慢病毒介导的下丘脑中过表达miR-505小鼠模型的构建  

Construction of a Hypothalamic miR-505 Overexpression Mouse Model

作　　者：常雪莹 邓倩云 王斯佳[1] 肖君华[1] 李凯[1] 周宇荀[1] 

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《现代生物医学进展》2015年第31期6005-6008,共4页Progress in Modern Biomedicine

基　　金：中央高校基本科研业务专项资金(13D110521)

摘　　要：目的:本文用慢病毒定点注射的方法构建了在下丘脑中过表达mi R-505的小鼠模型,并利用荧光原位杂交方法在冰冻切片组织上快速检测mi RNAs,以确认慢病毒载体介导的mi R-505在丘脑中的表达能力。方法:实验小鼠在脑立体定位仪下定位到下丘脑位置,采用原位注射的方式进行慢病毒注射,注射后采用实时荧光定量RCR和应用了LNA探针和TSA系统的FISH(fluorescence in situ hybridization)技术,完成在慢病毒介导的mi R-505过表达老鼠下丘脑区域细胞中的mi R-505检测和示踪。结果:mi R-505慢病毒注射未成年小鼠下丘脑区5、10、20和40天后,均可检测到mi R-505在下丘脑区域的表达,且实验结果表明在慢病毒介导的过表达小鼠下丘脑注射部位,mi R-505表达量有明显的提高。结论:利用慢病毒注射未成年小鼠下丘脑脑区的方法,成功的建立了下丘脑中过表达mi R-505的小鼠模型,使用LNA标记探针的FISH方法探索mi RNA表达规律较稳定,且重复率高。Objective： A hypothalamic mi R-505-3p overexpression mouse model was constructed by in suit injection, and a fast and effective fluorescence in situ hybridization method within the frozen section was employed in mi RNAs detection to validate the higher mi R-505-3p expression in the target site than the other area. Methods： The hypothalamus region was positioned by stereotaxic instruments and the lentiviral vector was administered by in suit injection. To illustrate the mi R-505 expression in mouse hypothalamus tissues,frozen sections of mouse hypothalamus were made and micro RNA FISH（fluorescence in situ hybridization） method with LNA（locked nucleic acids） probes and TSA（tyrosine signal amplification） system and RT-PCR was applied. Results： The stable expression of the mi R-505 was detected in juvenile mice region of hypothalamus at day 5, 10, 20 and 40 using RT-PCR. mi R-505 expression was elevated in the injected hypothalamus compared with the control tissues. Conclusion： A hypothalamic mi R-505 Overexpression mediated by lentivirus mouse model has been established successfully by in situ injection. LNA- fluorescence in situ hybridization（FISH） is a stable and repeatable method in the detection of micro RNA on frozen sections from mous brain.

关 键 词：荧光原位杂交 Micro RNA原位杂交 锁核酸 酪胺信号放大系统 

分 类 号：Q95-3[生物学—动物学] Q75