人嗜铬细胞瘤细胞原代培养及细胞分泌功能的相关研究
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摘要
[背景]嗜铬细胞瘤是一种罕见的起源于嗜铬细胞的肿瘤,其分泌儿茶酚胺,被认为是继发性高血压的重要病因。其临床表现错综复杂,常表现为难治性高血压、心律失常等症状,并常常伴随于某些特定的遗传疾病及内分泌疾病,如其可为Ⅱ、Ⅲ型多发性内分泌腺瘤综合症(MEN)的一部分,可伴发甲状腺髓样癌、甲状旁腺腺瘤、肾上腺腺瘤或增生；还可以并发其他神经细胞肿瘤如多发性神经纤维瘤、多发性神经血管母细胞瘤等。随着测定儿茶酚胺含量的生化技术和影像学的发展,我们对嗜铬细胞瘤诊断和治疗的能力有了显著的提高。计算机断层扫描(CT)及磁共振成像(MRI)等技术的成熟使得我们定位肾上腺及肾上腺外的嗜铬细胞瘤的能力取得长足进步。同时,对嗜铬细胞瘤病理生理学的认识,使得我们能更好的理解该肿瘤复杂多变的临床表现,并发展了药物处理、麻醉期管理的适当方法。外科手术技术的进步也使得嗜铬细胞瘤的手术切除成功率得到显著提升。但是目前在嗜铬细胞瘤的遗传学、生化诊断、定位和治疗方面,仍然存在着很多没有解决的问题,需要我们对其进行深入的研究。体外原代培养的人嗜铬细胞瘤细胞更符合嗜铬细胞瘤的临床特点,有利于临床研究的需要,应受到临床及基础研究的重视。
[目的]本研究应用原代培养人嗜铬细胞瘤细胞的方法对嗜铬细胞瘤进行体外培养,动态观察细胞的生长、分裂、分化、衰老与死亡等基本现象,并进一步研究该肿瘤激素的合成、分泌、释放及其影响因素,为围手术期治疗提供依据。
[方法]采用差速贴壁法培养人原代嗜铬细胞瘤细胞;检测细胞培养液中儿茶酚胺水平,并对细胞嗜铬粒蛋白A (CgA)用细胞免疫组化染色方法进行细胞性质鉴定；并用噻唑兰(MTT)法绘制原代培养的人嗜铬细胞瘤细胞的生长曲线；采用酶联免疫法(ELISA)检测不同时期细胞培养液中的儿茶酚胺(CA)浓度及物理刺激后细胞形态及分泌儿茶酚胺浓度的变化。
[结果]建立了人嗜铬细胞瘤细胞的原代培养方法。人嗜铬细胞瘤细胞在培养后的3-7天,细胞生长较快；7天后细胞生长减慢,出现丝状突起；2-3周后,突起相互连接成网状,细胞皱缩。物理刺激后,原代培养的人嗜铬细胞瘤细胞儿茶酚胺第3天分泌量有所增加,但无统计学意义(P>0.05)。
[结论]该方法可以成功原代培养人嗜铬细胞瘤细胞；培养的人嗜铬细胞瘤细胞具有生长和分泌功能,可为人嗜铬细胞瘤体外实验研究提供基本材料；该种物理刺激方法不影响原代培养的人嗜铬细胞瘤细胞儿茶酚胺分泌量。
[Background] Pheochromocytoma is a rare but important tumor of chromaffin cells that is frequently considered in the evaluation of hypertension, arrhythmias, or panic disorder and in the follow-up of patients with particular genetic diseases. By the development of sensitive and specific chemical techniques for assaying catecholamines in biological fluids, and by advances in noninvasive localizing techniques, our ability to diagnose and treat pheochromocytoma has been enhanced by striking advances in our knowledge of human catecholamine metabolism. We know that better understanding of the pathophysiology of pheochromocytoma and its varied clinical presentations, advances in antihypertensive drug therapy, and anesthetic and surgical techniques can radically change our overall approach to the diagnosis and treatment of pheochromocytoma. But it still remain many key questions in the genetics, biochemical diagnosis, localization, and management of pheochromocytoma. Primary culture method could offer material in vitro, and it is a direct way to investigate the cell.
