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3μl NMDA(30 nmol/L)构建青光眼模型,3μl PBS(10 nmol/L)作为对照组,24 h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(PKCα)、瞬时感受电位离子通道1(TRPM1)、G蛋白α亚单位(Gαo)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=0,P=0.001;U=0,P=0.002;U=36,P=0.002).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.

abstractsObjective: 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 3 μl 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 (Gαo). 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 PKCα (0.536±0.339), TRPM1 (0.337±0.271), and Gαo (4.36±1.83) were significantly different from the control eyes (U=0, P=0.001; U=0, P=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.