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目的 观察连续 5天体外培养人体肾上腺嗜铬细胞的生长情况和儿茶酚胺分泌功能的变化 ,以便选择细胞移植的适宜时机。方法 取 6例健康成人脑死亡的双侧肾上腺 ,按李氏改良方法 ,培养 5天。每 2 4小时记录培养细胞生长情况 ,更换培养液 ,并采用高效液相色谱技术检测培养液中儿茶酚胺含量。培养第 3天的细胞 ,采用免疫细胞化学 ( SABC)技术检测酪氨酸羟化酶表达阳性细胞 ,并计数阳性细胞百分率。结果 ( 1)培养第 3天的人体嗜铬细胞形态结构完整 ,生长良好 ;( 2 )免疫组化检测显示酪氨酸羟化酶表达阳性细胞为 72 % ,细胞内嗜铬颗粒清晰 ;( 3 )培养液去甲肾上腺素和肾上腺素含量 ,在培养第 3天均较第 1天明显升高 ( P<0 .0 1)。多巴胺含量在第 2、3天均有升高 ( P<0 .0 5 )。结论 本组人体嗜铬细胞体外培养第 3天是进行细胞移植的适宜时机
Objective To observe the growth of adrenal chromaffin cells cultured in vitro for 5 days and the changes of catecholamine secretion in order to select the appropriate timing of cell transplantation. Methods Bilateral adrenal brains from 6 healthy adults were killed and cultured for 5 days according to the improved method of Lee. The growth of cultured cells was recorded every 24 hours, the medium was changed, and the content of catecholamines in the culture medium was detected by high performance liquid chromatography. Day 3 cells were cultured, tyrosine hydroxylase expressing positive cells were detected by immunocytochemistry (SABC) technique and the percentage of positive cells was counted. Results (1) Human chromaffin cells were intact and grew well on the 3rd day of culture. (2) Immunohistochemistry showed 72% positive tyrosine hydroxylase positive cells and clear intracellular chromaffin granules. (3) ) Norepinephrine and epinephrine content in culture medium were significantly higher than the first day (P <0.01) on the third day. Dopamine increased in the first and second days (P <0.05). Conclusion This group of human chromaffin cells cultured in vitro is the appropriate time for cell transplantation