人垂体生长激素腺瘤体外培养及其生物学特性的研究
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摘要
目的：通过应用与ghrelin功能相同人工合成的生长激素释放剂GHRP-6对体外培养的人垂体生长激素腺瘤细胞GH分泌影响的观察，探讨GHRP-6对GH腺瘤细胞作用的生物学特征及多种细胞内第二信使调控GH分泌的机制，并分析其可能的临床意义。
材料和方法：用原代细胞培养技术，观察了10例人垂体GH腺瘤细胞和6例正常胚胎垂体细胞对GHRP-6、生长释放激素(GH-releasing hormone，GHRH)和生长抑素(somatostatin，SS)的体外分泌效应。
结果：体外细胞培养中，GHRP-6对所有10例GH腺瘤细胞GH分泌表现出明显的刺激效应(P＜0.01)，但远不如正常垂体细胞敏感，但GH腺瘤细胞对持续的GHRP-6刺激反应时间延长。GHRH和SS仅对部分GH腺瘤细胞有作用，且SS不能完全阻断GHRP-6对于GH分泌的刺激效应。
结论：在体外细胞培养中，正常垂体细胞大小形态一致，呈圆形或椭圆形，分散，悬浮生长，不易贴壁，而异常垂体细胞大小形态不一致，贴壁后的细胞呈椭圆形或梭形，成团状或片状生长，分裂旺盛，细胞周围有伪足，部分悬浮生长，增加了培养的难度，其帖壁时间在培养的24小时后，因此细胞培养的成功为进一步的垂体GH腺瘤的分子生物学的研究打下坚实的基础。GH腺瘤细胞对生长激素释放剂GHRP-6的GH分泌刺激效应远不如正常垂体细胞那样敏感，表现为一定的功能自主性。而腺瘤细胞对于体外GHRP-6持续刺激的异常反应，表明垂体GH腺瘤中可能存在GH释放剂受体(GHS-R)所固有的负反馈机制——“脱敏”机制的减弱，且与肿瘤发病机制有关。
Objective: To observe the effects of GH releasing peptide-6 (GHRP-6) on GH secretion of human pituitary somatotrophinoma cells in vitro,and to investigate the regulatory effects of multiple intracellular "second messengers" on GH secretion by human pituitary somatotrophinomas.
Materials and Methods: The response to GHRP-6, GH-releasing hormone(GHRH) and somatostatin(SS) in 6 somatotrophinomas and 3 embryo pituitaries in vitro were observed..
Results:In vitro cell culture, a stimulatory response to GHRP-6 was found in both groups of somatotrophinomas, and excitative effects is not as more sensitive as normal pituitary. Howerer, the GH response time to repeated administration of GHRP-6 to somatotrophinomas was prolonged. The GH-secreting effects of GHRH or SS was only obtained in parts of somatotrophinomas. Moreover, SS only attenuation but abolished GH response to GHRP-6.
Conclusion: In vitro cell culture, human pituitary cells have the same form and size, they grow dispersed and suspended, and not easy to adherence. On the contrary the abnomal human pituitary cells have different form and size, the attached cells were longer and shaped ellipse or shuttle and grown like a round or a flat under microscopy, they grow clumpy and desintegrate vigorous. The abnomal cells have parapodium and grow suspended partly, the adherence time of them is more than 24 hours, all of this increase the difficulty of cell culture. So, the success of cell culture is the compact base of studying the pituitary somatotrophinoma molecular biology. In vitro cell culture, the GH-secreting excitative effect of GHRP-6 to somatotrophinomas was not as far more sensitive as normal pituitary ,So GH-secreting pituitary adenomas manifestated autocrine characteristics. The abnormal response to repeated administration of GHRP-6 in vitro showed that the lack of desensition--negative feedback loops of GH releasing hormone receptors was associated with tumorigenesis of GH secreting adenomas.
