CGRP，SP，NPY在肾上腺疾病组织中表达特点及临床意义
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摘要
目的:为了探讨CGRP(Calcitoni rgene related peptide,降钙素基因相关肽)、SP(substance P,P物质)、NPY(Neurpeptide Y,神经肽Y)在肾上腺疾病发生过程中的所起的作用及临床意义,用免疫组化的方法,通过对肾上腺皮质增生、皮质腺瘤、髓质增生、嗜铬细胞瘤和正常肾上腺等不同组织,进行CGRP、SP、NPY免疫组化染色,分别观察其在肾上腺疾病组织中的分布特点。
方法:选取CGRP、SP、NPY作为标记物,运用免疫组化方法对广州军区总医院1980年12月～2006年12月提供的常规石蜡包埋组织块,其中肾上腺皮质增生、皮质腺瘤、髓质增生、嗜铬细胞瘤和正常肾上腺各15例,分别进行CGRP、SP、NPY染色。新鲜标木经10%福尔马林固定18-24h,常规石蜡包埋,连续切片厚5μm,1张作HE染色,其余分别作NPY、CGRP、SP免疫组化染色。正常肾上腺作为阳性对照,PBS代替一抗作为阴性对照。阳性物均位于胞浆内,呈棕黄色。判断标准:阳性细胞面积占细胞总面积的百分比。比较肾上腺皮质增生、皮质腺瘤、髓质增生、嗜铬细胞瘤和正常肾上腺等不同病例皮质和髓质中NPY、CGRP、SP染色阳性细胞占细胞总数百分比之间的差异。
统计数据用SPSS13.0处理,CGRP、SP、NPY阳性细胞面积数据行完全随机设计方差分析,CGRP、SP、NPY阳性细胞面积与患者入院血压行直线相关分析,检验水准均为α=0.05。
结果:CGRP、SP在正常肾上腺髓质、肾上腺髓质增生、嗜铬细胞瘤表达呈阴性,而在正常肾上腺皮质、肾上腺皮质增生、肾上腺皮质腺瘤均有不同程度的表达肾上腺皮质腺瘤、皮质增生、正常肾上腺皮质中均可见CGRP、SP染色阳性细胞,三者CGRP、SP染色面积有显著差异(P=0.002,P=0.006),其中肾上腺皮质腺瘤阳性细胞面积显著高于皮质增生、正常肾上腺皮质,肾上腺皮质增生表达高于正常肾上腺皮质;并且CGRP、SP阳性细胞面积与肾上腺皮质腺瘤、肾上腺皮质增生患者的血压存在正相关关系(相关系数分别为r=0.801,P=0.010;r=0.686,P=0.021;r=0.811,P=0.022;r=0.523,P=0.013)。NPY在正常肾上腺皮质、肾上腺皮质增生、肾上腺皮质腺瘤表达呈阴性,肾上腺髓质增生、肾上腺嗜铬细胞瘤和正常肾上腺髓质中均可见NPY染色阳性细胞,三者NPY染色面积有显著差异(P=0.018),其中肾上腺嗜铬细胞瘤阳性细胞面积显著高于肾上腺髓质增生和正常肾上腺髓质,肾上腺髓质增生表达高于正常肾上腺髓质;并且NPY阳性细胞面积与肾上腺嗜铬细胞瘤、肾上腺髓质增生患者的血压存在正相关关系(相关系数分别为r=0.856,P=0.031;r=0.792,P=0.009)。
结论:
1.CGRP、SP与肾上腺皮质疾病患者高血压有密切相关,对肾上腺皮质疾病激素的分泌可能起到到促进作用。NPY与肾上腺嗜铬细胞瘤、肾上腺髓质增生患者的血压密切相关,可能促进肾上腺髓质的激素分泌。
2.CGRP、SP和NPY在肾上腺疾病的发生发展中可能具有重要意义,由正常肾上腺组织向肾上腺增生和肾上腺腺瘤或肾上腺嗜铬细胞瘤的发展过程中CGRP、SP与NPY经历了由低表达到高表达的过程。这可能是治疗和诊断肾上腺疾病新的靶点。
3.以往的文献报道已经证明NPY由肾上腺嗜铬细胞生成分泌,并且NPY介导了直接的血管收缩作用;而我们的实验也已证明NPY在肾上腺嗜铬细胞瘤、肾上腺髓质增生疾病中是高表达的,所以我们推测NPY可能是肾上腺嗜铬细胞瘤、肾上腺髓质增生疾病中,除了NA(noradrenaline)和AD(adnephrin)之外,分泌的又一外周缩血管物质。
OBJECTIVE:For reveal the the roles and clinical significance of Neurpeptide Y (NPY),substance P(SP) and Calcitoni rgene related peptide(CGRP) in the in adrenal gland,we study the distribution of NPY,CGRP,SP in adrenal gland by immunohistochemistry and statistics percentage of colour positive cell to compare difference among adrenocortical adenoma,adrenocortical hyperplasia, pheochromocytoma,adrenal medullary hyperplasia and normal adrenal.
