Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction
Quantification of mitochondrial DNA in peripheral blood mononuclear cells and subcutaneous fat using real-time polymerase chain reaction
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DOI：
10.1016/s1386-6532(01)00195-0
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发表时间：
2001-10-01
影响因子：
8.8
通讯作者：
Wesselingh, SL
中科院分区：
文献类型：
--
作者：
Gahan, ME;Miller, F;Wesselingh, SL
背景资料：随着核苷类似物逆转录酶抑制剂（NRTI）治疗导致的HIV死亡率和疾病进展率降低，现在人们的注意力已经集中在这些治疗形式的毒性上。据信，NRTI由于其对DNA聚合酶γ的抑制而导致线粒体DNA（mtDNA）合成减少。这一假设得到了肌肉活检和人淋巴母细胞系的体外数据的支持。由此产生的线粒体毒性被认为表现为各种临床症状，包括疲劳、脂肪消耗和周围神经病变。需要对线粒体毒性进行非侵入性测试，以评估毒性并优化HIV治疗策略。外周血单个核细胞（PBMC）和皮下脂肪可能是理想的和可获得的mtDNA的来源，用于检测毒性。目的：本研究的目的是（a）开发一种定量PBMC的mtDNA拷贝数的方法，并获得可重复的结果;（B）建立皮下脂肪作为mtDNA定量来源的实用性。研究设计：通过在Ficoll-Paque上离心从血液中分离PBMC，并从两个3 mm打孔皮肤活组织检查中获得皮下脂肪。DNA提取后，通过实时聚合酶链反应（PCR）定量每个样品中的mtDNA拷贝数。结果如下：发现实时PCR测定产生一致且可重现的结果，其中在同一运行中以及在两个或更多个不同运行中进行的样品重复测定的平均变异系数分别为11.3%和17.2%。PBMC和皮下脂肪中每个细胞分别含有409 +/- 148和2042 +/- 391个mtDNA拷贝。结论：从所进行的工作可以得出结论，首先，实时PCR测定产生一致和可重复的结果，其次，mtDNA可以从PBMC和皮下脂肪中提取和定量。(C)2001 Elsevier Science B. V.保留所有权利。
Background: With decreased rates of HIV mortality and disease progression attributable to treatment with nucleoside analogue reverse transcriptase inhibitors (NRTIs), attention has now become focused on the toxicities of these forms of treatment. It is believed NRTIs cause a decrease in mitochondrial DNA (mtDNA) synthesis due to their inhibition of DNA polymerase gamma. This hypothesis is supported by in vitro data from muscle biopsies and human lymphoblastic cell lines. The resulting mitochondrial toxicity is thought to manifest itself in a variety of clinical symptoms including fatigue, fat wasting and peripheral neuropathy. A non-invasive test of mitochondrial toxicity is needed to assess toxicity and optimise HIV treatment strategies. Peripheral blood mononuclear cells (PBMC) and subcutaneous fat could be ideal and accessible sources of mtDNA for examining toxicity. Objectives: The objectives of this study were (a) to develop an assay to quantify the mtDNA copy number of PBMC and obtain reproducible results and (b) to establish the utility of subcutaneous fat as a source of mtDNA for quantification. Study Design: PBMC were isolated from blood by centrifugation over Ficoll-Paque((R)) and subcutaneous fat was obtained from two 3 mm, punch skin biopsies. Following DNA extraction, the mtDNA copy number in each sample was quantified by real-time polymerase chain reaction (PCR). Results: The real-time PCR assay was found to generate consistent and reproducible results with replicates of samples undertaken within the same run, and in two or more different runs, having a mean coefficient of variation of 11.3 and 17.2%, respectively. PBMC and subcutaneous fat contained 409 +/- 148 and 2042 +/- 391 copies of mtDNA per cell, respectively. Conclusions: From the work carried out it can be concluded that firstly, the real-time PCR assay generates consistent and reproducible results, and secondly that mtDNA can be extracted and quantified from PBMC and subcutaneous fat. (C) 2001 Elsevier Science B.V. All rights reserved.