材料和方法：用原代细胞培养技术，观察了10例人垂体GH腺瘤细胞和6例正常胚胎垂体细胞对GHRP-6、生长释放激素(GH-releasing hormone，GHRH)和生长抑素(somatostatin，SS)的体外分泌效应。
结果：体外细胞培养中，GHRP-6对所有10例GH腺瘤细胞GH分泌表现出明显的刺激效应(P＜0.01)，但远不如正常垂体细胞敏感，但GH腺瘤细胞对持续的GHRP-6刺激反应时间延长。GHRH和SS仅对部分GH腺瘤细胞有作用，且SS不能完全阻断GHRP-6对于GH分泌的刺激效应。
结论：在体外细胞培养中，正常垂体细胞大小形态一致，呈圆形或椭圆形，分散，悬浮生长，不易贴壁，而异常垂体细胞大小形态不一致，贴壁后的细胞呈椭圆形或梭形，成团状或片状生长，分裂旺盛，细胞周围有伪足，部分悬浮生长，增加了培养的难度，其帖壁时间在培养的24小时后，因此细胞培养的成功为进一步的垂体GH腺瘤的分子生物学的研究打下坚实的基础。GH腺瘤细胞对生长激素释放剂GHRP-6的GH分泌刺激效应远不如正常垂体细胞那样敏感，表现为一定的功能自主性。而腺瘤细胞对于体外GHRP-6持续刺激的异常反应，表明垂体GH腺瘤中可能存在GH释放剂受体(GHS-R)所固有的负反馈机制——“脱敏”机制的减弱，且与肿瘤发病机制有关。
Objective: To observe the effects of GH releasing peptide-6 (GHRP-6) on GH secretion of human pituitary somatotrophinoma cells in vitro,and to investigate the regulatory effects of multiple intracellular "second messengers" on GH secretion by human pituitary somatotrophinomas.
Materials and Methods: The response to GHRP-6, GH-releasing hormone(GHRH) and somatostatin(SS) in 6 somatotrophinomas and 3 embryo pituitaries in vitro were observed..
Results:In vitro cell culture, a stimulatory response to GHRP-6 was found in both groups of somatotrophinomas, and excitative effects is not as more sensitive as normal pituitary. Howerer, the GH response time to repeated administration of GHRP-6 to somatotrophinomas was prolonged. The GH-secreting effects of GHRH or SS was only obtained in parts of somatotrophinomas. Moreover, SS only attenuation but abolished GH response to GHRP-6.
Conclusion: In vitro cell culture, human pituitary cells have the same form and size, they grow dispersed and suspended, and not easy to adherence. On the contrary the abnomal human pituitary cells have different form and size, the attached cells were longer and shaped ellipse or shuttle and grown like a round or a flat under microscopy, they grow clumpy and desintegrate vigorous. The abnomal cells have parapodium and grow suspended partly, the adherence time of them is more than 24 hours, all of this increase the difficulty of cell culture. So, the success of cell culture is the compact base of studying the pituitary somatotrophinoma molecular biology. In vitro cell culture, the GH-secreting excitative effect of GHRP-6 to somatotrophinomas was not as far more sensitive as normal pituitary ,So GH-secreting pituitary adenomas manifestated autocrine characteristics. The abnormal response to repeated administration of GHRP-6 in vitro showed that the lack of desensition--negative feedback loops of GH releasing hormone receptors was associated with tumorigenesis of GH secreting adenomas.
引文
1. Korbonits M, Grossman AB. Growth hormone-releasing peptide and its analogues. Novel stimuli to growth hormone release. Trends Endocrinol Metab, 1995; 6: 43-49
2. Smith RG, Leonard R, Bailey AR, et al. Growth hormone secretagogue receptor family members and ligands. Endocrine 2001; 14(1): 9-14
3. KojimaM, Hosoda H, Date Y, er al. Ghrelin is a growth-hormone - releasing acylated peptide from stomach. Nature , 1999;402:656-660
4. Skinner MM, Nass R, Lopes B, et, al. Growth hormone secretagogue receptor expression in human pituitary tumors. J Clin Endocrinol Metab, 1998;83:4314-4320
5. Korbonits M, Kojina M, Kangawa K, Grossman AB. Presence of ghrelin in normal and adenomatous human pituitary. Endocrine 2001; 14(1): 101-4
6. Lei T, Adams EF, Buchfelder M, et al. Relationship between protein kinase C and adenylyl cyclase activity in the regulation of growth hormone secretion by human pituitary somatotrophinomas. Neurosurgery, 1996; 39: 569.
