人体嗜铬细胞培养的实验研究

Experimental study of culture of human chromaffin cell

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

Objective The growth condition of human chromaffin cells primary cultured in vitro for 5 days was observed successively in order to find the proper time to transplant Methods Bilateral adrenal glands of 6 healthy adults with dead cerebrum were taken out respectively According to the modified Lis method,the chromaffin cells were cultured in vitro for 5 days During culture,the growth of the cells was observed microscopically and the culture solution was replaced every 24 hours The supernatant was assessed with high performance liquid chromatography for determination of catecholamine production The cells cultured on the 3rd day were assessed immunocytochemically by labeling the cells with tyrosine hydroxylase antibody and the percentage of positive cells was counted Results (1)The growth of human chromaffin cells cultured on the 3rd day was better;(2) immunocytochemical results showed that positive cells expressed with tyrosine hydroxylase were 72% The granules in the cells were clear;(3) The levels of noradrenaline and adrenaline cultured on the 3rd day were significantly increased comparing with that on the 1st day and 5th day (P<0 01) The amount of dopamine was increased on both 2nd and 3rd day (P<0 05) Conclusions The 3rd day of human chromaffin cells primamry cultured in vitro is the proper time to transplant