一种碘-黄色荧光荧光蛋白-缝隙--细胞间通信方法
Summary
在这里, 我们提出了一种新的缝隙结细胞间通信检测的协议, 旨在为药物发现和毒理学评估的间隙结调节化学品的高通量筛选。
Abstract
间隙连接点 (gj) 是允许小于 1 kda 的分子在相邻细胞之间扩散的细胞膜通道。由于它们具有生理和病理作用, 因此需要进行高通量筛选 (hts) 检测, 以识别药物发现和毒理学检测中的 gj 调节剂。一种新的碘化荧光蛋白间隙连接细胞间通信 (i-yfp-gjic) 检测满足了这一需求。它是一种基于细胞的检测方法, 包括受体和供体细胞, 旨在稳定地表达黄色荧光蛋白 (yfp) 变体, 其荧光分别由碘化物或碘化物转运体 slc26a4 敏感地淬火。当碘化物被添加到两种细胞类型的混合培养中时, 它们通过 slc26a4 转运体进入供体细胞, 并通过 gj 扩散到相邻的受体细胞中, 在那里它们会淬火 yfp 荧光。yfp 荧光在动力学模式下得到了很好的测量。yfp 淬火率反映了 gj 活性。该检测可靠、快速, 可用于高温超导。介绍了利用 ln215 细胞 (人胶质瘤细胞) 进行 i-yfp-gjic 检测的方法。
Introduction
缝隙连接点 (gj) 作为细胞间通道, 允许 lt;1 kda 的小分子在相邻细胞之间扩散, 如营养物质、代谢物和信号分子。结合元素包括每个细胞中的血道或连接, 每个连接体构成六个连接 (cxs)1。gj 和 cx 已被用于毒理学检测致癌物质, 如多环芳烃 (pah), 这是 gj 抑制剂2,3,4。被破坏的 gjic 与非遗传毒性致癌 5,6有关。作为一个潜在的治疗目标, gj 的参与已报告特别是亚型的癫痫发作7, 8,保护心脏和大脑缺血再灌注损伤9, 偏头痛与光环 10,药物引起的肝损伤6,11, 和伤口愈合12。需要进行高通量筛选 (hts) 检测, 以识别用于药物发现、毒理学检测的 gj 调节化学品或抗体, 并识别 gj 活性的新型细胞调节剂。高温超导检测也可用于研究 gj 调制器 2、13、14、15的构效与活动关系。
一些 gjic 检测包括染料转移或双膜片钳技术。在染料转移试验中, 采用了路西法黄色 ch (ly) 和钙酸乙酰氧甲酯 (钙素-am)。细胞不能渗透到 ly, 它是由微注射, 刮加载, 或电穿孔引入。一旦进入细胞, ly 通过gj 扩散到邻近的细胞, gj 活动被 ly 迁移16的程度所攻击.钙素 am 检测通常涉及光漂白17,18后的跃点荧光恢复。钙素-am 是一种细胞渗透染料, 通过固有的酯酶在体内转化为不透水的钙蛋白。该检测需要一个共聚焦显微镜来观察激光光漂白后周围的细胞中钙蛋白-am 的转移。如果存在功能 gj, 相邻细胞中的钙蛋白 am 进入光细胞, 荧光被恢复。gj 活性是通过光细胞荧光恢复的程度来检测的。染料转移检测既费力又耗时, 或灵敏度低。双贴片夹紧是一种测量结合部电导的电生理方法。它是相对地敏感的, 以电导的直接依赖性在开放 gj的数量 19;然而, 它是技术要求高, 耗时, 和昂贵的20。i-yfp-gjic 检测方法是为高温超导系统中的应用而开发的。
图 1说明了 i-yfp gjic 分析的组成和步骤, 该分析利用表达具有 h148q 和 iodide-sensitive (yfpql)的碘化物敏感 yfp 变体的受体以及表达碘化物转运体 (slc26a4) 21 的供体细胞.yfpql 携带的两种突变允许碘化物22对荧光进行淬火。在共培养的受体和供体细胞中添加碘化物;它们不进入受体细胞, 而是被存在于供体细胞上的 slc26a4 转运体吸收。供体细胞中的碘化物通过功能 gj 扩散到相邻的受体细胞中, 在那里它们会淬火yfp ql 荧光。如果 gj 被抑制剂关闭或阻断, 碘化物不能进入受体细胞来抑制荧光。yfpql淬火率反映了 gj 活性。i-yfp gjic 检测程序既不复杂, 也不耗时。它与高温超导系统兼容, 可用于在相对较短的时间内测试大量化合物对 gj 活性的影响。它只需要受体和供体细胞, 以及两个平衡的盐溶液。下面描述的协议基于 ln215 细胞, 其主要 cx 为 cx4321。ln215-yfpql受体和 ln215-i–供体细胞是通过表达 yfpql 或slc26a421、23的慢病毒传导而产生的。
Protocol
Representative Results
Discussion
i-yfp-gjic 检测方法可用于高温超导系统, 因为它坚固、快速且价格低廉。如果 z ‘-因子高于 0.525,则 hts 检测被认为是鲁棒性。有关用于评估高温超导检测25适宜性的统计分析的说明,请参见 zhang 等人。当 ln215 细胞被使用时, z ‘-因子是 & gt;0.5 没有任何分析优化。如果在检测中使用其他细胞类型, 其 z ‘-因子为 & & lt;0.5, 则可以通过延长检测时间21来提…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究得到了由教育部资助的韩国国家研究基金会 (2011-0023701、2016r1d1a02929397 和 2016R1D1A1A02937397) 资助的基础科学研究方案。
