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Mar 2016
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Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay
利用改进的宿主细胞再活化反应进行人外周血单核细胞的DNA修复能力的离体检测   

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Abstract

The ability of humans to repair DNA damages decreases with increasing age. In order to be able to repair daily occurring DNA damages, it becomes more and more important to preserve repair capability of cells with aging. The preservation of DNA repair processes contributes to preventing DNA mutations and subsequently the onset of age-related diseases such as cancer. For the determination of DNA repair of human cells, mostly in vitro cell cultures are used. However, an ex vivo approach can provide a more accurate result compared with in vitro cell cultures, since the DNA repair ability is measured directly without the influence of prolonged culture time. Published protocols use in vitro cultured cells with a single reporter plasmid or a luciferase reporter. Our modified host cell reactivation assay enables the measurement of DNA repair capacity (nucleotide excision repair) of ex vivo isolated human peripheral blood mononuclear cells (PBMCs). For this purpose, PBMCs are isolated out of human anticoagulated blood by density gradient centrifugation. Directly after isolation, the PBMCs are co-transfected with two plasmids, one being previously damaged by UVC irradiation and one remaining undamaged. PBMCs are incubated for 24 h and subsequently analyzed by fluorescence activated cell sorting (FACS). The ability of cells to repair the DNA damages leads to a functional reactivation of the reporter gene. The assay presented here provides a solution to determine human DNA repair capacity ex vivo directly out of the human body. Furthermore, it can be used to research the ex vivo influence of different substances on DNA repair capacity of humans.

Keywords: Modified host cell reactivation assay (改进的宿主细胞再活化反应), DNA repair capacity (DNA修复能力), Human peripheral blood mononuclear cells (人外周血单核细胞), Ex vivo (离体), Nucleotide excision repair (核苷酸剪切修复), Aging (衰老)

Background

Maintaining the integrity of the human genome is a prerequisite for delaying or preventing the development of age-related diseases. Every day, up to 50,000 DNA-damaging events occur in every cell (reviewed in Shrinivas et al., 2017). Such damages arise because of exogenous influences like radiation or chemicals, or via the normal cellular metabolism. For this reason, functional DNA repair mechanisms are crucial for the survival of organisms. One of the most important DNA repair pathways is nucleotide excision repair (NER), which detects and eliminates bulky DNA lesions and helix-distorting DNA adducts like cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs) (for review see Nouspikel, 2009).

The modified host cell reactivation assay (mHCRA) is a reliable and reproducible method for the research of NER capacity of human cells based on the restoration of a previously damaged reporter gene. The principle of reporter gene restoration was first described in 1985 (Protić-Sabljić et al., 1985), subsequently adjusted by Athas et al. for an application with human lymphocytes (Athas et al., 1991) and further refined by using fluorescent reporter proteins (Roguev and Russev, 2000). Up to now, few researchers use the mHCRA, although it is a highly reproducible and reliable method. Qiao et al. used the modified HCRA with a luciferase reporter on human lymphocytes to study DNA repair (Qiao et al., 2002). In 2010, Burger et al. published an advanced method, which describes the mHCRA for studies on human skin cells. They used two reporter plasmids and fluorescence-activated cell sorting to determine DNA repair capacity (Burger et al., 2010). Furthermore, Mendez et al. researched DNA repair by carrying out nucleofection of cryopreserved human lymphocytes with one reporter plasmid (Mendez et al., 2011).

Our mHCRA enables the measurement of NER capacity of PBMCs as an ex vivo approach. By using two different plasmids, transfection efficiencies can be normalized by a transfected control plasmid. This procedure allows for comparing experiments with different transfection efficiencies with each other. We used the method described here to reveal the influence of calorie reduction on the DNA repair capacity of humans (Matt et al., 2016). Moreover, the use of this assay can provide a more accurate assessment of positive or negative effects of substances like dietary supplements or pharmaceutical agents on DNA repair ability of human cells than the use of in vitro cell cultures.

