仪方生物关于基因、蛋白表达分析的案例及策略浅析

常用的高通量基因和蛋白检测方法包括表达谱芯片、microRNA芯片、二代测序、蛋白质谱及代谢组学分析（2-D、MALDI-TOF-MS、iTRAQ），充分利用这些方法和工具能够起到事半功倍的效果，加速研究进程。

仪方生物会陆续推出一系列关于上述技术的案例介绍说明，希望能够为对高通量技术感兴趣的研究者提供有益的帮助，了解技术进展和具体应用，从而掌握相关的技术思路和方法。 miRNA（microRNA）表达谱芯片应用案例和说明因缺少黑素细胞诱发的白癜风是一种获得性皮肤色素功能紊乱疾病，在世界上的发病率约0.5-2%。临床上分为节段型白癜风和非节段型白癜风（泛发型白癜风），导致色素缺少的机制目前并不完全清楚。

目前有三个主要假说解释白癜风的病理机制：自身免疫、自体细胞毒性/代谢、神经功能紊乱。黑素细胞是来源于神经脊细胞的高度分化的细胞，负责色素合成。黑色素产生的依赖酪氨酸酶相关蛋白家族的参与，包括酪氨酸酶（Tyrosinase）、酪氨酸酶相关蛋白1（TYRP1）和酪氨酸酶相关蛋白1（TYRP1/DCT）等基因。

MITF调控的基因主要参与黑素合成、转运和黑素细胞的分化等。我们研究通过慢病毒介导进行MITF抑制，深入研究了microRNA表达谱的变化与TYR家族的关系。miRNA本身具有调控靶基因的作用，能够调控多种通路miRNA，可作为潜在药物靶点，在个性化治疗上有广阔的应用潜力。 The changes of microRNA expression profiles and tyrosinase related proteins in MITF knocked down melanocytesMol. BioSyst., 2012,8, 2924-2931. DOI: 10.1039/C2MB25228GAbstractMicrophthalmia-associated transcription factor (MITF) is a master regulator in melanocyte proliferation, development, survival and melanoma formation. In melanocyte dysfunction disease, it is observed that the expressions of MITF, tyrosinase (TYR), tyrosinase related protein 1 (TYRP1) and tyrosinase related protein 2 (TYRP2)/dopachrome tautomerase (DCT) are changed, the consequence of which remains unclear. In this study, we focused on the change of microRNA (miRNA) profiles and Tyrosinase Related Proteins (TRPs) in MITF knocked down melanocytes. For the first time, we assayed the MITF-KD miRNA profiles using a miRNA microarray and found that hsa-miR-1225-3p, hsa-miR-634, hsa-miR-197, hsa-miR-766, hsa-miR-574-5p and hsa-miR-328 were upregulated, and hsa-miR-720 and hsa-miR-1308 were downregulated in MITF knocked down melanocytes. These miRNAs were validated by miRNA real time qPCR. These miRNA potential targets, especially the TRPs, were analyzed according to the miRNA database (Sanger Center). By TargetScan prediction, the hsa-miR-634 and hsa-miR-328 have poorly conserved sites on TYR and hsa-miR-197 have poorly conserved sites on TYR1. Through qPCR and western blotting we found that the expression of TYR and TYRP1 were dramatically decreased and the expression of TYRP2 was increased in MITF knocked down melanocytes (MITF-KD). These results suggested that the miRNAs may be involved in MITF regulation of TYR, TYRP1 and TYRP2, which provides a new clue for understanding the role of miRNAs in melanocyte dysfunctional disease.

蛋白分析技术浅析酸敏感离子通道1a（ASIC1a）是神经系统中的关键质子受体，介导了一些酸中毒引起的神经元损伤。研究发现ASIC1a蛋白也存在于线粒体小鼠皮层神经元且与腺嘌呤核苷酸转位酶相关。ASIC1a(-/-)小鼠的纯化的线粒体表现出明显增加的Ca2 +保留能力和吸收率。

与过氧化氢（H2O2）竞争时，ASIC1a(-/-)神经元表现出抗细胞色素c释放和线粒体膜去极化，说明ASIC1a缺失可能影响线粒体通透性转换（MPT）。在ASIC1a(-/-)神经元中，依赖MPT的过氧化氢诱导的神经元死亡降低。

利用2-D二维差异凝胶电泳质谱技术分析发现线粒体中活性氧信号通路的蛋白显著上调。研究结果表明线粒体ASIC1a是MPT毛孔的重要调节器，促进氧化神经细胞死亡。 Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death. Cell Death Differ. 2013 Oct;20(10):1359-69. doi: 10.1038/cdd.2013.90. Abstract

Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a(-/-) mice exhibit significantly enhanced Ca(2+) retention capacity and accelerated Ca(2+) uptake rate. When challenged with hydrogen peroxide (H2O2), ASIC1a(-/-) neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H2O2-induced neuronal death, which is MPT dependent, is reduced in ASIC1a(-/-) neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a(-/-) mouse brain, which also displays marked changes (>2-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death. 关于仪方生物仪方生物一直致力于各种高通量技术的开发和技术服务，服务覆盖面广，技术稳定可靠，其提供的服务和产品得到了科研及临床诊断机构中的广泛应用。