7. Adams EF, Mashiter K. Secretion of growth hormone by normal and tumors human pituitary somatotrophs in cell cultrue. In: Endocrinology 85, Excerpta Medica, 1986;75
8. Chen K, Chan WWS, Butler B, et al. Evidence for a role of protein kinase C in His-D-Trp-Ala-Trp-D-Phe-Lys-NIt induced growth hormone release from rat primary pituitary cells. Endocrinology, 1991; 129: 3337
9. Ghigo E, Arvat E, Giordano R, et al. Biologic activities of growth hormone secretagogues in humans. Endocrine 2001; 14(1): 87-93
10. Dickson SL. Ghrelin: a newly discovered hormone. J Neuroendocrinol 2002; 14(1): 83-4
11. Bennett PA, Thomas GB, Howard AD, et al. Hypothalamic growth hormone secretagogue-receptor(GHS-R) expression is regulated by growth hormone in the rat. Endocrinology, 138; 4552-4557
12. Spada A, Reza-Elahi F, hrosio M, et al. Lack of desensitition of adenomatous somatotrophs to growth hormone releasing hormone-releasing hormone receptors in human pituitary adenomas. J Clin Endocrinol Metab, 1995; 80:2933
13. Adams EF, Lei T, Buchfelder M, et al. Biochemical characteristics of human pituitary somatotrophinomas with and without gsp mutations: in vitro cell culture studies. J Clin Endocrinol Metab, 1995; 80(7): 2077-2081
14．雷霆，蒋先惠，Adams EF，et al．Octreotide 治疗垂体生长激素腺瘤的体外分子学研究．临床神经外科杂志 1996；1：49
1．王忠诚主编 神经外科学 第一版 武汉：湖北科学技术出版社 1998：489
2. en-Shlomo A, Melmed S. Acromegaly. Endocrinol Metab Clin North Am. 2001 Sep;30(3):565-83
3. Alexander JM, Biller BM, Bikkal H, et, al. Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin Invest. 1990 Jul;86(1):336-40.
4. Spada A, Vallar L, Faglia G. Cellular alterations in pituitary tumors. Eur J Endocrinol. 1994 Jan;130(1):43-52.
5．谢启文主编 现代神经内分泌学 第一版 上海：上海医科大学出版社 1999：154
6. Spada A, Vallar L. G-protein oncogenes in acromegaly. Horm Res. 1992;38(1-2):90-3.
7. Vallar L, Spada A, Giannattasio G. Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas. Nature. 1987 Dec 10-16;330(6148):566-8.
8. Dhanasekaran N, Heasley LE, Johnson GL.G protein-coupled receptor systems involved in cell growth and oncogenesis. Endocr Rev. 1995 Jun;16(3):259-7.
9. Spiegel AM. Mutations in G proteins and G protein-coupled receptors in endocrine disease. J Clin Endocrinol Metab. 1996 Jul;81(7):2434-42.
10. Lyons J, Landis CA, Harsh G, et al. Two G protein oncogenes in human endocrine tumors. Science. 1990 Aug 10;249(4969):655-9
11. Landis CA, Masters SB, Spada A, et, al. Protein GTPase inhibitingmutations activate the alpha chain of Gs and stimulate adenylyl cyclase inhuman pituitary tumours. Nature. 1989 Aug 31;340(6236):692-6.
12. Yoshimoto K, Iwahana H, Fukuda A, Sano T, Itakura M. Rare mutations of the Gs alpha subunit gene in human endocrine tumors. Mutation detection by polymerase chain reaction-primer-introduced restriction analysis. Cancer. 1993 Aug 15;72(4):1386-93.