[Objective] The objective of the study is to establish the optical cell culture conditions of human pheochromocytoma cell and investigate the influence of physical stimulation on the human pheochromocytoma cell.
[Methods] The human pheochromocytoma cell were separated and cultured by differential adhesion, detect the secretion of catecholamine in cell culture medium and expression of ChromograninA with immunohistochemical method to identify the cell, we use MTT method to obtain the growth curve of primary culture human pheochromocytoma cell, and determine the level of catecholamine with enzyme-labeled immunosorbent assay (Elisa), and see whether the physical stimulations influence the culture cell.
[Results] We successfully established the method to primary culture the human pheochromocytoma cell, When first plated, the cells appeared as round, individual,phase-transparent cells, and the cells became firmly attached after three to four days in culture, the round cells frequently coalesced into clumps or groups, the cells grew quickly in three to seven days. We found this cell secreting catecholamine and ChromograninA, so it can be characterized as pheochromocytoma. With investigation of catecholamine concentration in cell culture medium, we found this physical stimulation method influence the cells, and made the cells secreting more catecholamine in vitro, but there is no statistical significance (P>0.05)
[Conlusion] We can obtain cultured human pheochromocytoma cell with this cell culture method; the cultured pheochromocytoma cells can synthesis and secreting catecholamine in vitro; this physical stimulation method does not influence the secretion of catecholamine.
[目的]本研究应用原代培养人嗜铬细胞瘤细胞的方法对嗜铬细胞瘤进行体外培养,动态观察细胞的生长、分裂、分化、衰老与死亡等基本现象,并进一步研究该肿瘤激素的合成、分泌、释放及其影响因素,为围手术期治疗提供依据。
[方法]采用差速贴壁法培养人原代嗜铬细胞瘤细胞;检测细胞培养液中儿茶酚胺水平,并对细胞嗜铬粒蛋白A (CgA)用细胞免疫组化染色方法进行细胞性质鉴定；并用噻唑兰(MTT)法绘制原代培养的人嗜铬细胞瘤细胞的生长曲线；采用酶联免疫法(ELISA)检测不同时期细胞培养液中的儿茶酚胺(CA)浓度及物理刺激后细胞形态及分泌儿茶酚胺浓度的变化。
[结果]建立了人嗜铬细胞瘤细胞的原代培养方法。人嗜铬细胞瘤细胞在培养后的3-7天,细胞生长较快；7天后细胞生长减慢,出现丝状突起；2-3周后,突起相互连接成网状,细胞皱缩。物理刺激后,原代培养的人嗜铬细胞瘤细胞儿茶酚胺第3天分泌量有所增加,但无统计学意义(P>0.05)。
[结论]该方法可以成功原代培养人嗜铬细胞瘤细胞；培养的人嗜铬细胞瘤细胞具有生长和分泌功能,可为人嗜铬细胞瘤体外实验研究提供基本材料；该种物理刺激方法不影响原代培养的人嗜铬细胞瘤细胞儿茶酚胺分泌量。
[Background] Pheochromocytoma is a rare but important tumor of chromaffin cells that is frequently considered in the evaluation of hypertension, arrhythmias, or panic disorder and in the follow-up of patients with particular genetic diseases. By the development of sensitive and specific chemical techniques for assaying catecholamines in biological fluids, and by advances in noninvasive localizing techniques, our ability to diagnose and treat pheochromocytoma has been enhanced by striking advances in our knowledge of human catecholamine metabolism. We know that better understanding of the pathophysiology of pheochromocytoma and its varied clinical presentations, advances in antihypertensive drug therapy, and anesthetic and surgical techniques can radically change our overall approach to the diagnosis and treatment of pheochromocytoma. But it still remain many key questions in the genetics, biochemical diagnosis, localization, and management of pheochromocytoma. Primary culture method could offer material in vitro, and it is a direct way to investigate the cell.
[Objective] The objective of the study is to establish the optical cell culture conditions of human pheochromocytoma cell and investigate the influence of physical stimulation on the human pheochromocytoma cell.
