全细胞膜片钳结合单细胞RT-PCR技术在NMDA诱导的小鼠青光眼模型视网膜双极细胞G蛋白通路研究中的应用

doi:DOI:10.3760/cma.j.issn.1674-845X.2017.08.003

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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3 μl NMDA（30 nmol/L）构建青光眼模型，3 μl 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。

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	沈吟

关键词 ：膜片钳, 单细胞逆转录聚合酶链式反应, 视网膜双极细胞, N-甲基-D-天冬氨酸, G蛋白, 小鼠

Abstract：

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 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 ONtype 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: NMDAinduced glaucoma model may affect the signal transduction of G protein-coupled channel of retinal ON bipolar cells.

Key words： patch clamp single-cell RT-PCR retina bipolar cell N-methyl-D-aspartate G-protein mice

收稿日期: 2017-03-29

基金资助:

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）

通讯作者: 沈吟，Email：yinshen@whu.edu.cn

引用本文:

高青,沈雨濛,娄翔峰,罗雪,卢苇,沈吟. 全细胞膜片钳结合单细胞RT-PCR技术在NMDA诱导的小鼠青光眼模型视网膜双极细胞G蛋白通路研究中的应用[J]. 中华眼视光学与视觉科学杂志, 2017, 19(8): 462-467. Qing Gao,Yumeng Shen,Xiangfeng Lou,Xue Luo,Wei Lu,Yin Shen. Application of the Whole-cell Patch Clamp Combined with Single-cellRT-PCR in an NMDA-induced Glaucoma Mouse Model of the G-proteinPathway in Retinal ON-type Bipolar Cells. Chinese Journal of Optometry Ophthalmology and Visual science, 2017, 19(8): 462-467. DOI: DOI:10.3760/cma.j.issn.1674-845X.2017.08.003

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[1]	Lambolez B, Audinat E, Bochet P, et al. AMPA receptor subunits expressed by single Purkinje cells. Neuron, 1992, 9(2): 247-258.
[2]	Ramakrishnan H, Dhingra A, Tummala SR, et al. Differential function of Gγ13 in rod bipolar and ON cone bipolar cells. J Physiol, 2015, 593(7): 1531-1550. DOI:10.1113/jphysiol.2014.281196.
[3]	Shen Y, Rampino MA, Carroll RC, et al. G-protein-mediated inhibition of the Trp channel TRPM1 requires the Gβγ dimer.Proc Natl Acad Sci USA, 2012, 109(22): 8752-8757. DOI: 10.
10	73/pnas.1117433109.
[4]	Tummala SR, Dhingra A, Fina ME, et al. Lack of mGluR6-related cascade elements leads to retrograde trans-synaptic effects on rod photoreceptor synapses via matrix-associated
	proteins. Eur J Neurosci, 2016, 43(11): 1509-1522. DOI: 10.1111/ejn.13243.
[5]	Devi S, Markandeya Y, Maddodi N, et al. Metabotropic glutamate receptor 6 signaling enhances TRPM1 calcium channel function and increases melanin content in human melanocytes.Pigment Cell Melanoma Res, 2013, 26(3): 348-356. DOI: 10.1111/pcmr.12083.
[6]	Xu Y, Orlandi C, Cao Y, et al. The TRPM1 channel in ONbipolar cells is gated by both the α and the βγ subunits of the G-protein Go. Sci Rep, 2016, 6: 20940. DOI:10.1038/srep20940.
[7]	Koike C, Obara T, Uriu Y, et al. TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade. Proc Natl Acad Sci U S A, 2010, 107(1): 332-337. DOI:10.1073/pnas.0912730107.
[8]	Dreyer EB, Zurakowski D, Schumer RA, et al. Elevated glutamate levels in the vitreous body of humans and monkeys with glaucoma. Arch Ophthalmol, 1996, 114(3): 299-305.
[9]	Euler T, Haverkamp S, Schubert T, et al. Retinal bipolar cells: elementary building blocks of vision. Nat Rev Neurosci, 2014,15(8): 507-519.
[10]	Ruether K, Feigenspan A, Pirngruber J, et al. PKC alpha is essential for the proper activation and termination of rod bipolar cell response. Invest Ophthalmol Vis Sci, 2010, 51(11): 6051-6058. DOI: 10.1167/iovs.09-4704.
[11]	Curinha A, Oliveira BS, Pereira-Castro I, et al. Implications of polyadenylation in health and disease. Nucleus, 2014, 5(6): 508-519. DOI: 10.4161/nucl.36360.
[12]	Audinat E, Lambolez B, Rossier J, et al. Activity-dependent regulation of N-methyl-D-aspartate receptor subunit expression in rat cerebellar granule cells. Eur J Neurosci, 1994, 6(12):1792-1800.
[13]	Kanumilli S, Tringham EW, Payne CE, et al. Alternative splicing generates a smaller assortment of CaV2.1 transcripts in cerebellar Purkinje cells than in the cerebellum. Physiol Genomics,2006, 24(2): 86-96. DOI: 10.1152/physiolgenomics.00149.2005.
[14]	Fries JE, Goczalik IM, Wheeler-Schilling TH, et al. Identification of P2Y receptor subtypes in human müller glial cells by physiology, single cell RT-PCR, and immunohistochemistry.
	Invest Ophthalmol Vis Sci, 2005, 46(8): 3000-3007. DOI: 10.1167/iovs.05-0043.
[15]	Shekhar K, Lapan SW, Whitney IE, et al. Comprehensive Classification of Retinal Bipolar Neurons by Single-Cell Transcriptomics. Cell, 2016, 166(5): 1308-1323. e30. DOI: 10.
10	16/j.cell.2016.07.054.