METHODS:
To collect 75 conventional paraffin wax tissues of adrenocortical adenoma, adrenocortical hyperplasia,pheochromocytoma,adrenal medullary hyperplasia and normal adrenal from our hospital from December 1980 toDecember 2006 and normal adrenal corned from corpse.Every group is 15 cases.Immunohistochemical technique was applied to detect the expression of NPY,CGRP and SP in routinely processed tissue specimens from human adrenal neoplasm and hyperplasia.All tissues were fixed by formalin,its concentration is 10 percentage.Fixed time is between eighteen hours and twenty-four hours.Specimens were embedded by ozocerite and chip to Sum thickness.One specimen was HE stain,another was NPY, CGRP,SP immunoreactivity.Normal adrenal was positive contrast and PBS replaced first antibody was negative contrast.Positive matter was found in cytolymph and its colour was brown.Judgement standard is percentage of positive cell.The destination was to study the characteristic of NPY,CGRP and SP in adrenal by immunohistochemistry and statistics percentage of colouer positive cell to compare difference to adrenocortical adenoma adrenocortical hyperplasia, pheochromocytoma,adrenal medullary hyperplasia and normal adrenal.
RESULTS:
CGRP,SP immunoreactivity was regularly seen in adrenocortical adenoma, adrenocorticohyperplasia and normal adrenal cortex and there was a significant difference among the three(P=0.002,P=0.006),but neither in the pheochromocytoma,adrenal medullary hyperplasia nor in the normal adrenal medulla.CGRP,SP expression was higher in adrenocortical adenoma tissue than that of adrenocorticohyperplasia and normal cortex,the same with SP.Besides,higher CGRP,SP expression were present in adrenocorticohyperplasia group than those of normal adrenal cortex group.There also was a positive correlation between CGRP,SP positive cell and blood pressure in adrenocortical adenoma and adrenocorticohyperplasia(r=0.801,P=0.010;r=0.686,P=0.021;r=0.811,P=0.022; r=0.523,P=0.013).
NPY positive cell were showed in pheochromocytoma,adrenal medullary hyperplasia and normal adrenal medulla and there was a significant difference among the three(P=0.018),but not both in the adrenocortical adenoma, adrenocorticohyperplasia and normal adrenal cortex.NPY expression was higher in pheochromocytoma tissue than that of adrenal medullary hyperplasia and adrenal medulla.Besides,higher NPY expression were present in adrenal medullary hyperplasia group than those of normal adrenal medulla group.There also was a positive correlation between NPY positive cell and blood pressure in adrenal medullary hyperplasia and adrenal Pheochromocytoma(r=0.856,P=0.031;r=0.792, P=0.009).
CONCLUSIONS:
1.Maybe CGRP and SP correspond to induces adrenal hormone release Besides,there was a positive correlation between CGRP,SP positive cell and blood pressure in adrenocortical adenoma and adrenocorticohyperplasia.The same with NPY for pheochromocytoma and adrenal medullary hyperplasia.
2.CGRP,SP and NPY may play an important role in the and the development of adrenal disease.The expression of CGRP,SP and NPY change from low to high in the process from normal adranal to adrenal hyperplasia and adrenal tumor.CGRP,SP and NPY are functional and may represent the molecular basis of adrenal Pheochromocytoma,as well as a potential new target for the diagnosis and therapy of adrenal disease.
