鸡卵泡细胞GnRH受体定位和定量分析

QUALITATIVE AND QUANTITATIVE ASSAY OF GnRH RECEPTORS IN CHICKEN FOLLICULAR CELLS

本实验采用免疫组织化学ＰＡＰ法对体外培养的鸡卵泡细胞进行ＧｎＲＨ受体定位检查，证明了卵泡颗粒细胞（ＧＣ）和膜细胞（ＴＣ）上均存在能与鸡ＧｎＲＨ结合的阳性物质。应用放射配体结合法对ＧＣ和ＴＣ的ＧｎＲＨ受体定量分析，获得如下结果：１２５ⅠＧｎＲＨⅡ与ＧＣ或ＴＣ结合率随未标记配体的增加而减少，表现出专一的竞争抑制结合；ＧＣ的ＧｎＲＨ受体亲和常数ＫＡ＝１１１×１０８Ｍ－１，结合位点为４９ｆｍｏｌ／ｍｇ蛋白；ＴＣ的ＧｎＲＨ受体ＫＡ＝１１３×１０８Ｍ－１，结合位点为５３ｆｍｏｌ／ｍｇ蛋白。鸡的两型ＧｎＲＨ（Ⅰ型和Ⅱ型）与受体竞争结合有较强的交叉结合反应（交叉反应率为８８４％）。ＧｎＲＨ类似物ＬＲＨＡ３与ＧＣ的ＧｎＲＨ受体竞争结合反应率很低，几乎无结合活性。

Quality of GnRH receptors in cultured follicular cells from chicken were examined by immunocell chemical method(peroxidose antiperoxidose complex method).It was demonstrated that the positive immunostaining substance existed at the granular cells(GC) and theca(TC).Using radioligand receptor assay,the quantity analysis of GnRH receptors in the GC and TC were determined.The results indicated that (1)binding of 125 Ⅰ GnRH Ⅱ to GC or TC was progressively reduced with increasing concentration of unlabelled ligand.It showed a specific competitive inhibitory binding.The binding affinity(Ka)in GC and TC was determined to be 1 11×10 8M -1 and 1 13×10 8M -1 respectively;The binding capacity(equivalent to receptor sits)was 49fmol/mg and 53fmol/mg.(2)The cross reaction were significant in GnRH Ⅰ and GnRH Ⅱ(the ratio of cross reactions 88 4%).(3)The specificity binding of LRH A 3 to GnRH receptor in GC was very low in the assay.