13. Hosoi E, Yokogoshi Y, Hosoi E, et al. Analysis of the Gs alpha gene in growth hormone-secreting pituitary adenomas by the polymerase chain reaction-direct sequencing method using paraffin-embedded tissues. Acta Endocrinol (Copenh). 1993 Oct;129(4):301-6
14．白祥军，雷霆，刘暌，等 Gsp癌基因在国人肢端肥大症患者垂体肿瘤中的表达中华实验外科杂志 2000；17：53-54
15. Adams EF, Brockmeier S, Friedmann E, et, al. Clinical and biochemical characteristics of acromegalic patients harboring gsp-positive and gsp-negative pituitary tumors. Neurosurgery. 1993 Aug;33(2):198-203
16. Adams EF, Lei T, Buchfelder M, Petersen B, Fahlbusch R. Biochemical characteristics of human pituitary somatotropinomas with and without gsp mutations: in vitro cell culture studies. J Clin Endocrinol Metab. 1995 Jul;80(7):2077-81.
17. Spada A, Arosio M, Bochicchio D, et, al. Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase. J Clin Endocrinol Metab. 1990 Dec;71(6):1421-6
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29. Adams EF, Lei T, Buchfelder M, Bowers CY, Fahlbusch R. Protein kinase C-dependent growth hormone releasing peptides stimulate cyclic adenosine 3',5'-monophosphate production by human pituitary somatotropinomas expressing gsp oncogenes: evidence for crosstalk between transduction pathways. Mol Endocrinol. 1996 Apr;10(4):432-8
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34. Ghigo E, Arvat E, Giordano R, et, al. Biologic activities of growth hormone secretagogues in human. Endocrine, 2001;14:87-93
35. Akman MS, Giard M,O Brien LF, et, al. Mechanisms of action of a second generation growth hormone releasing peptide (Ala-His-D-beta-Nal-Ala-Trp-D-Phe-Lys-NH_2) in rat anterior pituitary cells. Endocrionology;1993,132:1286
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37. Adams EF, Petersen B, Lei T, Buchfelder M, Fahlbusch R. The growth hormone secretagogue, L-692,429, induces phosphatidylinositol hydrolysis and hormone secretion by human pituitary tumors. Biochem Biophys Res Commun 1995 Mar 17;208(2):555-61
38. Bertherat J. Cloning of the growth hormone secretagogues receptor cDNA: new evidence for a third endocrine pathway controlling growth hormone release. Eur J Endocrinol. 1997 Jan;136(1):37-8
39. Howard AD,. Feighner SD, Cully DF, et al . A receptor in pituitary and hypothalamus that functions in grouth hormone release. Scienec,1996;273:974-7
40. KojimaM, Hosoda H, Date Y, er al. Ghrelin is a growth-hormone - releasing acylated peptide from stomach. Nature , 1999;402:656-660
41. Tolle V, Zizzari P, Tomasetto C, et, al. In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat. Neuroendocrin, 2001;73:54-61
42. Bertherat J. Cloning of the GHSs-R cDNA: new evidence for a third endocrine pathway controlling growth hormone release .Eur J Endocrinol, 1997; 136:37
43. Korbonits M, Bustin SA, Kojima M, et al. The expression of the growth hormone secretagogue receptor ligand ghrelin in normal and abnormal human pituitary and other neuroendocrine tumors. J Clin Endocrinol Metab. 2001 Feb;86(2):881-7
44. Kim K, Arai K, Sanno N, et al. Ghrelin and growth hormone secretagogue receptor mRNA expression in human pituitary adenomas. Clin Endocrinol;2001;54:759-768
45. Skinner MM, Nass R, Lopes B, et, al. Growth hormone secretagogue receptor expression in human pituitary tumors. J Clin Endocrinol Metab,1998;83:4314-4320
46. Chaidorum SS, Eggo MC, Sheppard MC, et al. Expression of epidermal growth factor , its receptor , and related oncoprotein in human pituitary tumors and response to EGFin vitro. Endocrinology. 1994;135:2012
47. Sawada T, Koike K, Kanda Y, et, al. Interleukin-6 stimulates cell proliferation of rat pituitary clonal cell lines in vitro. J Endocrinol Invest, 1995;18:83
48. Bowers CY, Granda-Ayala R. Growth hormone/insulin-like growth factor-1 response to acute and chronic growth hormone-releasing peptide-2, growth hormone-releasing hormone 1-44NH2 and in combination in older men and women with decreased growth hormone secretion. Endocrine 2001 Feb;14(1):79-86
49. Jones TH, Justice SK, Price A. Suppression of tyrosine kinase activity inhibits [3H]thymidine uptake in cultured human pituitary tumor cells. J Clin Endocrinol Metab. 1997 Jul;82(7):2143-7.
