分离纯化牛嗜铬细胞方法改进  

Modified method of isolating and purifying bovine chromaffin cells

作　　者：雄鹰[1] 张晓慧[2] 梁丰[1] 郭昕[2] 王为[2] 马小军[2] 

机构地区：[1]解放军第二一O医院神经内科,辽宁省大连市116021 [2]中科院大连化学物理研究所生物医学材料工程组,辽宁省大连市116023

出　　处：《中国组织工程研究与临床康复》2007年第12期2219-2222,2232,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30472102);大连市科技计划资助项目(2004B3SFF161)~~

摘　　要：目的:建立一种稳定、经济、简单的牛肾上腺髓质嗜铬细胞体外分离、纯化方法,为临床移植提供充足、健康的细胞来源。方法:实验于2004-01/2005-12在中科院大连化学物理研究所生物医学材料工程组实验室完成。将胶原酶消化法和机械分离法结合,分离新生牛嗜铬细胞;采用Percoll非连续密度梯度离心法纯化新生牛嗜铬细胞。采用相差显微镜观察牛嗜铬细胞形态,椎虫蓝排斥染色观察牛嗜铬细胞活性,组织化学染色鉴定牛嗜铬细胞属性和纯度,免疫组化染色观察牛嗜铬细胞的功能活性。结果:①健康的牛嗜铬细胞集中在一定密度的两层分离介质之间,红细胞完全沉降于离心管底部,而皮质细胞和细胞碎屑则分散在特定密度的分离介质中。②平均每个牛肾上腺可分离得到(0.7～3.1)×108个牛嗜铬细胞。③将分离得到的牛嗜铬细胞进行椎虫蓝排斥染色,显示新鲜分离的牛嗜铬细胞存活率大于99%,具有合成儿茶酚胺的功能活性。结论:机械分离结合胶原酶灌注消化法分离牛嗜铬细胞后,经Percoll非连续等密度梯度离心可以有效纯化牛嗜铬细胞,分离的细胞数量多,纯度高,有合成儿茶酚胺的功能活性。AIM： To establish a more stable, economic and convenient method of in vitro isolation and purification bovine chromaffin calls （BCCs）, and provide a plenteous and healthy source of viable calls for clinical transplantation, METHODS： The experiment was carried out in the Biomedical Material Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences between January 2004 and December 2005. BCCs of new-born calves were isolated by collagenase digestion combined with mechanical isolation and then purified by Percoll discontinuous density gradient cantrifugation. Phase contrast microscope was used to determine the morphology of BCCs. The functional activity and purification of BCCs were identified by immunocytochemical/histochemical staining, respectively, Trypan blue assay were used to detect the viability of BCCs. RESULTS： ①Healthy BCCs concentrated between the two separating medium, and erythrocytes settled on the bottom of centrifuge tube. Cortex cells and cellular debris scattered on the separating medium of specific density,②On average, （0.7-3.1）×10^8 calls could be isolated from per adrenal gland with 99% viability, ③The isolated BCC had the functional activity of catecholamine-secretion in culture. CONCLUSION： Collagenase perfusion digestion combined with mechanical dissociation and discontinuous density gradient centrifugation can be used to isolate and purify BCCs. The purity of the cells depends on the amount of isolated calls and has general biological characteristics of catecholamine.

关 键 词：肾上腺髓质/细胞学 嗜铬颗粒 细胞分离 

分 类 号：R617[医药卫生—外科学]