Machnik Grzegorz, Skudrzyk Estera, Bułdak Łukasz, Łabuzek Krzysztof, Ruczyński Jarosław, Alenowicz Magdalena, Rekowski Piotr, Nowak Piotr Jan, Okopień Bogusław
Department of Pharmacology, Medical University of Silesia, Medyków 18, 40-752, Katowice, Poland,
Mol Biotechnol. 2015 Sep;57(9):801-13. doi: 10.1007/s12033-015-9873-2.
HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1 (coding syncytin-1, which is a glycoprotein essential in the formation of the placenta) and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV env gene and a peptide nucleic acid (PNA) probe, fully complementary to the ERVWE1 sequence fragment that efficiently blocks the polymerase action on ERVWE1 templates. The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e(2) to 1 × 10 e(8) copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1% in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.
人内源性逆转录病毒W（HERV-W）是一类多位点的人内源性逆转录病毒家族，已发现其在人类生理和病理过程中发挥重要作用。HERV-W家族的两个特定成员尤其令人关注：ERVWE1（编码合胞素-1，一种在胎盘形成中必不可少的糖蛋白）和MSRV（多发性硬化症相关逆转录病毒，由于其在多发性硬化症（MS）患者的脑组织和血细胞中表达增加，被认为在人类病理过程中起重要作用）。ERVWE1和MSRV的mRNA具有高度相似性，因此，需要一种能够专门定量临床样本中MSRV表达的方法。我们开发了一种定量聚合酶链反应（QPCR）技术，用于检测和定量多发性硬化症相关逆转录病毒。该检测方法利用了荧光标记的寡核苷酸探针，其与MSRV env基因的保守片段互补，以及一种肽核酸（PNA）探针，该探针与ERVWE1序列片段完全互补，能有效阻断聚合酶对ERVWE1模板的作用。如果在QPCR分析中与水解探针平行使用，PNA分子即使在所分析样本中ERVWE1相对于MSRV大量存在的情况下，也能极大地提高MSRV的检测效率。我们实现了1×10²至1×10⁸拷贝/反应的宽且可测量的范围；即使在MSRV相对于ERVWE1为1%的低水平下，该技术的线性也得以保持。使用我们新开发的方法，我们证实MSRV在正常人星形胶质细胞和人脐静脉内皮细胞中体外表达。我们还发现，刺激人单核细胞不会影响MSRV的特异性表达，但会导致不同HERV-W模板的mRNA水平发生变化。
