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Determination of Mitochondrial DNA Upon Drug Treatment
药物处理后的线粒体DNA测定   

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Abstract

Drug-induced mitochondrial injury can be caused by many different mechanisms including inhibition of mitochondrial DNA replication, transcription, translation, and altered protein function. Determination of the level of mitochondrial DNA relative to the nuclear DNA levels provides important information on potential mitochondrial toxicity.

Keywords: Mitochondrial toxicity(线粒体毒性), Nucleoside analogs(核甘酸类似物), Inhibitor of DNA synthesis(DNA合成抑制剂)

Materials and Reagents

  1. HepG2 cells (ATCC, catalog number: HB 8065 )
  2. DMSO (cell culture grade) (Sigma-Aldrich, catalog number: D2650 )
  3. Phosphor-buffered saline (PBS) (Life Sciences, catalog number: 10010049 )
  4. QIAamp DNA mini kit (QIAGEN, catalog number: 51304 )
  5. TaqMan universal mastermix (Life Technologies, Applied Biosystems®, catalog number: 4352042 )
  6. β-actin Assay-on-Demand kit (Life Technologies, Applied Biosystems®, catalog number: 4331182 )
  7. Eagle’s minimum essential medium (Life Technologies, Gibco®, catalog number: 41090 )
  8. GlutaMAXTM
  9. Fetal bovine serum (FBS) (HyClone, catalog number: SH30071.03 )
  10. 100 units/ml penicillin, 100 units/ml streptomycin (Life Technologies, Gibco®, catalog number: 15140 )
  11. Sodium pyruvate (Life Technologies, Gibco®, catalog number: 11360 )
  12. Cells were cultured in Eagle’s minimum essential medium (see Recipes)
    Note: Cells were cultured in Eagle’s minimum essential medium.

Equipment

  1. 12-well plates (Corning, catalog number: 3513 )
  2. ABI Prism 7900HT Fast Real-Time PCR system (Life Technologies, Applied Biosystems®)

Procedure

  1. HepG2 cells were seeded into 12-well plates at a density of 2 x 105 cells per well and allowed to attach overnight. The volume of medium was 1.0 ml in each well.
  2. After the overnight incubation, the media in each well was replaced with 1.0 ml of fresh media containing tested compounds and controls, and incubated for 10 more days. The media was replaced with fresh media and compounds every 3 to 4 days. The DMSO concentration was kept at 1.0% for all treatments including control samples (no drug, DMSO only).
  3. Following the incubation, the cells were washed once with PBS and the total DNA was extracted from the cells using the QIAamp DNA Mini Kit according to the manufacturer’s protocol.
  4. Real-time PCR reactions were performed using TaqMan universal mastermix in an ABI Prism 7900HT Fast Real-Time PCR System.
  5. Quantification of mtDNA was achieved by amplification of a fragment of the mitochondrial specific cytochrome b gene using the primers and probe described in Table 1. Chromosomal DNA was quantified by the amplification of a fragment of the β-actin gene using a β-actin Assay-on-Demand kit.
  6. Amplification reactions for the quantification of mitochondrial and chromosomal DNA were performed independently using approximately 25 ng of total DNA in a volume of 20 μl.

    Table 1. Real-Time PCR primers and probe used in the quantification of the cytochrome b gene from HepG2 cells
    Primer/probe name
    Oligonucleotide sequence
    Concentration in qPCR
    Cytochrome b forward
    CCTTCCACCCTTACTACACAATCAA
    0.9 μM
    Cytochrome b reverse
    GGTCTGGTGAGAATAGTGTTAATGTCA
    0.9 μM
    Cytochrome b probe
    FAM-ACGCCCTCGGCTTAC-BHQ1
    0.2 μM

  7. Data analysis
    1. The relative amount of mtDNA in treated samples was determined using a relative quantification method based upon the 2-ΔΔCT formula (Livak and Schmittgen, 2001).
    2. The amount of mtDNA (% mtDNA) in compound treated samples relative to the DMSO treated controls was calculated based upon the following formula:
      % mtDNA = 100 x 2-ΔΔCT
      ΔΔCT = ΔCT, treated – ΔCT, control
      ΔCT, treated = (CT, cyt b – CT, β-actin) treated
      ΔCT, control = (CT, cyt b – CT, β-actin) control
      CT, cyt b and CT, β-actin represent the cycle threshold values for the amplification of cytochrome b and β-actin, respectively, as determined by the computational analysis of amplification curves using the ABI Prism software. The final results are presented as the mean % mtDNA ± SD from 3 independent experiments, each performed in triplicate.
    3. The 2-ΔΔCT method was validated for cytochrome b and β-actin genes by determining the ΔCT values for amplification reactions containing various amounts of total cellular DNA. Minimal differences were observed in the ΔCT values in samples containing 5 to 40 ng of total cellular DNA; indicating that neither the amplification nor detection efficiencies of cytochrome b and β-actin were affected by the amount of DNA template within the dilution range relevant for the quantitative analysis performed in this study Table 2.
    4. The effect of a positive control compound ddC (dideoxy cytidine) is shown in Table 3.