3.Previous literature has proved that NPY secrete from adrenal chromaffin cell and our experiment also has proved NPY high expression in adrenal medulla disease,so, we think that maybe NPY is the another vasoconstriction substance except norepinephrine and adnephrin,which pheochromocytoma or adrenal medullary hyperplasia secreted.
方法:选取CGRP、SP、NPY作为标记物,运用免疫组化方法对广州军区总医院1980年12月～2006年12月提供的常规石蜡包埋组织块,其中肾上腺皮质增生、皮质腺瘤、髓质增生、嗜铬细胞瘤和正常肾上腺各15例,分别进行CGRP、SP、NPY染色。新鲜标木经10%福尔马林固定18-24h,常规石蜡包埋,连续切片厚5μm,1张作HE染色,其余分别作NPY、CGRP、SP免疫组化染色。正常肾上腺作为阳性对照,PBS代替一抗作为阴性对照。阳性物均位于胞浆内,呈棕黄色。判断标准:阳性细胞面积占细胞总面积的百分比。比较肾上腺皮质增生、皮质腺瘤、髓质增生、嗜铬细胞瘤和正常肾上腺等不同病例皮质和髓质中NPY、CGRP、SP染色阳性细胞占细胞总数百分比之间的差异。
统计数据用SPSS13.0处理,CGRP、SP、NPY阳性细胞面积数据行完全随机设计方差分析,CGRP、SP、NPY阳性细胞面积与患者入院血压行直线相关分析,检验水准均为α=0.05。
结果:CGRP、SP在正常肾上腺髓质、肾上腺髓质增生、嗜铬细胞瘤表达呈阴性,而在正常肾上腺皮质、肾上腺皮质增生、肾上腺皮质腺瘤均有不同程度的表达肾上腺皮质腺瘤、皮质增生、正常肾上腺皮质中均可见CGRP、SP染色阳性细胞,三者CGRP、SP染色面积有显著差异(P=0.002,P=0.006),其中肾上腺皮质腺瘤阳性细胞面积显著高于皮质增生、正常肾上腺皮质,肾上腺皮质增生表达高于正常肾上腺皮质;并且CGRP、SP阳性细胞面积与肾上腺皮质腺瘤、肾上腺皮质增生患者的血压存在正相关关系(相关系数分别为r=0.801,P=0.010;r=0.686,P=0.021;r=0.811,P=0.022;r=0.523,P=0.013)。NPY在正常肾上腺皮质、肾上腺皮质增生、肾上腺皮质腺瘤表达呈阴性,肾上腺髓质增生、肾上腺嗜铬细胞瘤和正常肾上腺髓质中均可见NPY染色阳性细胞,三者NPY染色面积有显著差异(P=0.018),其中肾上腺嗜铬细胞瘤阳性细胞面积显著高于肾上腺髓质增生和正常肾上腺髓质,肾上腺髓质增生表达高于正常肾上腺髓质;并且NPY阳性细胞面积与肾上腺嗜铬细胞瘤、肾上腺髓质增生患者的血压存在正相关关系(相关系数分别为r=0.856,P=0.031;r=0.792,P=0.009)。
结论:
1.CGRP、SP与肾上腺皮质疾病患者高血压有密切相关,对肾上腺皮质疾病激素的分泌可能起到到促进作用。NPY与肾上腺嗜铬细胞瘤、肾上腺髓质增生患者的血压密切相关,可能促进肾上腺髓质的激素分泌。
2.CGRP、SP和NPY在肾上腺疾病的发生发展中可能具有重要意义,由正常肾上腺组织向肾上腺增生和肾上腺腺瘤或肾上腺嗜铬细胞瘤的发展过程中CGRP、SP与NPY经历了由低表达到高表达的过程。这可能是治疗和诊断肾上腺疾病新的靶点。
3.以往的文献报道已经证明NPY由肾上腺嗜铬细胞生成分泌,并且NPY介导了直接的血管收缩作用;而我们的实验也已证明NPY在肾上腺嗜铬细胞瘤、肾上腺髓质增生疾病中是高表达的,所以我们推测NPY可能是肾上腺嗜铬细胞瘤、肾上腺髓质增生疾病中,除了NA(noradrenaline)和AD(adnephrin)之外,分泌的又一外周缩血管物质。
OBJECTIVE:For reveal the the roles and clinical significance of Neurpeptide Y (NPY),substance P(SP) and Calcitoni rgene related peptide(CGRP) in the in adrenal gland,we study the distribution of NPY,CGRP,SP in adrenal gland by immunohistochemistry and statistics percentage of colour positive cell to compare difference among adrenocortical adenoma,adrenocortical hyperplasia, pheochromocytoma,adrenal medullary hyperplasia and normal adrenal.