Materials and Reagents

  1. Centrifuge tubes 15, 50 ml (SARSTEDT, catalog numbers: 62.554.001, 62.547.254)
  2. 1.5 ml reaction tube (SARSTEDT, catalog number: 72.706)
  3. Petri dishes (SARSTEDT, catalog number: 83.3902)
  4. Pipette tips 10 μl, 20 μl, 200 μl, 1,000 μl (SARSTEDT, catalog numbers: 70.1130, 70.116, 70.760.002, 70.762)
  5. Serological pipettes 5 ml and 10 ml (SARSTEDT, catalog numbers: 86.1253.001 and 86.1254.001)
  6. LeucosepTM, with porous barrier, 50 ml, pre filled with LeucosepTM separation medium (Greiner Bio-One GmbH, catalog number: 227288)
  7. Sterile filter 0.2 μm (SARSTEDT, catalog number: 83.1826.001)
  8. Cell culture plate, 6-well, Cell+ (SARSTEDT, catalog number: 83.3920.300)
  9. 0.4 cm electroporation cuvettes (Molecular BioProducts, Inc., purchased via Thermo Fisher Scientific GmbH, catalog number: 5540-11)
  10. FACS tubes 5 ml and 0.5 ml (SARSTEDT, catalog numbers: 55.1579, 55.673)
  11. Plasmid-DNA solution pEGFP-N1 (Clontech, catalog number: 6085-1) and pDsRedExpress-N1 (Clontech, catalog number: 632429), each 900 ng/µl in Qiagen EB buffer (storage temperature for longer periods: -80 °C, otherwise keep at 4 °C)
  12. Human venous blood, anticoagulated
  13. KCl (Carl Roth, catalog number: 6781.3)
  14. KH2PO4 (Carl Roth, catalog number: 3904.2)
  15. Na2HPO4·12H2O (Carl Roth, catalog number: N350.1)
  16. NaCl (Carl Roth, catalog number: 3957.3)
  17. S-Monovette® K3 EDTA 4.9 ml and 7.5 ml (SARSTEDT, catalog numbers: 04.1931.001 and 01.1605.001)
  18. RPMI 1640 without Phenol Red (Gibco® by Life Technologies, purchased via Fisher Scientific GmbH, catalog number: 11835-063, storage temp. 4 °C)
  19. Fetal Bovine Serum (Gibco® by Life Technologies, chased via Fisher Scientific GmbH, catalog number: 10270-106)
  20. EB Buffer, part of the QIAGEN Plasmid Plus Giga Kit (Qiagen GmbH, Hilden, catalog number: 12991)
  21. BD FACS Sheath Solution with Surfactant (BD Biosciences, catalog number: 336911)
  22. 1x PBS (see Recipes)
  23. 0.9% NaCl solution (see Recipes)
  24. PBMC medium (see Recipes)

Equipment

  1. Pipettes, Eppendorf Research® Plus 10 μl, 20 μl, 200 μl, 1,000 μl (Eppendorf, catalog numbers: 3123000020, 3123000039, 3123000055, 3123000063)
  2. Stratalinker® UV Crosslinker (Stratagene, catalog number: 400072, model: Model 1800)
  3. Centrifuge with swinging bucket rotor (Eppendorf, catalog numbers: 5805000010, 5804709004)
  4. Neubauer counting chamber improved (Carl Roth, catalog number: PC72.1)
  5. Gene Pulser II with Capacitance extender plus and Pulse controller plus (Bio-Rad Laboratories, Inc., catalog numbers: 165-2106, 165-2108, 165-2110)
  6. CO2 Incubator Hera cell 150 (Kendro Laboratory Products, catalog number: 51013568)
  7. BD FACSCalibur Flow Cytometry System with 488 nm laser (Becton, Dickinson and Company)
  8. -80 °C freezer HFU600TV (Thermo Fisher ScientificTM, catalog number: 11670823)