      Table 2. Validation of the 2-ΔΔCT method for cytochome b and β-actin target genes
      Amount of total cellular DNA (ng/reaction)
       CT Valuea
      ΔCT Value
      Cytochome b
      β-actin
      5
      15.7 ± 0.4
      22.2 ± 0.3
      -6.6 ± 0.1
      10
      17.3 ± 0.4
      23.9 ± 0.3
      -6.7 ± 0.1
      20
      18.8 ± 0.3
      25.7 ± 0.3
      -6.9 ± 0.1
      40
      20.3 ± 0.4
      27.3 ± 0.3
      -7.0 ± 0.1
      aThe data represent the mean ± SD of 3 independent experiments performed in triplicate

      Table 3. Effect of positive control ddC on the levels of mtDNA in HepG2 cells
      Compound
      Concentration
      (μM)
      Relative amount of
      mtDNA (% mtDNA)a
      p-value compared to DMSO (control)b
      DMSO (control)
      -
      100.0 ± 8.8
      -
      ddC
      0.2
      57.0 ± 10.4
      < 0.0001
      2.0
      25.1 ± 7.8
      < 0.0001
      20
      6.9 ± 2.9
      < 0.0001
      aThe data represent the mean ± SD of 3 independent experiments performed in triplicate
      bPaired, two-tailed Student’s t-test

Recipes

  1. Eagle’s minimum essential medium
    GlutaMAXTM
    10% fetal bovine serum
    100 units/ml penicillin
    100 units/ml streptomycin
    1 mM sodium pyruvate

Acknowledgments

All of the work was sponsored by Gilead Sciences, Inc. This protocol was adapted from Feng et al. (2014).

References

  1. Feng, J. Y., Cheng, G., Perry, J., Barauskas, O., Xu, Y., Fenaux, M., Eng, S., Tirunagari, N., Peng, B., Yu, M., Tian, Y., Lee, Y. J., Stepan, G., Lagpacan, L. L., Jin, D., Hung, M., Ku, K. S., Han, B., Kitrinos, K., Perron, M., Birkus, G., Wong, K. A., Zhong, W., Kim, C. U., Carey, A., Cho, A. and Ray, A. S. (2014). Inhibition of hepatitis C virus replication by GS-6620, a potent C-nucleoside monophosphate prodrug. Antimicrob Agents Chemother 58(4): 1930-1942.
  2. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408.

简介

药物诱导的线粒体损伤可以由许多不同的机制引起,包括线粒体DNA复制,转录,翻译和改变的蛋白质功能的抑制。 相对于核DNA水平测定线粒体DNA的水平提供了关于潜在线粒体毒性的重要信息。

关键字:线粒体毒性, 核甘酸类似物, DNA合成抑制剂

材料和试剂

  1. HepG2细胞(ATCC,目录号:HB 8065)
  2. DMSO(细胞培养级)(Sigma-Aldrich,目录号:D2650)
  3. 磷酸缓冲盐水(PBS)(Life Sciences,目录号:10010049)
  4. QIAamp DNA mini试剂盒(QIAGEN,目录号:51304)
  5. TaqMan通用主混合物(Life Technologies,Applied Biosystems ,目录号:4352042)
  6. β-生物素测定试剂盒(Life Technologies,Applied Biosystems ,目录号:4331182)
  7. Eagle的最低必需培养基(Life Technologies,Gibco ,目录号:41090)
  8. GlutaMAX TM
  9. 胎牛血清(FBS)(HyClone,目录号:SH30071.03)
  10. 100单位/ml青霉素,100单位/ml链霉素(Life Technologies,Gibco ,目录号:15140)。
  11. 丙酮酸钠(Life Technologies,Gibco ,目录号:11360)
  12. 细胞在Eagle's最低必需培养基(参见Recipes)中培养 注意:细胞在Eagle's最低必需培养基中培养。

设备

  1. 12孔板(Corning,目录号:3513)
  2. ABI Prism 7900HT快速实时PCR系统(Life Technologies,Applied Biosystems )

程序

  1. 将HepG2细胞以2×10 5个细胞/孔的密度接种到12孔板中,并使其附着过夜。每孔中培养基的体积为1.0ml
  2. 过夜温育后,用1.0ml含有测试化合物和对照的新鲜培养基替换每个孔中的培养基,并孵育10天以上。每3至4天用新鲜培养基和化合物替换培养基。对于包括对照样品(无药物,仅DMSO)的所有处理,DMSO浓度保持在1.0%
  3. 孵育后,用PBS洗涤细胞一次,并使用QIAamp DNA Mini试剂盒根据制造商的方案从细胞中提取总DNA。
  4. 使用TaqMan通用主混合物在ABI Prism 7900HT快速实时PCR系统中进行实时PCR反应。
  5. 通过使用表1中描述的引物和探针扩增线粒体特异性细胞色素b基因的片段来实现mtDNA的定量。通过使用β-肌动蛋白测定 - 扩增β-肌动蛋白基因的片段来扩增染色体DNA,随需应变套件。
  6. 使用大约25ng体积为20μl的总DNA独立地进行用于线粒体和染色体DNA的定量的扩增反应。