50. Tahara S, Sanno N, Teramoto A, Osamura RY. Expression of Rab3, a Ras-related GTP-binding protein, in human nontumorous pituitaries and pituitary adenomas. Mod Pathol. 1999 Jun;12(6):627-34
51. Ezzat S. The role of hormones, growth factors and their receptors in pituitary tumorigenesis Brain Pathol 2001 Jul;11(3):356-370
52. Bowers CY. Unnatural growth hormone -releasing peptide begets natural ghrelin . J Clin Endocrinol Metab 2001;86(4):1464-1469
53. Tolle V, Zizzari P, Tomasetto C, et, al. In vivo and in vitro effects of ghrelin/motilin-related peptide on growth hormone secretion in the rat. Neuroendocrin, 2001;73:54-61
54. Hannew K, Utsumi A, Tanaka A, et, al. Secretory mechanisms of growth hormone - releasing peptide-, GH-releasing hormone -, and thyrotropin-releasing hormone-induced GH release in patients with acromegaly. J Clin Endocrinol Metab. 1998:83:3578-3583 state, 1996, 29:371-380
2. Smith RG, Leonard R, Bailey AR, et al. Growth hormone secretagogue receptor family members and ligands. Endocrine 2001; 14(1): 9-14
3. KojimaM, Hosoda H, Date Y, er al. Ghrelin is a growth-hormone - releasing acylated peptide from stomach. Nature , 1999;402:656-660
4. Skinner MM, Nass R, Lopes B, et, al. Growth hormone secretagogue receptor expression in human pituitary tumors. J Clin Endocrinol Metab, 1998;83:4314-4320
5. Korbonits M, Kojina M, Kangawa K, Grossman AB. Presence of ghrelin in normal and adenomatous human pituitary. Endocrine 2001; 14(1): 101-4
6. Lei T, Adams EF, Buchfelder M, et al. Relationship between protein kinase C and adenylyl cyclase activity in the regulation of growth hormone secretion by human pituitary somatotrophinomas. Neurosurgery, 1996; 39: 569.