METHODS:
To collect 75 conventional paraffin wax tissues of adrenocortical adenoma, adrenocortical hyperplasia,pheochromocytoma,adrenal medullary hyperplasia and normal adrenal from our hospital from December 1980 toDecember 2006 and normal adrenal corned from corpse.Every group is 15 cases.Immunohistochemical technique was applied to detect the expression of NPY,CGRP and SP in routinely processed tissue specimens from human adrenal neoplasm and hyperplasia.All tissues were fixed by formalin,its concentration is 10 percentage.Fixed time is between eighteen hours and twenty-four hours.Specimens were embedded by ozocerite and chip to Sum thickness.One specimen was HE stain,another was NPY, CGRP,SP immunoreactivity.Normal adrenal was positive contrast and PBS replaced first antibody was negative contrast.Positive matter was found in cytolymph and its colour was brown.Judgement standard is percentage of positive cell.The destination was to study the characteristic of NPY,CGRP and SP in adrenal by immunohistochemistry and statistics percentage of colouer positive cell to compare difference to adrenocortical adenoma adrenocortical hyperplasia, pheochromocytoma,adrenal medullary hyperplasia and normal adrenal.
RESULTS:
CGRP,SP immunoreactivity was regularly seen in adrenocortical adenoma, adrenocorticohyperplasia and normal adrenal cortex and there was a significant difference among the three(P=0.002,P=0.006),but neither in the pheochromocytoma,adrenal medullary hyperplasia nor in the normal adrenal medulla.CGRP,SP expression was higher in adrenocortical adenoma tissue than that of adrenocorticohyperplasia and normal cortex,the same with SP.Besides,higher CGRP,SP expression were present in adrenocorticohyperplasia group than those of normal adrenal cortex group.There also was a positive correlation between CGRP,SP positive cell and blood pressure in adrenocortical adenoma and adrenocorticohyperplasia(r=0.801,P=0.010;r=0.686,P=0.021;r=0.811,P=0.022; r=0.523,P=0.013).
NPY positive cell were showed in pheochromocytoma,adrenal medullary hyperplasia and normal adrenal medulla and there was a significant difference among the three(P=0.018),but not both in the adrenocortical adenoma, adrenocorticohyperplasia and normal adrenal cortex.NPY expression was higher in pheochromocytoma tissue than that of adrenal medullary hyperplasia and adrenal medulla.Besides,higher NPY expression were present in adrenal medullary hyperplasia group than those of normal adrenal medulla group.There also was a positive correlation between NPY positive cell and blood pressure in adrenal medullary hyperplasia and adrenal Pheochromocytoma(r=0.856,P=0.031;r=0.792, P=0.009).
CONCLUSIONS:
1.Maybe CGRP and SP correspond to induces adrenal hormone release Besides,there was a positive correlation between CGRP,SP positive cell and blood pressure in adrenocortical adenoma and adrenocorticohyperplasia.The same with NPY for pheochromocytoma and adrenal medullary hyperplasia.
2.CGRP,SP and NPY may play an important role in the and the development of adrenal disease.The expression of CGRP,SP and NPY change from low to high in the process from normal adranal to adrenal hyperplasia and adrenal tumor.CGRP,SP and NPY are functional and may represent the molecular basis of adrenal Pheochromocytoma,as well as a potential new target for the diagnosis and therapy of adrenal disease.
3.Previous literature has proved that NPY secrete from adrenal chromaffin cell and our experiment also has proved NPY high expression in adrenal medulla disease,so, we think that maybe NPY is the another vasoconstriction substance except norepinephrine and adnephrin,which pheochromocytoma or adrenal medullary hyperplasia secreted.