[Methods] The human pheochromocytoma cell were separated and cultured by differential adhesion, detect the secretion of catecholamine in cell culture medium and expression of ChromograninA with immunohistochemical method to identify the cell, we use MTT method to obtain the growth curve of primary culture human pheochromocytoma cell, and determine the level of catecholamine with enzyme-labeled immunosorbent assay (Elisa), and see whether the physical stimulations influence the culture cell.
[Results] We successfully established the method to primary culture the human pheochromocytoma cell, When first plated, the cells appeared as round, individual,phase-transparent cells, and the cells became firmly attached after three to four days in culture, the round cells frequently coalesced into clumps or groups, the cells grew quickly in three to seven days. We found this cell secreting catecholamine and ChromograninA, so it can be characterized as pheochromocytoma. With investigation of catecholamine concentration in cell culture medium, we found this physical stimulation method influence the cells, and made the cells secreting more catecholamine in vitro, but there is no statistical significance (P>0.05)
[Conlusion] We can obtain cultured human pheochromocytoma cell with this cell culture method; the cultured pheochromocytoma cells can synthesis and secreting catecholamine in vitro; this physical stimulation method does not influence the secretion of catecholamine.
引文
1、Lucon AM, Falci R Jr, Praxedes JN. Multicentric pheochromocytoma and involvement of the inferior vena cava. Sao Paulo Med J,2001,119:86—88.
2、Smythe GA, Edwards G, Graham P, Lazarus L 1992 Biochemical diagnosis of pheochromocytoma by simultaneous measurement of urinary excretion of epinephrine and norepinephrine. Clin Chem 38:486-492.
3、Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. ProcNatl Acad Sci USA, 1976,73:2424-2428.
4、Su F, Kozak KR, Herschman H, et al. Characterization of the rat urokinase plasminogen activator receptor promoter in PC 12 cells. J Neurosci Res.2007, 85:1952-1958.
5、童安莉,曾正陪,李汉忠等.人嗜铬细胞瘤细胞的原代培养及鉴定.基础医学与临床,2003,23：447-450.
6、omlinson A, Durbin J, Coupland RE. A quantitative analysis of rat adrenal chromaffin tissue:morphometric analysis at tissue and cellular level correlated with catecholamine content. Neuroscience,1987,20:895-904.
7、兰蓉,周珍辉等,细胞培养技术.化学工业出版社,2007.8
8 Liu J, Kahri AI, Heikkila P, et al. Glucocorticoids increase insulin-like growth factor-Ⅱ mRNA accumulation in cultured human pheochromocytoma cells.
9 Wong DL, Siddall B, Wang W. Hormonal control of rat adrenal phenoletrranolamine /V-methyltransferase. Enzyme activity, the final critical pathway. Neuropsychopharinacology 1995; 13:223-234.
10 Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism:a contemporary view with implications for physiology and medicine. Pharmacol Rev 2004; 56:331-349
11 Herrera MF, Stone E, Deitel M. Pheochromocytoma producing multiple vasoactive peptides. Arch Surg,1992,127:105-108.
12 Canale MP, Bravo EL 1994 Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction. J Clin Endocrinol Metab 78:1139-1144
13 Canale MP, Bravo EL 1994 Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction. J Clin Endocrinol Metab 78:1139-1144
14 J. F. Murphy, D. H. Davies and C. J. Smith The development of enzyme-linked immunosorbent assays (ELISA) for the catecholamines adrenalin and noradrenalin Journal of Immunological Methods Volume 154, Issue 1,18 September 1992, Pages 89-98
15 Coupiand RE. The Natural History of the Chromaffin Cell. New York:Longman, 1965.
16 Choi AY. Fukui H, Perlman RL. Glucocorticoids enhance histaminc-evoked catecholamine secretion from bovine chromaffin cells. J Neurochem 1995; 64: 206-212.
17 Starikova AM, Pogorelaya NC,Kostyuk PG. Long-term depolarization changes morphological paremeters of PC12 cells.Neuroscience,2000,95:923-926.