Materials
| 96-well plate | SPL | 30096 | |
| Calcium chloride (CaCl2) | Sigma | C5670 | I-solution |
| D-(+)-Glucose | Sigma | G7021 | C-solution, I-solution |
| Dimethyl sulfoxide (DMSO) | sigma | 276855 | |
| HEPES | Sigma | RES6003H-B7 | C-solution, I-solution |
| Lipofectamine 2000 | Invitrogen | 11668-027 | transfection reagent |
| Magnesium chloride hexahydrate (MgCl2 6H2O) | Sigma | M2393 | C-solution |
| Microplate reader | BMG LabTech | POLARstar Omega 415-1618 | |
| pMD2.G | Addgene | #12259 | |
| Polybrene | sigma | H9268 | |
| Poly-L-lysine solution | sigma | P4707 | |
| Potassium chloride (KCl) | Sigma | P5405 | C-solution, I-solution |
| psPAX2 | Addgene | #12260 | |
| Puromycin Dihydrochloride | sigma | P8833 | |
| Sodium chloride (NaCl) | Sigma | S5886 | C-solution, I-solution |
| Sodium hyroxide (NaOH) | Sigma | S2770 | |
| Sodium Iodide (NaI) | Sigma | 383112 | I-solution |
Riferimenti
- Goodenough, D. a., Goliger, J. a., Paul, D. L. Connexins, connexons, and Intercellular Communication. Annual Review of Biochemistry. 65, 475-502 (1996).
- Upham, B. L., Weis, L. M., Trosko, J. E. Modulated Gap Junctional Intercellular Communication as a Biomarker of PAH Epigenetic Toxicity: Structure-Function Relationship. Environmental Health Perspectives. 106, 975 (1998).
- U, J. E. T., Chang, C., Upham, B., Wilson, M. Epigenetic toxicology as toxicant-induced changes in intracellular signalling leading to altered gap junctional intercellular communication. Toxicology Letters. , 71-78 (1998).
- Yamasaki, H. Role of disrupted gap junctional intercellular communications in detection and characterization of carcinogens. Mutation Research – Reviews in Genetic Toxicology. , (1996).
- Yamasaki, H., et al. Gap junctional intercellular communication and cell proliferation during rat liver carcinogenesis. Environmental Health Perspectives. 101, 191-197 (1993).
- Vinken, M., et al. Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity. Critical Reviews in Biochemistry and Molecular Biology. 44, 201-222 (2009).
- Fonseca, C. G., Green, C. R., Nicholson, L. F. B. Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy. Brain Research. 929 (1), 105-116 (2002).