7. Adams EF, Mashiter K. Secretion of growth hormone by normal and tumors human pituitary somatotrophs in cell cultrue. In: Endocrinology 85, Excerpta Medica, 1986;75
8. Chen K, Chan WWS, Butler B, et al. Evidence for a role of protein kinase C in His-D-Trp-Ala-Trp-D-Phe-Lys-NIt induced growth hormone release from rat primary pituitary cells. Endocrinology, 1991; 129: 3337
9. Ghigo E, Arvat E, Giordano R, et al. Biologic activities of growth hormone secretagogues in humans. Endocrine 2001; 14(1): 87-93
10. Dickson SL. Ghrelin: a newly discovered hormone. J Neuroendocrinol 2002; 14(1): 83-4
11. Bennett PA, Thomas GB, Howard AD, et al. Hypothalamic growth hormone secretagogue-receptor(GHS-R) expression is regulated by growth hormone in the rat. Endocrinology, 138; 4552-4557
12. Spada A, Reza-Elahi F, hrosio M, et al. Lack of desensitition of adenomatous somatotrophs to growth hormone releasing hormone-releasing hormone receptors in human pituitary adenomas. J Clin Endocrinol Metab, 1995; 80:2933
13. Adams EF, Lei T, Buchfelder M, et al. Biochemical characteristics of human pituitary somatotrophinomas with and without gsp mutations: in vitro cell culture studies. J Clin Endocrinol Metab, 1995; 80(7): 2077-2081
14．雷霆，蒋先惠，Adams EF，et al．Octreotide 治疗垂体生长激素腺瘤的体外分子学研究．临床神经外科杂志 1996；1：49
1．王忠诚主编 神经外科学 第一版 武汉：湖北科学技术出版社 1998：489
2. en-Shlomo A, Melmed S. Acromegaly. Endocrinol Metab Clin North Am. 2001 Sep;30(3):565-83
3. Alexander JM, Biller BM, Bikkal H, et, al. Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin Invest. 1990 Jul;86(1):336-40.
4. Spada A, Vallar L, Faglia G. Cellular alterations in pituitary tumors. Eur J Endocrinol. 1994 Jan;130(1):43-52.
5．谢启文主编 现代神经内分泌学 第一版 上海：上海医科大学出版社 1999：154
6. Spada A, Vallar L. G-protein oncogenes in acromegaly. Horm Res. 1992;38(1-2):90-3.
7. Vallar L, Spada A, Giannattasio G. Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas. Nature. 1987 Dec 10-16;330(6148):566-8.
8. Dhanasekaran N, Heasley LE, Johnson GL.G protein-coupled receptor systems involved in cell growth and oncogenesis. Endocr Rev. 1995 Jun;16(3):259-7.
9. Spiegel AM. Mutations in G proteins and G protein-coupled receptors in endocrine disease. J Clin Endocrinol Metab. 1996 Jul;81(7):2434-42.
10. Lyons J, Landis CA, Harsh G, et al. Two G protein oncogenes in human endocrine tumors. Science. 1990 Aug 10;249(4969):655-9
11. Landis CA, Masters SB, Spada A, et, al. Protein GTPase inhibitingmutations activate the alpha chain of Gs and stimulate adenylyl cyclase inhuman pituitary tumours. Nature. 1989 Aug 31;340(6236):692-6.
12. Yoshimoto K, Iwahana H, Fukuda A, Sano T, Itakura M. Rare mutations of the Gs alpha subunit gene in human endocrine tumors. Mutation detection by polymerase chain reaction-primer-introduced restriction analysis. Cancer. 1993 Aug 15;72(4):1386-93.
13. Hosoi E, Yokogoshi Y, Hosoi E, et al. Analysis of the Gs alpha gene in growth hormone-secreting pituitary adenomas by the polymerase chain reaction-direct sequencing method using paraffin-embedded tissues. Acta Endocrinol (Copenh). 1993 Oct;129(4):301-6
14．白祥军，雷霆，刘暌，等 Gsp癌基因在国人肢端肥大症患者垂体肿瘤中的表达中华实验外科杂志 2000；17：53-54
15. Adams EF, Brockmeier S, Friedmann E, et, al. Clinical and biochemical characteristics of acromegalic patients harboring gsp-positive and gsp-negative pituitary tumors. Neurosurgery. 1993 Aug;33(2):198-203
16. Adams EF, Lei T, Buchfelder M, Petersen B, Fahlbusch R. Biochemical characteristics of human pituitary somatotropinomas with and without gsp mutations: in vitro cell culture studies. J Clin Endocrinol Metab. 1995 Jul;80(7):2077-81.
17. Spada A, Arosio M, Bochicchio D, et, al. Clinical, biochemical, and morphological correlates in patients bearing growth hormone-secreting pituitary tumors with or without constitutively active adenylyl cyclase. J Clin Endocrinol Metab. 1990 Dec;71(6):1421-6
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