冻干重组人促黄体激素释放激素-绿脓杆菌外毒素A融合蛋白与不同细胞受体结合竞争试验

The Assay of LHRH-PE40 Binding and competition to Some Species Cells Receptor

目的为了验证重组人促黄体激素释放激素-绿脓杆菌外毒素A融合蛋白(LHRH-PE40)对癌细胞的靶向性,对LHRH-PE40进行125I标记,并进行了与多种细胞受体结合试验和竞争试验。为该药物的导向治疗提供可靠的依据。方法利用体外细胞培养方法将人胃癌细胞系BGC-823,人结肠癌细胞系Lovo,人T细胞淋巴瘤Hut102,人白血病细胞系Jurket,狗肾细胞系DK 5种细胞系,分别大量培养至单层,收获细胞,破碎,制备细胞膜。同时制备鼠肺组织的细胞膜进行试验。利用125I标记的LHRH-PE40与不同细胞膜进行放射性配基结合分析,以及与LHRH竞争结合分析。确定这些细胞表面有无LHRH受体,并用Scatchard作图方法计算出其亲和常数及最大结合量。结果小鼠肺组织细胞膜、人T细胞淋巴瘤Hut102、人白血病细胞系Jurket、狗肾细胞系DK未见配基-受体特异性结合及竞争结果;而人胃癌细胞系BGC-823,人结肠癌细胞系Lovo与LHRH-PE40的结合竞争符合特异性配基-受体结合、竞争特性。其中LHRH-PE40与Lovo细胞的Kd=10.6±2.33 nmol/L,Bmax=345±7.59 pmol/mg;与BGC-823细胞的Kd=37.82±1.42nmol/L,Bmax=475±17.86 pmol/mg。结论受试的6种细胞膜蛋白中正常细胞(鼠肺、狗肾)和两种肿瘤细胞(Jurket、Hut102)表面无LHRH受体表达。而结肠癌和胃癌肿瘤细胞表面存在LHRH受体的表达。另外本试验还证实了重组毒素LHRH-PE40保留了天然LHRH与其受体结合的高亲和力。

Objective To determine the recombinant human luteinizing hormone releasing hormone pseudomonas aeruginosa exotoxin40（LHRH-PE40）can be specific identified by the LHRH receptor of the tumor cellular membrane, so radioactive ligand of LHRH-PE40 marked by^125I,and using the radioactivity ligand receptor assay method to test the some tumor cells and normal cells. Methods The gastric carcinoma BGC-823,colon carcinoma Lovo,T lymphoma Hut102,leukemia Jurket and dog kidney DK Cultured by monolayer, the cells acquired and crushed by homogenizer and prepare the cellular membrane,and prepare mouse lung fissue＇s cell membrane for test,The different cells membrane carries on the radioactive ligand binding assay to determine whether have the LHRH receptor at these cells membrane.Results The mouse lung tissue, T lymphoma Hut102,leukemia Jurket and dog kidney DK no lig- and-receptor specific binding and competition reaction, but the gastric carcinoma BGC-823,colon carcinoma Lovo cellular membrane reactive with the^125I- LHRH-PE40 have the specific binding and competition, the affmity constant of LHRH-PE40 with Lovo cellular membrane was Kd= 10.6 ＋ 2.33 nmol/L,the maximum specific binding amount was Bmax = 345 ＋ 7.59 pmol/mg;Ihe gastric carcinoma BGG823 cellular membrane affinity eanstant of LHRH-PE40 was Kd = 37.82 ± 1.42 nmol/L, the maximum specific binding amount was Bmax = 475 ± 17.86 pmol/mg. Conclusion The tested normal tissues or cells （meuse lung and dog kidney） and tumor cells（Jurket,Hut102）no expression the receptor of the LHRH,But colon and gastric carcinoma cell have the receptor of the LHRH.