【作者】 章杨；

【导师】 李汉忠；

【作者基本信息】 北京协和医学院 ， 临床医学， 2010， 博士

【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。更多还原

【Abstract】 [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.更多还原