CAJ | 학술논문

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Foxn1nu.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EG FP mRNA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EGFP mRNA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.
목적 분석증강형록색형광단백(EGFP)전기인라소서(록색라소서)뇌조직중EGFP적표체정황,위록색라소서적뇌내이식응용제공의거. 방법 이본실험실자건적록색라소서Foxn1nu.B6-CAG-EGFP/SU위실험대상,채용반정량RT-PCR화형광정량PCR분석소서주요기관조직/세포화뇌불동부위조직중EG FP mRNA표체수평,통과형광현미경관찰소서전신주요기관조직화전뇌불동층면조직중록색형광강도,응용형광분광광도계정량분석、면역조화염색정성관찰소서뇌불동부위조직중EGFP단백함량급양성세포적표체. 결과 반정량RT-PCR결과현시,록색라소서주요기관조직/세포균불동정도표체EGFP.형광현미경하관찰도뇌불동층면조직균능현시록색형광,차량도정현부위차이,기중소뇌피층、해마、후구등부위형광교량.형광정량PCR화형광분광광도계정량분석표명,여대뇌피질상비,소뇌피층、해마화후구등조직EGFP mRNA화단백적표체수평교고.면역조화염색현시,소뇌포긍야세포、해마추체세포、후구과립세포등정현EGFP강양성반응. 결론 록색라소서뇌조직균불동정도표체EGFP,기중소뇌피층、해마화후구등부위표체교고,인차뇌야괄합주뇌내이식실험연구.
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 Foxn1nu.B6-CAG-EGFP/SU green nude mice were chosen;the EGFPmRNA 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 frozen 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 and fluorescence 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.