Software

  1. CellQuest Pro (Becton, Dickinson and Company, version 4.0.2)
  2. Prism (GraphPad Software, Inc.)

Procedure

Day 1

  1. Prepare plasmid solutions
    1. pDsRedExpress-N1: Adjust concentration to a maximum of 900 ng/µl using EB buffer.
      Note: The higher the plasmid concentration is, the more likely plasmids lie above each other during irradiation and irradiation becomes less effective. This results in higher DNA repair capacities in the end. Irradiate half of the volume by pipetting droplets of 40 µl into a Petri dish (10 cm diameter). Irradiate with 5 kJ/m2 UVC (254 nm) without lid of the Petri dish using Stratalinker® UV Crosslinker. After irradiation, collect droplets in a 1.5 ml reaction tube. The other half of the plasmid solution remains non-irradiated.
    2. pEGFP-N1 needs no further treatment as it serves as transfection control. The concentration of plasmid solution should be > 600 ng/µl in order to avoid too high volumes. The ratio of absorbance at 260 nm and 280 nm should be ~1.8.
    3. For the measurement of DNA repair, mix 10 µg of pEGFP-N1 and 30 µg of irradiated pDsRedExpress-N1. For calculation of the correction factor, mix 10 µg of pEGFP-N1 and 30 µg of non-irradiated pDsRedExpress-N1 for each electroporation. Note that the volumes needed depend on the concentration of the plasmid solutions. Adjust the volume of the mixture to 100 µl using EB buffer. Prepare enough plasmid-DNA mixtures for the entire study planned. Store mixtures at 4 °C for short-term storage or at -80 °C for longer periods.
  2. Isolate PBMCs from 12 ml of human blood using LeucosepTM tubes according to the manufacturer’s instructions as follows:
    1. Dilute 12 ml anticoagulated blood 1:2 using 0.9% NaCl solution (24 ml final volume) and transfer into LeucosepTM tubes.
    2. Centrifugate at 1,000 x g for 10 min in a swinging bucket rotor and switch off brakes. Figure 1A shows the LeucosepTM tube before centrifugation and Figure 1B shows the tube after centrifugation.


      Figure 1. LeucosepTM tube before and after centrifugation. A. LeucosepTM tube filled with anticoagulated blood, which was previously diluted 1:2 with 0.9% NaCl solution. B. LeucosepTM tube after density gradient centrifugation. The different layer can be seen clearly from top to bottom: plasma, enriched cells, separation medium, the porous barrier, again separation medium and finally erythrocytes and granulocytes.

    3. Pipet approximately 10 ml of plasma from the top in order to reach the enriched cell fraction more easily.
    4. Harvest the enriched cell fraction using a 1,000 µl Eppendorf pipette and transfer cells into a 50 ml centrifuge tube.
    5. Wash cells with 10 ml PBS and centrifuge at 250 x g for 10 min.
    6. Repeat the washing step two times each with 5 ml PBS. When adding 5 ml for the second time, count cells using a Neubauer counting chamber.
  3. For each electroporation, fill 3 ml PBMC medium per well into a 6-well cell culture plate.
  4. Centrifuge 2 x 106 cells at 250 x g for 10 min in a 15 ml centrifuge tube for each electroporation. Prepare two tubes in order to be able to calculate DNA repair capacity of one sample properly. One tube will contain the irradiated pDsRedExpress-N1 and pEGFP-N1 and the other one the non-irradiated pDsRedExpress-N1 and pEGFP-N1. Resuspend pellet in 150 µl RPMI media without phenol red.
  5. Add 100 µl of the previously prepared plasmid mixture to the cell suspension, so that the suspension contains 10 µg pEGFP-N1 and 30 µg pDsRedExpress-N1 (either irradiated or non-irradiated).
  6. Transfer 250 µl of the cell suspension to electroporation cuvettes with 4 mm gap, place it into the shocking chamber of Gene Pulser II and start electroporation at 400 V and 500 µF.
    Note: Always electroporate one irradiated batch and one non-irradiated batch directly one after the other. These two will be used to calculate the DNA repair capacity of one sample.
  7. Transfer electroporation batches in the order of electroporation into the prepared 6-well cell culture plate containing RPMI medium.
  8. Incubate for 24 h at 37 °C, 5 % CO2 and relative humidity of 95%.