引文
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[1]Tatemoto K,Carlquist M,Mutt V.Neuropeptide Y a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide[J].Nature,1982,296:659-660.
[2] Renshaw D, Thomson LM, Carroll M, et al. Actions of neuropeptide Y on the rat adrenal cortex[J]. Endocrinology, 2000, 141:169-173.
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[4] Nussdorfer GG, Mazzocchi G, Malendowicz LK. The possible involvement of pancreatic polypeptide in the paracrine regulation of human and rat adrenal cortex[J]. Endocr Res, 1998,24:695-702.
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[6] Claudia Cavadas, Antonio P. Silva, Francois Mosimann, et al.NPY Regulates Catecholamine Secretion from Human Adrenal Chromaffin Cells[J] . The Journal of Clinical Endocrinology &Metabolism ,2001, 86 (12) : 5956-5963.
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[1]Tatemoto K,Carlquist M,Mutt V.Neuropeptide Y a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide[J].Nature,1982,296:659-660.
[2] Renshaw D, Thomson LM, Carroll M, et al. Actions of neuropeptide Y on the rat adrenal cortex[J]. Endocrinology, 2000, 141:169-173.
[3] Ehrhart-Bornstein M, Hinson JP, Bornstein S, et al. Intraadrenal interactions in the regulation of adrenocortical steroidogenesis[J]. Endocr Rev, 1998, 19:101-143.
[4] Nussdorfer GG, Mazzocchi G, Malendowicz LK. The possible involvement of pancreatic polypeptide in the paracrine regulation of human and rat adrenal cortex[J]. Endocr Res, 1998,24:695-702.
[5] Cavadas C, Silva AP, Mosimann F, et al. NPY regulates catecholamine secretion from human adrenal chromaffin cells[J]. J Clin Endocrinol Metab, 2001,86:5956-63.
[6] Claudia Cavadas, Antonio P. Silva, Francois Mosimann, et al.NPY Regulates Catecholamine Secretion from Human Adrenal Chromaffin Cells[J] . The Journal of Clinical Endocrinology &Metabolism ,2001, 86 (12) : 5956-5963.
[7] Capaldo A, Sciarrillo R, Falco MD,et al. Immunocytochemical localization of substance P in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae):evidence for its involvement in the modulation of adrenal activity[J]. Gen Comp Endocrinol,2003 ,134(3):229-36.
[8] J. P. Hinson , S. Kapas. Effect of sodium depletion on the response of rat adrenal zona glomerulosa cells to stimulation by neuropeptides: actions of vasoactive intestinal peptide,enkephalins, substance P, neuropeptide Y and corticotrophin-releasing hormone[J]. J. Endocr, 1995, 146 :209-214.
[9] T. Yoshida, M. Mio , K. Tasaka. Cortisol secretion induced by substance P from bovine adrenocortical cells and its inhibition by calmodulin inhibitors[J]. Biochem Pharmacol, 1992, 433: 513-517.
[10] H. Houchi, K. Teraoka, M. Oka, et al.Substance P inhibits catecholamine biosynthesis stimulated by carbamylcholine in cultured adrenal chromaffin cells [J]. Biochem Pharmacol, 1993, 45:1165-1167.
[11] A. V. Edwards , C. T. Jones . Effects of substance P on adrenal responses to acetylcholine in conscious calves [J]. Am. J. Physiol,1994, 267 (9): 447-453.
[12] A. Capaldo, V. Laforgia, R. Sciarrillo, et al. Localization and role of serotonin in the adrenal gland of Podarcis sicula (Reptilia, Lacertidae) [J]. Gen. Comp Endocrinol, 2003, 132(1):66-76.
[13] C. Manzo, A. Capaldo, V. Laforgia, et al. Inhibin in the testis and adrenal gland of the male lacertid, Podarcis sicula Raf.: a light immunocytochemical study[J]. Eur J. Histochem, 2000, 44:285-293.
[14] V. Laforgia , R. Muoio. Effects of ACTH and corticosteroids on phenylethanolamine-N-methyltransferase (PNMT) expression as determined by immunocytochemical localization in the adrenal gland of the lizard Podarcis sicula[J]. Ital. J. Zool , 1997, 64:301 - 306.