1. Crout JR, Pisano JJ, Sjoerdsma A 1961 Urinary excretion of catecholamines and their metabolites in pheochromocytoma. Am Heart J 61:375-381
2. Beard CM, Sheps SG, Kurland LT, Carney JA, Lie JT 1983 Occurrence of pheochromocytoma in Rochester, Minnesota,1950 through 1979. Mayo Clin Proc 58:802-804
3. Smythe GA, Edwards G, Graham P, Lazarus L 1992 Biochemical diagnosis of pheochromocytoma by simultaneous measurement of urinary excretion of epinephrine and norepinephrine. Clin Chem 38:486-492.
4、McNeil AR, Blok BH, Koelmeyer TD, Burke MP, Hilton JM 2000 Pheochromocytomas discovered during coronial autopsies in Sydney, Melbourne and Auckland. Aust N Z J Med 30:648-652
5. Relationship between the height of arterial blood pressure and circulating catecholamine (NE and E) levels in pheochromocytoma. [Reproduced with permission from E. L. Bravo et al.:N Engl J Med 301:682-686..
6. Williams RH, Larsen PR. Williams Textbook of Endocrinology.10th ed. Philadelphia, Pa:Saunders; 2003
7. Rchard EG, James AO, George WH et al. Clinical Experience over 48 years with pheochromocytomas. Annol of Surgery,1999,229:755-766.
8. Keiser HR. Phochromocytoma and related tumors. In DeGroot L. Endocrinology. Ed 3.vol. Philadelphia, WB Saunders Co.1995.
9. 王吉耀,廖二元,胡品津等内科学人民卫生出版社2006 P935.
10. Sutton MG, Sheps SG, Lie JT 1981 Prevalence of clinically unsuspected pheochromocytoma. Review of a 50-year autopsy series. Mayo Clin Proc 56:354-360.
11. Karel Pacak, W.Marston Linehan, Graeme Eisenhofer, McClellan M.Walther, David S.Goldstein Recent Advances in Genetics, Diagnosis, Localization, and Treatment of Pheochromocytoma, Ann Intern Med February 20,2001 vol.134 no.4 315-329
12. Lenders JW, Pacak K, Walther MM, Linehan WM, Mannelli M, Friberg P, Keiser HR, Goldstein DS, Eisenhofer G 2002 Biochemical diagnosis of pheochromocytoma:which test is best? JAMA 287:1427-1434
13. Eisenhofer G, Goldstein DS, walther MM。 et al. Biochemical diagnosis of pheochromocytoma:how to distinguish true—from false—positive test re— suits. J Clin Endocrinol Metab.2003,88:2656-2666.
14. O'Connor DT, Bernstein KN 1984 Radioimmunoassay of chromogranin A in plasma as a measure of exocytotic sympathoadrenal activity in normal subjects and patients with pheochromocytoma. N Engl J Med 311:764-770
15. Michele d'Herbomez, Catherine Bauters, Philippe Caron, et.al. Serum chromogranin A assay in the biological diagnosis of pheochromocytomas and/or paragangliomas:results in 146 patients. Endocrine Abstracts (2009) 20 P190
16. Canale MP, Bravo EL 1994 Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction. J Clin Endocrinol Metab 78:1139-1144
17. Arnold F. Jacobson*, Hsiaowei Deng, et.al.1231-Meta-Iodobenzylguanidine Scintigraphy for the Detection of Neuroblastoma and Pheochromocytoma: Results of a Meta-Analysis. Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2009-2604
18. Shern L. Chew. Diagnosis:Imaging of pheochromocytomas and paragangliomas. Nature Reviews Endocrinology 6,193-194 (April 2010).