    表1.用于定量HepG2细胞的细胞色素b基因的实时PCR引物和探针
    引物/探针名称
    寡核苷酸序列
    qPCR中的浓度
    细胞色素b向前移动
    CCTTCCACCCTTACTACACAATCAA
    0.9μM
    细胞色素b反转
    GGTCTGGTGAGAATAGTGTTAATGTCA
    0.9μM
    细胞色素b探针
    FAM-ACGCCCTCGGCTTAC-BHQ1
    0.2μM

  7. 数据分析
    1. 处理样品中mtDNA的相对量使用a 相对定量方法基于2 -ΔΔC公式(Livak和 Schmittgen,2001)。
    2. 化合物中mtDNA的量(%mtDNA) 计算相对于DMSO处理的对照的处理的样品 基于以下公式:
      %mtDNA = 100 x 2 -ΔΔC
      ΔΔC =ΔC,处理 - △C ,控制
      ΔC T,处理的(C T,cyt b-C ,β-肌动蛋白) /> ΔC T,对照 =(C T,cyt b-C ,β-肌动蛋白) /> C th,cyt b和C sub,β-肌动蛋白代表对于 分别扩增细胞色素b和β-肌动蛋白,如所确定的 通过使用ABI的扩增曲线的计算分析 Prism软件。最终结果表示为平均%mtDNA±SD  来自3个独立实验,每个实验一式三份
    3. 通过以下方法验证了细胞色素b和β-肌动蛋白基因的2 -ΔΔC 确定包含的扩增反应的ΔC值 各种量的总细胞DNA。观察到最小差异  在含有5至40ng总细胞的样品中的ΔC值 脱氧核糖核酸;表明既不进行扩增也不检测 细胞色素b和β-肌动蛋白的效率受量的影响 DNA模板在稀释范围内与定量相关 本研究中进行的分析表2
    4. 阳性对照化合物ddC(双脱氧胞苷)的效果示于表3中
      表2.验证2 -ΔΔC T 细胞色素b和 β - 肌动蛋白靶基因
      细胞总DNA量(ng /反应)
        C T 价值 a
      Δ C T 价值
      细胞素b
      β-肌动蛋白
      5
      15.7±0.4
      22.2±0.3
      -6.6±0.1
      10
      17.3±0.4
      23.9±0.3
      -6.7±0.1
      20
      18.8±0.3
      25.7±0.3
      -6.9±0.1
      40
      20.3±0.4
      27.3±0.3
      -7.0±0.1
      a 数据代表三次重复进行的3次独立实验的平均值±SD
      表3.阳性对照ddC对HepG2细胞中mtDNA水平的影响
      化合物
      集中
      (μM)
      相对金额
      mtDNA(%mtDNA) a
      与DMSO(对照)相比的p值 b
      DMSO(对照)
      -
      100.0±8.8
      -
      ddC
      0.2
      57.0±10.4
      < 0.0001
      2.0
      25.1±7.8
      < 0.0001
      20
      6.9±2.9
      < 0.0001
      a 数据代表三次重复进行的3次独立实验的平均值±SD b 成对的双尾学生t检验

食谱

  1. 鹰的最低必需培养基
    G1utaMAX TM
    10%胎牛血清 100单位/ml青霉素
    100单位/ml链霉素 1mM丙酮酸钠

致谢

所有的工作是由吉利德科学公司赞助的。该协议改编自Feng等人(2014)。

参考文献

  1. Feng,JY,Cheng,G.,Perry,J.,Barauskas,O.,Xu,Y.,Fenaux,M.,Eng,S.,Tirunagari,N.,Peng,B.,Yu,M.,Tian Y.,Lee,YJ,Stepan,G.,Lagpacan,LL,Jin,D.,Hung,M.,Ku,KS,Han,B.,Kitrinos,K.,Perron,M.,Birkus, ,Wong,KA,Zhong,W.,Kim,CU,Carey,A.,Cho,A.and Ray,AS(2014)。 通过GS-6620(一种有效的C-核苷单磷酸酯前药)抑制丙型肝炎病毒复制。 a> Antimicrob Agents Chemother 58(4):1930-1942。
  2. Livak,K.J.and Schmittgen,T.D。(2001)。 使用实时定量PCR分析相对基因表达数据,并使用2(-Delta Delta C( T))方法 方法 25(4):402-408
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Perron, M. and Feng, J. Y. (2015). Determination of Mitochondrial DNA Upon Drug Treatment. Bio-protocol 5(2): e1377. DOI: 10.21769/BioProtoc.1377.
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