- Garbelli, R., et al. Expression of connexin 43 in the human epileptic and drug-resistant cerebral cortex. Neurology. 76 (10), 895-902 (2011).
- Schulz, R., et al. Connexin 43 is an emerging therapeutic target in ischemia/reperfusion injury, cardioprotection and neuroprotection. Pharmacology and Therapeutics. 153, 90-106 (2015).
- Sarrouilhe, D., Dejean, C., Mesnil, M. Involvement of gap junction channels in the pathophysiology of migraine with aura. Frontiers in Physiology. , (2014).
- Patel, S. J., et al. Gap junction inhibition prevents drug-induced liver toxicity and fulminant hepatic failure. Nature Biotechnology. 30 (2), 179-183 (2012).
- Kandyba, E. E., Hodgins, M. B., Martin, P. E. A murine living skin equivalent amenable to live-cell imaging: analysis of the roles of connexins in the epidermis. Journal of Investigative Dermatology. 128 (4), 1039-1049 (2008).
- Upham, B. L., et al. Differential roles of 2 , 6 , and 8 carbon ceramides on the modulation of gap junctional communication and apoptosis during carcinogenesis. Cancer Letters. 191, 27-34 (2003).
- Upham, B. L., et al. Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems. Environmental Health Perspectives. 117 (4), 545-551 (2009).
- Weis, L. M., et al. Bay or Baylike Regions of Polycyclic Aromatic Hydrocarbons Were Potent Inhibitors of Gap Intercellular Communication. Environmental Health Perspectives. 106 (1), 17-22 (1998).
- el-Fouly, M. H., Trosko, J. E., Chang, C. C. Scrape-Loading and Dye Transfer: A Rapid and Simple Technique to Study Gap Junctional Intercellular Communication. Experimental Cell Research. 168 (2), 422-430 (1987).
- Wade, M. H., Trosko, J. E., Schindler, M. Photobleaching Assay of Gap Junction- Mediated Communication Between Human Cells. Advancement of Science. 232 (4749), 525-528 (2010).
- Abbaci, M., et al. In vitro characterization of gap junctional intercellular communication by gap-FRAP technique. Proceedings of SPIE. , 585909 (2005).
- Neyton, J., Trautmann, A. Single-channel currents of an intercellular junction. Nature. 317 (6035), 331-335 (1985).
- Wilders, R., Jongsma, H. J. Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophysical Journal. 63 (4), 942-953 (1992).
- Lee, J. Y., Choi, E. J., Lee, J. A new high-throughput screening-compatible gap junctional intercellular communication assay. BMC Biotechnology. 15 (1), 1-9 (2015).
- Galietta, L. J. V., Haggie, P. M., Verkman, A. S. Green fluorescent protein-based halide indicators with improved chloride and iodide affinities. FEBS Letters. 499 (3), 220-224 (2001).
- Lee, J. Y., Yoon, S. M., Choi, E. J., Lee, J. Terbinafine inhibits gap junctional intercellular communication. Toxicology and Applied Pharmacology. 307, 102-107 (2016).
- Hughes, J., Rees, S., Kalindjian, S., Philpott, K. Principles of early drug discovery. British Journal of Pharmacology. 162 (6), 1239-1249 (2011).
- Zhang, J. H., Chung, T. D. Y., Oldenburg, K. R. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening. 4 (2), 67-73 (1999).
- Upham, B. L. Role of Integrative Signaling Through Gap Junctions in Toxicology. Current Protocols in Toxicology. , (2011).
- Schalper, K. A., et al. Modulation of gap junction channels and hemichannels by growth factors. Molecular BioSystems. 8 (3), 685-698 (2012).
- Kulkarni, G. V., Mcculloch, C. A. G. Serum deprivation induces apoptotic cell death in a subset of Balb / c 3T3 fibroblasts. Journal of Cell Science. 1179, 1169-1179 (1994).
- Harris, A. L., Locke, D. Permeability of Connexin Channels. Connexins. , 165-206 (2009).
- Choi, E. J., Yeo, J. H., Yoon, S. M., Lee, J. Gambogic Acid and Its Analogs Inhibit Gap. Junctional Intercellular Communication. 9, 1-10 (2018).
