甘氨酸延伸型胃泌素受体在结肠和结直肠肿瘤中的研究

Study on the Receptor for Glycine-extended Gastrin on Colon and Colorectal Neoplasms

目的：建立甘氨酸延伸型胃泌素胞浆膜受体结合试验，并应用该方法检测甘氨酸延伸型胃泌素受体的部分特 性及其在正常结肠和人结直肠肿瘤中的表达。方法：用人结直肠肿瘤细胞株ＤＬＤ－１建立胞浆膜制备方法，１２５Ｉ标记 的甘氨酸延伸型胃泌素进行受体结合试验。结果：胞浆膜受体结合试验的条件为： ５～１０ ｍｇ胞浆膜（离心２５ ０００ ｒ／ ｍｉｎ ４５ ｍｉｎ制备），３７ ℃温育６０ ｍｉｎ。正常Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠结肠粘膜和从结肠粘膜制备的胞浆膜有特异结合， 而从完整结肠制备的胞浆膜无特异结合。胞浆膜甘氨酸延伸型胃泌素受体抑制５０％结合的非标记配体浓度（ＩＣ５０）为 ３．６４ μｍｏｌ／Ｌ±１．９３μｍｏｌ／Ｌ；特异性胆囊收缩素（ＣＣＫ）－Ａ受体拮抗剂Ｌ３６４和特异性ＣＣＫＢ受体拮抗剂Ｌ３６５不能 拮抗该受体；非特异性受体拮抗剂氯苯酰色氨酸则能拮抗。结论：甘氨酸延伸型胃泌素受体位于正常大鼠结肠粘 膜、结肠粘膜和结直肠肿瘤细胞膜上，属单结合位点、低亲和力受体，不能被特异性ＣＣＫ－Ａ或ＣＣＫ－Ｂ受体拮抗剂拮 抗，是一种新受体。

Background/Aims: To set up a binding assay of glycine-extended gastrin with plasma membranes and examine some of the characteristics of its receptor on colorectal tumor cell line DLD-1 and detect the expression of the receptor on normal colon and colorectal tumors. Methods: To prepare the plasma membranes from DLD-1 cells and label glycine-extended gastrin with 125I, then set up the binding assay with plasma membranes. Results: The conditions for the binding assay of plasma membranes were 5-10 mg protein (centrifugation of 25 000 r/min 45 min) incubated at 37℃ for 60 minutes. The membranes prepared from normal rats' colon showed no specific binding, while the colonic crypts of normal rats and the membranes so prepared showed specific binding. The receptor for glycine-extended gastrin (IC50= 3.64 μmol/LI± 1.93 μmol/L) was not inhibited by specific cholecystokinin (CCK)-A antagonist L364 or specific CCK-B antagonist L365, but inhibited by nonspecific antagonist benzotript. Conclusions: The receptor for glycine-extended gastrin is located at the mucosa of normal rats' colon and plasma membranes of normal colonic mucosa or colorectal tumors. It is a low affinity, on-site receptor which is different from CCK-A/CCK-B.