常用的高通量基因和蛋白檢測方法包括表達譜芯片、microRNA芯片、二代測序、蛋白質譜及代謝組學分析（2-D、MALDI-TOF-MS、iTRAQ），充分利用這些方法和工具能夠起到事半功倍的效果，加速研究進程。

儀方生物會陸續推出一系列關于上述技術的案例介紹說明，希望能夠為對高通量技術感興趣的研究者提供有益的幫助，了解技術進展和具體應用，從而掌握相關的技術思路和方法。 miRNA（microRNA）表達譜芯片應用案例和說明因缺少黑素細胞誘發的白癜風是一種獲得性皮膚色素功能紊亂疾病，在世界上的發病率約0.5-2%。臨床上分為節段型白癜風和非節段型白癜風（泛發型白癜風），導致色素缺少的機制目前并不完全清楚。

目前有三個主要假說解釋白癜風的病理機制：自身免疫、自體細胞毒性/代謝、神經功能紊亂。黑素細胞是來源于神經脊細胞的高度分化的細胞，負責色素合成。黑色素產生的依賴酪氨酸酶相關蛋白家族的參與，包括酪氨酸酶（Tyrosinase）、酪氨酸酶相關蛋白1（TYRP1）和酪氨酸酶相關蛋白1（TYRP1/DCT）等基因。

MITF調控的基因主要參與黑素合成、轉運和黑素細胞的分化等。我們研究通過慢病毒介導進行MITF抑制，深入研究了microRNA表達譜的變化與TYR家族的關系。miRNA本身具有調控靶基因的作用，能夠調控多種通路miRNA，可作為潛在藥物靶點，在個性化治療上有廣闊的應用潛力。 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.

蛋白分析技術淺析酸敏感離子通道1a（ASIC1a）是神經系統中的關鍵質子受體，介導了一些酸中毒引起的神經元損傷。研究發現ASIC1a蛋白也存在于線粒體小鼠皮層神經元且與腺嘌呤核苷酸轉位酶相關。ASIC1a(-/-)小鼠的純化的線粒體表現出明顯增加的Ca2 +保留能力和吸收率。

與過氧化氫（H2O2）競爭時，ASIC1a(-/-)神經元表現出抗細胞色素c釋放和線粒體膜去極化，說明ASIC1a缺失可能影響線粒體通透性轉換（MPT）。在ASIC1a(-/-)神經元中，依賴MPT的過氧化氫誘導的神經元死亡降低。

利用2-D二維差異凝膠電泳質譜技術分析發現線粒體中活性氧信號通路的蛋白顯著上調。研究結果表明線粒體ASIC1a是MPT毛孔的重要調節器，促進氧化神經細胞死亡。 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. 關于儀方生物儀方生物一直致力于各種高通量技術的開發和技術服務，服務覆蓋面廣，技術穩定可靠，其提供的服務和產品得到了科研及臨床診斷機構中的廣泛應用。