Analysis of ACTH receptor in adrenal tumor and tumor-like lesion.

我们从ACTH受体蛋白中选择了15个氨基酸序列。将所合成的多肽作为载体蛋白偶联到KLH上，免疫兔。在1：25,600倍稀释度时，酶联免疫吸附试验显示该肽呈阳性反应。我们检测了人肾上腺组织石蜡切片上的免疫组织化学反应。其中一种抗体与肾上腺皮质细胞的细胞膜发生反应，Western印迹分析显示一条约60 kDa的条带。两种阳性反应均被免疫多肽预吸收所消除。对肾上腺皮质癌、腺瘤和增生症的石蜡切片进行免疫组织化学染色。PCNA和Ki-67在癌组织中的阳性反应性分别为3.43~20.08和0~20.9，在腺瘤中分别为0.14~1.33和0~1.03，在增生性疾病中分别为0.19~0.7和0~0.05。肿瘤的生物学恶性程度与增殖活性显著相关。ACTHR在癌组织中的表达低于…腺瘤和增生症患者较多。P53在3例癌组织中呈阳性表达，在腺瘤和增生性病变中均为阴性。肿瘤的发生与P53的关系已被提出，但其他因素仍有待进一步研究。RAS均为阴性。Myc抗体(9E10)可与正常肾上腺皮质细胞的核、增生和腺瘤的核及部分胞浆反应，在癌中以胞浆为主。斑点杂交检测到10pg。在人肾上腺组织石蜡切片上，用TT二聚体探针进行原位杂交(ISH)，经甲基绿派罗宁染色和28S rRNA原位杂交证实RNA保存。为了获得更高的反应性，我们尝试了两种不同的寡核苷酸，并用地高氧素探针进行了ISH。它们的结果与TT二聚化探针相似。肿瘤性病变尚未表现出特异性反应。较少

We selected 15 amino acid sequences from the ACTH receptor protein. The symtthesized peptides were coupled to KLH as carrier protein and was immunized to rabbits. ELISA showed positive reaction to the peptide at the dilution of 1 : 25,600. We evaluated the immunohistochemical reactivity on the paraffin sections of human adrenal tissue. One of the antibody reacted with the cell membrane of adrenocortical cells and the Western blot analysis revealed one band at approximately 60 kDa. Both positive reactions were abolished by preabsorption with the immunizing peptide.Immunohistochemical reactivity was evaluated on the paraffin sections of the adrenocortical carcinoma, adenoma and hyperplasia. The reactivity for the PCNA and Ki-67 were 3.43-20.08 and 0-20.9 in carcinoma, 0.14-1.33 and 0-1.03 in adenoma, 0.19-0.7 and 0-0.05 in hyperplasia, respectively. The correlation of the biological malignant grading and proliferative activity was significant. ACTHR expression in carcinoma was lower than … More those in adenoma and hyperplasia. p53 was positive in three of carcinoma and negative in all of adenoma and hyperplasia. The relation between the carcinogenesis and p53 was suggested, however, the other factors are still to be considered. Ras was negative in all cases. The myc antibody (9E10) reacted with nuclei of normal adrenal cortical cells, nuclei and some cytoplasm in hyperplasia and adenoma, and predominantly cytoplasm in carcinoma.We synthesized the oligo-DNA of 60mer with a selected sequence from the ACTHR DNA with additional 5 repeater of ATT sequence. Dot blot hybridization detected l0pg. We examined the in situ hybridization (ISH) using TT dimerized probe, on the paraffin section of the human adrenal tissue that was confirmed RNA preservation by methylgreen-pyronin stain and ISH of 28s rRNA.Compact cells in zona glomerulosa and zona fasciculata showed weak positivity. For more reactivity, we tried 2 different oligo-DNAs by ISH with digoxygenin-probe. They showed similar result to TT dimerized probe. Neoplastic lesion have not showed specific reactivity yet. Less