全脑稳定表达绿色荧光蛋白的胶质瘤模型裸鼠的制备与鉴定  

The establishment and verification of a model mice express enhanced green fluorescent protein for human glioma research

作　　者：王麒龙 王爱东[2] 芮琴[2] 代兴亮[1] 陈金生[1] 沈艳华[3] 董军[1] 黄强[1] 

机构地区：[1]苏州大学附属第二医院神经外科,215004 [2]苏州大学附属第二医院实验中心,215004 [3]苏州大学实验动物中心

出　　处：《中华实验外科杂志》2016年第5期1412-1416,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81472739、81172400）;江苏省自然科学基金（BK20151214）

摘　　要：目的 以同源导入法建立近交系增强型绿色荧光（EGFP）裸小鼠并分析脑组织各部位EGFP的表达,旨在为建立稳定表达EGFP示踪的脑肿瘤原位移植模型奠定基础.方法 通过杂交-回交的方法将EGFP基因导入BALB/c背景裸小鼠,并通过基因及表型分步筛选子代,繁育新的同源导入近交系.以免疫荧光法检测模型鼠不同区域脑组织及神经干细胞EGFP表达情况.颅内接种U87-红色荧光蛋白（RFP）细胞并建立胶质瘤模型.结果 在杂交-回交第10代及其以后各代,模型鼠T淋巴细胞比例低下（小于外周血淋巴细胞总数的0.3％）,14个生化位点皆为纯合型,H-2表型为单倍型（d型）,交替植皮成功率是100％,表明已达到EGFP基因同源导入法建立近交系BALB/c荧光裸小鼠标准.模型鼠在荧光激发下全身均可见到明亮的绿色荧光,各脏器组织细胞（红细胞除外）均表达EGFP,但表达强度有差异,脑组织中EGFP呈中度表达,显著高于肝组织（肝组织中EGFP呈低度表达）.原位移植U87-RFP胶质瘤细胞株的移植瘤组织中可清晰显示其中存在的红色、绿色等颜色的荧光细胞.结论 利用同源导入法建立的新品系近交稳定表达EGFP的胶质瘤模型荧光裸小鼠,为人胶质瘤原位移植示踪研究胶质瘤及其肿瘤微环境奠定基础.Objective To establish a new congenic inbred nude mice and analyzed the expression of enhanced green fluorescent protein （EGFP） in various parts of the brain,it is designed for the study of brain tumor orthotopic transplantation.Methods Female C57BL/6-Tg （CAG-EGFP） mice were backcrossed with male BALB/c nude mice for ten generations.The genotype and phenotype were identified by biochemical locus detection,skin transplantation and flow cytometry.The U87-red fluorescent protein （RFP） cells were inoculated in mice＆#39; s brain to establish a human glioma model.Results In the 10th generation,T lymphocytes accounted for 0.3% of peripheral blood lymphocytes,retained the parental BALB/c nude mice of T lymphocytes lack of features;14 biochemical loci are homozygous,H-2 phenotype haplotype （d type）,success rate of skin transplantation is 100%,reached the standard of congenic inbred strain.There are differences in expression of brain tissue,cerebellar cortex,hippocampus,olfactory bulb,striatum,and neural stem cells showed moderate level of EGFP expression.The orthotopic glioma model show red brain tumor with green background.Conclusion The establishment of new strain into BALB/c inbred line stably expressing EGFP fluorescence,and lay a solid foundation for the study of human glioma orthotopic transplantation.

关 键 词：绿色荧光蛋白 胶质瘤模型 同源导入近交系 裸小鼠 

分 类 号：R739.4[医药卫生—肿瘤] R-332[医药卫生—临床医学]