19. Henri J. L. M. Timmers, Clara C. Chen, Jorge A. Carrasquillo, Comparison of 18F-Fluoro-L-DOPA,18F-Fluoro-Deoxyglucose, and 18F-Fluorodopamine PET and 123I-MIBG Scintigraphy in the Localization of Pheochromocytoma and Paraganglioma,2009 The Journal of Clinical Endocrinology & Metabolism Vol. 94, No.12 4757-4767
20. Gimenez—Roqueplo AP, Favier J, Rustin P, et al. Mutations in the SDHB gene are associated with extra-adrenal and/or malignant pheoehromocytomas. Cancer Res.2003.63:5615-5621
21. KlIlke MH。 Smart K. Enzlnger PC. Et.al. Phase II study of temozolomide and thalidomide in patients with metastatic ncuroendocrine tumors. J Clin Oncol 2006.24:401-406.
22. Tarek Ezzatl, James Skipworthl, et.al. Open or laparoscopic surgery for phaeochromocytoma. Endocrine Abstracts (2010) 21 P223
2、Smythe GA, Edwards G, Graham P, Lazarus L 1992 Biochemical diagnosis of pheochromocytoma by simultaneous measurement of urinary excretion of epinephrine and norepinephrine. Clin Chem 38:486-492.
3、Greene LA, Tischler AS. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. ProcNatl Acad Sci USA, 1976,73:2424-2428.
4、Su F, Kozak KR, Herschman H, et al. Characterization of the rat urokinase plasminogen activator receptor promoter in PC 12 cells. J Neurosci Res.2007, 85:1952-1958.
5、童安莉,曾正陪,李汉忠等.人嗜铬细胞瘤细胞的原代培养及鉴定.基础医学与临床,2003,23：447-450.
6、omlinson A, Durbin J, Coupland RE. A quantitative analysis of rat adrenal chromaffin tissue:morphometric analysis at tissue and cellular level correlated with catecholamine content. Neuroscience,1987,20:895-904.
7、兰蓉,周珍辉等,细胞培养技术.化学工业出版社,2007.8
8 Liu J, Kahri AI, Heikkila P, et al. Glucocorticoids increase insulin-like growth factor-Ⅱ mRNA accumulation in cultured human pheochromocytoma cells.
9 Wong DL, Siddall B, Wang W. Hormonal control of rat adrenal phenoletrranolamine /V-methyltransferase. Enzyme activity, the final critical pathway. Neuropsychopharinacology 1995; 13:223-234.
10 Eisenhofer G, Kopin IJ, Goldstein DS. Catecholamine metabolism:a contemporary view with implications for physiology and medicine. Pharmacol Rev 2004; 56:331-349
11 Herrera MF, Stone E, Deitel M. Pheochromocytoma producing multiple vasoactive peptides. Arch Surg,1992,127:105-108.
12 Canale MP, Bravo EL 1994 Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction. J Clin Endocrinol Metab 78:1139-1144
13 Canale MP, Bravo EL 1994 Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction. J Clin Endocrinol Metab 78:1139-1144
14 J. F. Murphy, D. H. Davies and C. J. Smith The development of enzyme-linked immunosorbent assays (ELISA) for the catecholamines adrenalin and noradrenalin Journal of Immunological Methods Volume 154, Issue 1,18 September 1992, Pages 89-98
15 Coupiand RE. The Natural History of the Chromaffin Cell. New York:Longman, 1965.
16 Choi AY. Fukui H, Perlman RL. Glucocorticoids enhance histaminc-evoked catecholamine secretion from bovine chromaffin cells. J Neurochem 1995; 64: 206-212.
17 Starikova AM, Pogorelaya NC,Kostyuk PG. Long-term depolarization changes morphological paremeters of PC12 cells.Neuroscience,2000,95:923-926.
1. Crout JR, Pisano JJ, Sjoerdsma A 1961 Urinary excretion of catecholamines and their metabolites in pheochromocytoma. Am Heart J 61:375-381
2. Beard CM, Sheps SG, Kurland LT, Carney JA, Lie JT 1983 Occurrence of pheochromocytoma in Rochester, Minnesota,1950 through 1979. Mayo Clin Proc 58:802-804
3. Smythe GA, Edwards G, Graham P, Lazarus L 1992 Biochemical diagnosis of pheochromocytoma by simultaneous measurement of urinary excretion of epinephrine and norepinephrine. Clin Chem 38:486-492.
