绿色裸小鼠脑组织中增强型绿色荧光蛋白表达分析

Enhanced green fluorescent protein expression in brain tissues of green nude mice

目的分析增强型绿色荧光蛋白（EGFP）转基因裸小鼠（绿色裸小鼠）脑组织中EGFP的表达情况，为绿色裸小鼠的脑内移植应用提供依据。方法以本实验室自建的绿色裸小鼠FoxnlTnu．B6-CAG-EGFP／SU为实验对象，采用半定量RT-PCR和荧光定量PCR分析小鼠主要器官组织／细胞和脑不同部位组织中EGFPmRNA表达水平，通过荧光显微镜观察小鼠全身主要器官组织和全脑不同层面组织中绿色荧光强度．应用荧光分光光度计定量分析、免疫组化染色定性观察小鼠脑不同部位组织中EGFP蛋白含量及阳性细胞的表达。结果半定量RT-PCR结果显示，绿色裸小鼠主要器官组织／细胞均不同程度表达EGFP。荧光显微镜下观察到脑不同层面组织均能显示绿色荧光，且亮度呈现部位差异，其中小脑皮层、海马、嗅球等部位荧光较亮。荧光定量PCR和荧光分光光度计定量分析表明，与大脑皮质相比，小脑皮层、海马和嗅球等组织EGFPmRNA和蛋白的表达水平较高。免疫组化染色显示，小脑浦肯野细胞、海马锥体细胞、嗅球颗粒细胞等呈现EGFP强阳性反应。结论绿色裸小鼠脑组织均不同程度表达EGFP，其中小脑皮层、海马和嗅球等部位表达较高．因此脑也适合做脑内移植实验研究。

Objective To analyze the expression levels of enhanced green fluorescent protein （EGFP） in brain tissues ofEGFP transgenic mice for providing the basis of brain transplantation in green nude mice. Methods Foxnlr^nu.B6-CAG-EGFP/SU green nude mice were chosen; the EGFP mRNA expression level in various organ tissues/cells and different parts of the brain were analyzed by semi-quantitation real time-PCR or quantitative PCR （qPCR）; fluorescence microscopy was used to detect the green fluorescence in main organ tissues and 12 brain coronal frozen tissues were directly observed to survey the patterns of EGFP expression. Furthermore, the EGFP protein expression and EGFP positive cells in different parts of brain were detected by fluorescence spectrophotometer assay and immunohistochemistry. Results The level of EGFP expression varied among main organs tissues/cells analyzed by semi-quantitation real time-PCR. The green fluorescence could be observed in 12 coronal fi-ozen sections of brain with intensity differences under the fluorescence microscope, and the fluorescence intensity was higher in the cerebellar cortex, hippocampus and olfactory bulb tissue. As compared with those in the cerebral cortex tissue, the EGFP mRNA and protein expression levels in the cerebellum, hippocampus and olfactory bulb tissues were obviously higher showed by qPCR andfluorescence spectrophotometer assay. Meanwhile, the immunohistochemistry results showed that there were some cells which strongly expressed EGFP in the cerebellum, hippocampus and olfactory bulb tissues. Conclusions EGFP is positively expressed in the brain of green fluorescence nude mice, especially in the cerebellum, hippocampus and olfactory bulb tissues, which indicates that the brain may be useful for human cancer transplantation research.