Day 2
  1. Prepare one 1.5 ml reaction tube for each electroporation approach.
  2. Mix cells in the wells of the cell culture plate using a 1,000 µl Eppendorf pipette before transferring 1.5 ml of the cell suspension into the 1.5 ml reaction tubes prepared previously. 
  3. Centrifuge at 250 x g for 10 min. 
  4. Remove 1.4 ml of the supernatant, resuspend cells in the remaining 100 µl, and transfer into 0.5 ml FACS tubes.
  5. Place the 0.5 ml tube into a 5 ml tube and carry out FACS analysis using 488 nm laser.

Data analysis

FACS analysis was carried out using FACSCalibur, CellQuest Pro software and the 488 nm laser. Gating of the cells of interest (lymphocytes) was carried out with the control sample. For analysis of the sample containing the irradiated pDsRedExpress-N1 the same gates were used. Gating of the transfected control samples and the samples containing the irradiated pDsRedExpress-N1 are shown in Figures 2 and 3 respectively.


Figure 2. FACS analysis of the control sample. A. Control sample containing the non-irradiated pDsRedExpress-N1 with gated lymphocytes. B. Transfected cells gated with gate R1. The area lower right (R2) displays cells expressing only GFP, the area upper left (R3) displays cells expressing only DsRed and the area upper right (R4) displays cells expressing GFP as well as DsRed.


Figure 3. FACS analysis of the samples containing the irradiated pDsRedExpress-N1. A. Sample containing irradiated pDsRedExpress-N1 with gated lymphocytes. B. Transfected cells gated with gate R1. The area lower right (R2) displays cells expressing only GFP, the area upper left (R3) displays cells expressing only expressing DsRed and the area upper right (R4) displays cells expressing GFP as well as DsRed.

For calculation of DNA repair capacity, percentages of cells expressing GFP and DsRed are added up as follows. All GFP-expressing cells are the cells in gates R2 and R4. All DsRed-expressing cells are the cells in gates R3 and R4.
  For the correction of repair capacity with regard to efficacy of transfection, the correction factor F is needed. F is always calculated from the percentages of GFP and DsRed of the control sample. Hence, a control is needed for each transfection with irradiated pDsRedExpress-N1. F is the ratio of percentages of GFP-expressing cells to the DsRed-expressing cells of the control sample. DNA repair capacity is calculated according to Matt et al. (2016) as follows:



For statistical analyses, any version of Prism (GraphPad Software, Inc.) can be used. We recommend triplicates for the calculation of DNA repair capacity.

Notes

  1. Pay attention to the time constants during electroporation. In our experiments, time constants are usually between 20 and 25 ms. Furthermore, too high time constants resulted in low transfection efficiencies or cell death.
  2. To allow the laser to warm up, switch on flow cytometer approximately 30 min before FACS analysis.
  3. To compensate for the non-optimal excitation of DsRed by the 488 nm laser, three times the amount of DsRed compared to GFP is used for electroporation. 
  4. We recommend triplicates for each sample (three control approaches and three matching approaches containing the irradiated pDsRedExpress-N1).

Recipes

  1. 1x PBS
    8 g NaCl
    0.20 g KCl
    2.88 g Na2HPO4·12H2O
    1.24 g KH2PO4
    Add 1 L ddH2O, pH 7.4
  2. 0.9% NaCl solution
    9 g NaCl
    Add 1 L ddH2O, sterile filter solution using 0.2 µm sterile filter
  3. PBMC medium
    RPMI 1640 without phenol red
    20% fetal bovine serum
    Store at 4 °C

Acknowledgments

The authors thank Dr. med. Karin Rupprecht (Center for Traditional Chinese Medicine–TCM Sigmaringen) and Dr. med. Adrian Schulte (F. X. Mayr Bodensee Center Überlingen) for drawing of the blood samples and informing of the volunteers.

Competing interests

One of the authors has consulting contracts with MSE Pharmazeutika GmbH, Bad Homburg, Germany.

Ethics

All experiments were conducted in accordance with the declaration of Helsinki as well as approved by the ethics committee of the Landesärztekammer Baden-Württemberg, Germany. The volunteers were informed before collection of the samples and gave their written consent to the use of their blood samples.

