摘要
目的：介绍一种视网膜全细胞膜片钳联合单细胞逆转录聚合酶链式反应（RT-PCR）技术在视网膜单细胞信号成分分析中的应用，并研究N-甲基-D-天冬氨酸（NMDA）对ON型双极细胞中G蛋白相关mRNA表达的影响。方法：实验研究。3～5周龄C57BL／6J／小鼠20只随机分为2组，每组各10只，分别玻璃体腔注射3μlNMDA（30nmol／L）构建青光眼模型，3μl PBS（10nmol／L）作为对照组，24h后取视网膜，膜片钳下钳取单个ON型双极细胞，并做RT-PCR观察几种目的mRNA的表达差异。2组间各参数比较采用Mann．Whitney检验。结果：RT-PCR结果显示蛋白激酶Cα亚基（PKca）、瞬时感受电位离子通道1（TRPM1）、G蛋白α亚单位（Gα0）的表达在NMDA青光眼模型构建后分别为0．536±0．339、0．337±0．271、4．36±1．83，构建前后差异有统计学意义（U=O，P=-O．001；U=O，P=O．002；U=36，P=O．002o结论：NMDA诱导的青光眼模型可影响视网膜ON型双极细胞G蛋白耦联通道的信号传导。
Objective： To introduce the application of a retinal whole-cell patch clamp combined with a single-cell reverse transcription polymerase chain reaction in retinal single-cell component analysis and to investigate the effects of N-methyl-D-aspartate （NMDA） on G-protein related mRNA expression by ON-type bipolar cells of the retina. Methods： In this experimental study, twenty 3-5 weeks old C57BL/6J mice were randomly and equally divided into two groups. They then received a 3μl intravitreal injection of 30 nmol/L NMDA for the induced-glaucoma group or 10 nmol/L phosphate-buffered saline （PBS） for the control group. Twenty-four hours later, ON-type bipolar cells were collected by patch clamp electrode, and RT-PCR was used to determine the relative expression of mRNAs for protein kinase C alpha （PKCα）, transient receptor potential melastatin 1 （TRPM1）, and G-protein alpha subunit （Gtto）. The two groups were compared by Mann-Whitney tests. Results： After intravitreal injection of NMDA, the relative mRNA expression in ON- type bipolar cells of PKCct （0.536-1-0.339）, TRPM1 （0.337±0.271）, and Gcto （4.36±1.83） were significantly different from the control eyes （U=0, P=0.001; U=0,/9=-0.002; U=36, P=0.002）. Conclusion： NMDA- induced glaucoma model may affect the signal transduction of G protein-coupled channel of retinal ON bipolar cells.
出处
《中华眼视光学与视觉科学杂志》
CAS
CSCD
2017年第8期462-467,共6页
Chinese Journal Of Optometry Ophthalmology And Visual Science
基金
国家自然科学基金面上项目（81470628、81270998）
湖北省自然科学基金杰出青年项目（2014CFA019）
湖北省卫计委青年人才项目（WJ2015Q014）
