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在肾脏高效导入miR-483-5p的方法

摘要:目的:研究在肾脏高效导入目的基因miR-483-5p的方法。方法肾皮质注射miR-483-5p慢病毒:取35只C57BL/6J小鼠,随机分为空白对照组、慢病毒低剂量组(每侧肾皮质注射5 μL慢病毒)和慢病毒高剂量组(每侧肾皮质注射20 μL慢病毒),注射后7 d和21 d处死取材;构建可诱导全身过表达miR-483-5p的转基因小鼠;利用cre-loxp系统构建肾小管特异过表达miR-483-5p的转基因小鼠。3种模型小鼠利用全自动生化分析仪检测血清中尿素氮(BUN)水平判断肾功能,组织切片HE 染色观察肾脏组织结构,TUNEL法检测肾脏细胞凋亡。Real-time qPCR 检测miR-483-5p在肾脏的表达。结果3种过表达miR-483-5p小鼠肾功能均正常,肾脏组织结构无明显变化,肾脏细胞无凋亡。肾皮质注射20 μL LV3-miR-483-5p 后21 d表达最高(1.2±0.43 vs 8.6±1.09,P<0.001)。可诱导全身过表达miR-483-5p 的转基因动物在肾脏表达效率较低(0.9±0.09 vs 1.7±0.19,P<0.05),Creloxp 转基因小鼠可实现在肾脏特异性表达,且效率较高(1.6±1.13 vs 12.36±3.89,P<0.05)。结论首次构建肾小管特异过表达miR-483-5p的转基因小鼠模型,实现在肾小管特异高效表达,且不影响肾脏结构及功能,可以作为研究miR-483-5p在肾脏中的作用及其机制的良好模型;C57BL/6J小鼠每侧肾皮质注射20 μL LV3-miR-483-5p 后21 d过表达miR-483-5p效率较高,不影响肾功能,对肾组织无损伤,且模型构建时间较短,为miR-483-5p在肾脏中的作用及其机制研究提供良好的实验模型。

英文摘要:Objective To establish a method for gene delivery in murine renal tissue using lentivirus vector encoding miR-483-5p. Methods Thirty-five C57BL/6J mice were randomly divided into control group, low-dose treatment group (5 μL each kidney) , and high-dose treatment group (20 μL each kidney), and in the latter two groups, the lentivirus vector encoding miR-483-5p were injected in the renal cortex. The tissue samples were collected at 7 and 21 days after the injection. A transgenic mouse model with inducible systemic overexpression of miR-483-5p was established in TG483 mice. The Cre-loxp system was used to create a mouse model with renal tubule-specific expression of miR-483-5p. The levels of BUN in the mice were detected and HE staining and fluorometric TUNEL assay were used to observe the morphological changes of the kidneys; real-time qPCR was used to detect miR-483-5p expression in the renal cortex. Results The mice with overexpression of miR-483-5p had normal renal function without obvious pathological changes or apoptosis in the renal tissue. Renal cortex injection of 20 μL lentivirus resulted in obviously increased level of miR-483-5p at 21 days (1.2 ± 0.43 vs 8.6 ± 1.09, P<0.001). miR-483-5p showed a low expression (0.9±0.09 vs 1.7±0.19, P<0.05) in TG483 mice and a high expression in the kidney of the transgenic mice established using the Cre-loxp system (1.6±1.13 vs 12.36±3.89, P<0.05). Conclusion The transgenic mice with renal tubule-specific expression of miR-483-5p show normal renal function, and this model facilitates further study of the role of miR-483-5p in the kidney.

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[V1] 2018-06-15 21:41:27 chinaXiv:201806.00140V1 下载全文
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