently expressing the human melanocortin-1 （MC1）, MC3, MC4, and MC5 receptors. The human MC1 and MC5 receptor genes were cloned into the expression vector pcDNA3. 1/ myc-his（ - ）B. The vectors were transferred to HEK-293 cells by the calcium phosphate method. Stable receptor populations were generated using G418 selection （900 μg · mL^-1） for subsequent bioassay analysis. Ki values of the novel α-MSH analogue for MC1 , MC3, MC4, and MC5 receptors were obtained in competition with [ ^125I]-NDP-MSH for binding studies. The cyclic AMP level was tested by using [ ^3H] - cyclic AMP kit. It is showed that Ki values of the novel α-MSH analogue for MC1, MC3, MC4, and MC5 receptors were （0. 159 ± 0.040）, （35.430 ± 6. 743）, （19.293 ± 2. 780） and （2.230 ± 0.670） nmol · L^-1 , respectively. Its EC50 values for MC1 , MC3, MC4, and MC5 receptors were （0.45 ±0.07） , （7.80±0.65）, （2.55 ±0.23） and （0.33 ±0.09） nmol · L^-1, respectively. In these tests, the novel α-MSH analogue is a MC1R and MC5R selective agonist.