4、McNeil AR, Blok BH, Koelmeyer TD, Burke MP, Hilton JM 2000 Pheochromocytomas discovered during coronial autopsies in Sydney, Melbourne and Auckland. Aust N Z J Med 30:648-652
5. Relationship between the height of arterial blood pressure and circulating catecholamine (NE and E) levels in pheochromocytoma. [Reproduced with permission from E. L. Bravo et al.:N Engl J Med 301:682-686..
6. Williams RH, Larsen PR. Williams Textbook of Endocrinology.10th ed. Philadelphia, Pa:Saunders; 2003
7. Rchard EG, James AO, George WH et al. Clinical Experience over 48 years with pheochromocytomas. Annol of Surgery,1999,229:755-766.
8. Keiser HR. Phochromocytoma and related tumors. In DeGroot L. Endocrinology. Ed 3.vol. Philadelphia, WB Saunders Co.1995.
9. 王吉耀,廖二元,胡品津等内科学人民卫生出版社2006 P935.
10. Sutton MG, Sheps SG, Lie JT 1981 Prevalence of clinically unsuspected pheochromocytoma. Review of a 50-year autopsy series. Mayo Clin Proc 56:354-360.
11. Karel Pacak, W.Marston Linehan, Graeme Eisenhofer, McClellan M.Walther, David S.Goldstein Recent Advances in Genetics, Diagnosis, Localization, and Treatment of Pheochromocytoma, Ann Intern Med February 20,2001 vol.134 no.4 315-329
12. Lenders JW, Pacak K, Walther MM, Linehan WM, Mannelli M, Friberg P, Keiser HR, Goldstein DS, Eisenhofer G 2002 Biochemical diagnosis of pheochromocytoma:which test is best? JAMA 287:1427-1434
13. Eisenhofer G, Goldstein DS, walther MM。 et al. Biochemical diagnosis of pheochromocytoma:how to distinguish true—from false—positive test re— suits. J Clin Endocrinol Metab.2003,88:2656-2666.
14. O'Connor DT, Bernstein KN 1984 Radioimmunoassay of chromogranin A in plasma as a measure of exocytotic sympathoadrenal activity in normal subjects and patients with pheochromocytoma. N Engl J Med 311:764-770
15. Michele d'Herbomez, Catherine Bauters, Philippe Caron, et.al. Serum chromogranin A assay in the biological diagnosis of pheochromocytomas and/or paragangliomas:results in 146 patients. Endocrine Abstracts (2009) 20 P190
16. Canale MP, Bravo EL 1994 Diagnostic specificity of serum chromogranin-A for pheochromocytoma in patients with renal dysfunction. J Clin Endocrinol Metab 78:1139-1144
17. Arnold F. Jacobson*, Hsiaowei Deng, et.al.1231-Meta-Iodobenzylguanidine Scintigraphy for the Detection of Neuroblastoma and Pheochromocytoma: Results of a Meta-Analysis. Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2009-2604
18. Shern L. Chew. Diagnosis:Imaging of pheochromocytomas and paragangliomas. Nature Reviews Endocrinology 6,193-194 (April 2010).
19. Henri J. L. M. Timmers, Clara C. Chen, Jorge A. Carrasquillo, Comparison of 18F-Fluoro-L-DOPA,18F-Fluoro-Deoxyglucose, and 18F-Fluorodopamine PET and 123I-MIBG Scintigraphy in the Localization of Pheochromocytoma and Paraganglioma,2009 The Journal of Clinical Endocrinology & Metabolism Vol. 94, No.12 4757-4767
20. Gimenez—Roqueplo AP, Favier J, Rustin P, et al. Mutations in the SDHB gene are associated with extra-adrenal and/or malignant pheoehromocytomas. Cancer Res.2003.63:5615-5621
21. KlIlke MH。 Smart K. Enzlnger PC. Et.al. Phase II study of temozolomide and thalidomide in patients with metastatic ncuroendocrine tumors. J Clin Oncol 2006.24:401-406.
22. Tarek Ezzatl, James Skipworthl, et.al. Open or laparoscopic surgery for phaeochromocytoma. Endocrine Abstracts (2010) 21 P223
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