References

  1. Athas, W. F., Hedayati, M. A., Matanoski, G. M., Farmer, E. R. and Grossman, L. (1991). Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Cancer Res 51(21): 5786-5793.
  2. Burger, K., Matt, K., Kieser, N., Gebhard, D. and Bergemann, J. (2010). A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts. BMC Biotechnol 10: 46.
  3. Matt, K., Burger, K., Gebhard, D. and Bergemann, J. (2016). Influence of calorie reduction on DNA repair capacity of human peripheral blood mononuclear cells. Mech Ageing Dev 154: 24-29.
  4. Mendez, P., Taron, M., Moran, T., Fernandez, M. A., Requena, G. and Rosell, R. (2011). A modified host-cell reactivation assay to quantify DNA repair capacity in cryopreserved peripheral lymphocytes. DNA Repair (Amst) 10(6): 603-610.
  5. Nouspikel, T. (2009). DNA repair in mammalian cells-Nucleotide excision repair: variations on versatility. Cell Mol Life Sci 66(6): 994–1009.
  6. Protić-Sabljić, M., Whyte, D., Fagan, J., Howard, B. H., Gorman, C. M., Padmanabhan, R. and Kraemer, K. H. (1985). Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line. Mol Cell Biol 5(7): 1685-1693.
  7. Qiao, Y. W., Spitz, M. R., Guo, Z. Z., Hadeyati, M., Grossman, L., Kraemer, K. H. and Wei, Q. Y. (2002). Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes. Mutat Res 509(1-2): 165-174.
  8. Roguev, A. and Russev, G. (2000). Two-wavelength fluorescence assay for DNA repair. Anal Biochem 287(2): 313-318.
  9. Shrinivas, S. A., Shanta, S. H., Prajakta, B. B. (2017). DNA: damage and repair mechanisms in humans. Glob J Pharmaceu Sci 3(2): 555613.

简介

人类修复DNA损伤的能力随着年龄的增长而降低。为了能够修复日常发生的DNA损伤,保持细胞老化的修复能力变得越来越重要。 DNA修复过程的保存有助于防止DNA突变并随后发生与癌症等年龄相关的疾病。为了测定人细胞的DNA修复,主要使用体外细胞培养物。然而,与体外细胞培养物相比,离体方法可以提供更准确的结果,因为直接测量DNA修复能力而没有延长培养时间的影响。公布的方案使用体外培养的细胞和单一报告质粒或荧光素酶报告基因。我们修改的宿主细胞再激活测定能够测量离体离体分离的人外周血单核细胞(PBMC)的DNA修复能力(核苷酸切除修复)。为此目的,通过密度梯度离心从人抗凝血中分离PBMC。分离后直接将PBMC与两种质粒共转染,一种质粒先前被UVC辐射损伤,另一种未受损。将PBMC孵育24小时,随后通过荧光激活细胞分选(FACS)进行分析。细胞修复DNA损伤的能力导致报告基因的功能性再激活。这里提供的测定提供了一种直接测定人体DNA离体外修复能力的解决方案。此外,它可用于研究不同物质对人类DNA修复能力的离体影响。
【背景】保持人类基因组的完整性是延迟或预防与年龄有关的疾病发展的先决条件。每天,每个细胞中都会发生多达50,000个DNA损伤事件(在Shrinivas et al。,2017中进行了综述)。这种损害是由于辐射或化学物质等外源性影响或正常细胞代谢引起的。因此,功能性DNA修复机制对于生物体的存活至关重要。最重要的DNA修复途径之一是核苷酸切除修复(NER),其检测并消除体积大的DNA损伤和螺旋扭曲的DNA加合物,如环丁烷嘧啶二聚体(CPD)和嘧啶 - (6-4) - 嘧啶酮光产物(6- 4PPs)(供审查见Nouspikel,2009年)。

