欢迎访问《现代生物医学进展》编辑部！

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miR-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miRNAs，以确认慢病毒载体介导的miR-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miR-505 过表达老鼠下丘脑区域细胞中的miR-505 检测和示踪。结果： miR-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miR-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，miR-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miR-505 的小鼠模型，使用LNA 标记探针的FISH 方法探索miRNA 表达规律较稳定，且重复率高。

英文摘要:

Objective:A hypothalamic miR-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 miRNAs detection to validate the higher miR-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 miR-505 expression in mouse hypothalamus tissues, frozen sections of mouse hypothalamus were made and microRNA 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 hypothalamus region was positioned by stereotaxic instruments and the lentiviral vector was administered by in suit injection. To illustrate the miR-505 expression in mouse hypothalamus tissues, frozen sections of mouse hypothalamus were made and microRNA FISH (fluorescence in situ hybridization) method with LNA(locked nucleic acids) probes and TSA (tyrosine signal amplification) system and RT-PCR was applied.Conclusion:A hypothalamic miR-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 microRNA on frozen sections frommous brain.

查看全文查看/发表评论下载PDF阅读器

关闭