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本研究拟建立人嗜铬细胞瘤细胞的原代培养方法。采用连续分次胶原酶消化法分离培养人嗜铬细胞瘤细胞 ,采用细胞培养液中儿茶酚胺水平检测、多聚甲醛诱发荧光及细胞嗜铬粒蛋白A (CgA)和神经元特异性烯醇化酶(NSE)的免疫组化染色等方法进行细胞性质和功能鉴定 ,并用噻唑兰 (MTT)法观察原代培养的人嗜铬细胞瘤细胞的生长状况。结果表明 ,人嗜铬细胞瘤细胞在培养 3～ 7天时生长较快 ,7天后细胞开始分化。经检测细胞培养液中的儿茶酚胺浓度、多聚甲醛诱发荧光等 ,证明该细胞有合成和分泌儿茶酚胺的功能。并且培养的细胞CgA和NSE免疫组化染色阳性。因此 ,本研究成功建立了人嗜铬细胞瘤细胞的原代培养方法 ,并鉴定其具有嗜铬细胞瘤的分泌和表达功能 ,国内尚未见报告
This study intended to establish a primary culture method of human pheochromocytoma cells. The human pheochromocytoma cells were isolated and cultured by continuous collagenase digestion. The levels of catecholamine in cell culture medium, the fluorescence induced by paraformaldehyde and the levels of CgA and NSE NSE) immunohistochemical staining and other methods of cell characterization and functional identification, and the use of thiazide blue (MTT) method to observe the growth of primary cultured human pheochromocytoma cells. The results showed that human pheochromocytoma cells grew rapidly during 3 to 7 days of culture and began to differentiate after 7 days. The concentration of catecholamines, paraformaldehyde-induced fluorescence, etc. in the test cell culture medium proved that the cells have the function of synthesizing and secreting catecholamines. The cultured cells were positive for immunohistochemical staining of CgA and NSE. Therefore, this study successfully established the primary culture method of human pheochromocytoma cells, and identified its secretion and expression of pheochromocytoma function has not been reported