人体胚胎肾上腺嗜铬细胞原代培养和生长特征的研究.DOCVIP

人胚早期肾上腺嗜铬细胞体外培养和生长特征 周华芳 （贵阳医学院公共卫生学院 贵阳 550004） 摘要：目的 建立人体胚胎早期肾上腺嗜铬细胞体外培养。方法 取胎龄为7-12周的肾上腺用剪切分离法分离培养胚胎肾上腺嗜铬细胞，用嗜铬粒蛋白A（CgA）免疫组化染色鉴定细胞的性质，用增殖细胞核抗原（PCNA）的表达来分析细胞 增殖能力。结果 细胞培养5-15天后，贴壁型细胞生长良好；培养3或4周后出现悬浮类似神经球细胞；培养8-10周后，细胞逐渐减少。培养的两型细胞CgA免疫组化染色均呈阳性，贴壁型细胞在本实验条件下具有一定增殖能力，并与体内的增殖能力进行比较。结论 本研究成功的建立了人胚早期肾上腺嗜铬细胞的体外培养方法，培养的两型细胞均具有嗜铬细胞特异型抗原标志物CgA的表达和增殖能力，国内未见报告。 关键词：人体胚胎肾上腺嗜铬细胞 体外培养 增殖能力 Characteristies of growth and culture of human fetal chromaffin cell Zhou huafang (Guiyang medical college, Guiyang 550004) Abstract: Objective To establish a method of culturing of human fetal chromaffin cell. Methods Chromaffin celll from human fetal adrenal glands by surgical operation between 7-12 gestational week (GW) were cultured. The cell identification was performed with immunohistochemical staining of chromogranin A(CgA), expression of PCNA at nuclei was used to proliferative analysis. Results The growth of adherent colonies was better from 5th-15th days after plating. Neurosphere-like colonies appeared after three to four weeks in the same culture. After 8th-10th weeks of culture, the cell number is decreasing, two types of populations showed CgA positive reaction. The adherent colonies under the condition of this experiment has a certain proliferation, comparing with the proliferative in vivo at the same period. Conclusion The method of human fetal chromaffin cell culturing has been well establish, the isolated and cultured cells possess the expression of CgA and the capability of proliferation. Key words human fetal chromaffin cell culture proliferation 第一作者：电话：0851-6908908，E-mail: zhouhuafang@ 肾上腺嗜铬细胞来源于外胚神经嵴细胞，与外周交感神经元在生物化学，生理学特征上有很多相似的特征[1]。目前临床通过蛛网膜下腔移植或脑移植肾上腺嗜铬细胞可治疗慢性顽固性疼痛和帕金森病[2,3]，然而由于健康成人肾上腺源有限，研究人员趋向于使用人体胚胎肾上腺嗜铬细胞，并建立细胞库以供临床使用。本文拟建立人体胚胎肾上腺嗜铬细胞体外培养的方法，并对该细胞进行生物学、形态学鉴定及生长特征的研究，为进一步的研究提供依据。 材料与方法 材料 HBSS缓冲液，DMEM/F12培养基, bFGF, EGF, 胰蛋白酶和EDTA均购自Invitrogen公司；胎牛血清；谷氨酰胺；胰岛素；20ug/ml纤维结合素；0.05%胰蛋白酶；0.02%EDTA和4%甲醛缓冲液均购自Sigma公司；嗜铬粒蛋白A（CgA）抗体；PCNA抗体；LSAB II+ kit；Envisi