[15] Sherwood NM, Krueckl SL, McRory JE . The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily[J]. Endocr Rev , 2000, 21:619-670.
[16] Vaudry D, Gonzales BJ, Basille M, et al .Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions[J]. Pharmacol Rev, 2000, 52:269-324.
[17] Takahashi K, Totsune K, Murakami O, et al. Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in pheochromocytomas[J]. Peptides, 1993,14:365-369
[18] Yon L, Breault L, Contesse V, et al. Localization, characterization,and second messenger coupling of pituitary adenylate cyclase-activating polypeptide receptors in the fetal human adrenal gland during the second trimester of gestation[J]. J Clin Endocrinol Metab , 1998 , 83:1299-1305.
[19] Bornstein SR, Haidan A, Ehrhart M. Cellular communication in the neuroadrenocortical axis: role of vasoactive intestinal polypeptide (VIP ) [J]. Endocr Res , 1996 ,22:819-829.
[20] Neri G, Andreis PG, Prayer-Galetti T, et al. Pituitary adenylate cyclase activating peptide enhances aldosterone secretion of human adrenal gland: evidence for an indirect mechanism, probably involving the local release of catecholamines [J]. J Clin Endocrinol Metab , 1996, 81:169-273.
[21] K. Isobe, N. Yukimasa, T. Nakai , et al.Pituitary adenylate cyclase-activating polypeptide induces gene expression of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine beta hydroxylase, through 3,5-cyclic adenosine monophosphate-and protein kinase Odependent mechanisms in cultured porcine adrenal medullary chromaffin cells[J].Neuropeptides , 1996, 30: 167 - 175.
[22] G. Mazzocchi, L. K. Malendowicz, P. Rebuffat, et al.The Impact of the Human Genome on Endocrinology: Original Articles[J]. The Journal of Clinical Endocrinology & Metabolism, 2002, 87 (6) :2575-2580.
[23] Lamouche S, Yamaguchi N . Role of PAC, receptor in adrenal catecholamine secretion induced by PACAP and in vivo[J]. Am J Physiol, 2001 , 280:R510 - R518 .
[24] Inoue M, Fujishiro N, Ogawa K, et al. Pituitary adenylate cyclase-activating polypeptide may function as a neuromodulator in guinea pig adrenal medulla[J]. J Physiol, 2000,528:473-487.
[25] Vaudry D, Gonzales BJ, Basille M, et al. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions[J]. Pharmacol Rev , 2000,52:269-324.
[26] Bornstein SR, Ehrhart-Bornstein M, Scherbaum WA. Morphological and functional studies of the paracrine interaction between cortex and medulla in the adrenal gland[J]. Microsc Res Tech , 1997,36:520-533.
[27] Yin Shuyi, Heym CH. An ultrastructural study of peptidergic nerve fiber in the human adrenal gland[J]. Chinese journal of histochemistry and cytochemistry, 1994,3(1):46-51.
[28] P. Chakravarty, T. P. Suthar, H. A. Coppock, et al. CGRP and adrenomedullin binding correlates with transcript levels for calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins in rat tissues[J]. Brit J Pharmacol, 2000, 130:189-195.
[29]Supriya Kapas,Derek Renshaw,Mark Carroll,et al.Adrenomedullin and calcitonin gene-related peptide receptors in the rat adrenal cortex[J].Peptides,2001,22(11):Pages 1903-1907.
[30]A.S.Belloni,V.Meneghelli,H.C.Champion,et al.Autoradiographic evidence that zona glomerulosa and capsular vessels of the human adrenal cortex are provided with different subtypes of adrenomedullin receptors[J].Peptides,1998,19:1581-1584.
[31]彭永德,高国峰,顾彦杰,等.人肾上腺基因表达谱的建立及其功能的新认识[J].生命科学研究,2005,9(3):272-277.
[1]方竹培,王怀才.肾上腺的应用解剖学[J].解剖学杂志,1983,26(2):108-110.
[2]Williams Peter L.Gray' s Anatomy[M].1996,:1901-1903.
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