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目的:观察大鼠肺间质巨噬细胞(PIMs)胆囊收缩素(CCK)受体的表达亚型和结合特性。方法:用酶消化法结合肺泡耗竭灌洗和肺循环灌洗技术分离纯化大鼠PIMs,超速离心法提取细胞膜,与[~3H]标记的硫酸化CCK-8([~3H]-CCK-8S)进行放射配基结合实验,用非标记的CCK-8S、CCK-A受体(CCK-AR)特异性拮抗剂CR1409及CCK-B受体(CCK-BR)特异性拮抗剂CR2945进行竞争抑制实验,观察配体受体结合的特异性及CCK受体表达亚型,观察孵育时间和温度对特异性结合的影响。结果:正常大鼠PIMs未能检出特异性结合,静脉注射脂多糖(LPS)48h出现特异性结合,且对孵育时间与温度有依赖性。经Scatchard分析,平衡解离常数(Kd)值为:(0.68±0.28)nmol·L~(-1),最大结合容量(Bmax)值为(32.50±2.70)pmol·g~(-1)蛋白。通过竞争抑制实验,[~3H]-CCK-8S与膜的结合可被CCK-8S、CR1409、CR2945所抑制,其IC_(50)值分别为:(3.20±1.13)nmol·L~(-1),(0.19±0.06)μmol·L~(-1)和(2.30±0.80)nmol·L~(-1)。结论:大鼠PIMs存在CCK-A和CCK-B两种受体亚型,为CCK对生理及病理条件下巨噬细胞发挥效应提供了直接的结构依据。
Objective: To observe the expression subtypes and binding characteristics of cholecystokinin (CCK) receptors in rat pulmonary interstitial macrophages (PIMs). Methods: PIMs were isolated and purified by enzymatic digestion combined with alveolar depletion lavage and pulmonary circulation lavage. Cell membrane was extracted by ultracentrifugation and incubated with [~ 3H] labeled sulfated CCK-8 ([~ 3H] -CCK-8S) Radioligand binding assay was performed using a non-labeled CCK-8S, a CCK-A receptor antagonist CR1409 and a CCK-B receptor antagonist CR2945 , Observe the specificity of ligand receptor binding and CCK receptor expression subtypes, observe the incubation time and temperature on the specific binding. Results: The specific binding of PIMs was not detected in normal rats. Specific binding was observed 48h after intravenous injection of lipopolysaccharide (LPS), and the incubation time was temperature-dependent. The results of Scatchard analysis showed that the equilibrium dissociation constant (Kd) was (0.68 ± 0.28) nmol·L -1 and the maximum binding capacity (Bmax) was (32.50 ± 2.70) pmol · g -1 . The inhibitory effect of [~ 3H] -CCK-8S on the membrane was inhibited by CCK-8S, CR1409 and CR2945, and the IC 50 values were (3.20 ± 1.13) nmol·L -1 ), (0.19 ± 0.06) μmol·L -1 and (2.30 ± 0.80) nmol·L -1, respectively. Conclusion: CCK-A and CCK-B receptor subtypes exist in rat PIMs, which provide a direct structural basis for the effect of CCK on macrophages in physiological and pathological conditions.