改良的宿主细胞再激活试验(mHCRA)是一种可靠且可重复的方法,用于基于先前受损的报告基因的恢复来研究人细胞的NER能力。报告基因修复的原理首先在1985年描述(Protić-Sabljić et al。,1985),随后由Athas 等人调整用于人淋巴细胞的应用( Athas et al。,1991)并通过使用荧光报告蛋白进一步精制(Roguev和Russev,2000)。到目前为止,很少有研究人员使用mHCRA,尽管它是一种高度可重复且可靠的方法。 Qiao 等在人淋巴细胞上使用修饰的HCRA和荧光素酶报告基因来研究DNA修复(Qiao et al。,2002)。 2010年,Burger 等人发表了一种先进的方法,该方法描述了用于人体皮肤细胞研究的mHCRA。他们使用两种报告质粒和荧光激活细胞分选来确定DNA修复能力(Burger et al。,2010)。此外,Mendez 等人通过用一种报告质粒对Menopz进行核转染来研究DNA修复(Mendez et al。,2011)。

我们的mHCRA可以测量PBMC的NER容量,作为离体方法。通过使用两种不同的质粒,转染效率可以通过转染的对照质粒标准化。该程序允许将具有不同转染效率的实验相互比较。我们使用此处描述的方法揭示了卡路里减少对人类DNA修复能力的影响(Matt et al。,2016)。此外,与使用体外细胞培养物相比,使用该测定法可以更准确地评估诸如膳食补充剂或药剂之类的物质对人类细胞的DNA修复能力的正面或负面影响。

关键字:改进的宿主细胞再活化反应, DNA修复能力, 人外周血单核细胞, 离体, 核苷酸剪切修复, 衰老

材料和试剂

  1. 离心管15,50 ml(SARSTEDT,目录号:62.554.001,62.547.254)
  2. 1.5 ml反应管(SARSTEDT,目录号:72.706)
  3. 培养皿(SARSTEDT,目录号:83.3902)
  4. 移液器吸头10μl,20μl,200μl,1,000μl(SARSTEDT,目录号:70.1130,70.116,70.760.002,70.762)
  5. 血清移液器5 ml和10 ml(SARSTEDT,目录号:86.1253.001和86.1254.001)
  6. Leucosep TM ,带有多孔屏障,50 ml,预填充Leucosep TM 分离介质(Greiner Bio-One GmbH,目录号:227288)
  7. 无菌过滤器0.2μm(SARSTEDT,目录号:83.1826.001)
  8. 细胞培养板,6孔,Cell +(SARSTEDT,目录号:83.3920.300)
  9. 0.4cm电穿孔小杯(Molecular BioProducts,Inc。,通过Thermo Fisher Scientific GmbH购买,目录号:5540-11)
  10. FACS试管5 ml和0.5 ml(SARSTEDT,目录号:55.1579,55.673)
  11. 质粒-DNA溶液pEGFP-N1(Clontech,目录号:6085-1)和pDsRedExpress-N1(Clontech,目录号:632429),Qiagen EB缓冲液各900 ng /μl(储存温度较长时间:-80°C) ,否则保持在4°C)
  12. 人体静脉血,抗凝
  13. KCl(Carl Roth,目录号:6781.3)
  14. KH 2 PO 4 (Carl Roth,目录号:3904.2)
  15. Na 2 HPO 4 ·12H 2 O(Carl Roth,目录号:N350.1)
  16. NaCl(Carl Roth,目录号:3957.3)
  17. S-Monovette ® K3 EDTA 4.9 ml和7.5 ml(SARSTEDT,目录号:04.1931.001和01.1605.001)
  18. 不含酚红的RPMI 1640(Life Technologies的Gibco ®,通过Fisher Scientific GmbH购买,目录号:11835-063,储存温度4°C)
  19. 胎牛血清(Life Technologies的Gibco ®,通过Fisher Scientific GmbH追踪,目录号:10270-106)
  20. EB Buffer,QIAGEN Plasmid Plus Giga Kit的一部分(Qiagen GmbH,Hilden,目录号:12991)
  21. 具有表面活性剂的BD FACS护套溶液(BD Biosciences,目录号:336911)
  22. 1x PBS(见食谱)
  23. 0.9%NaCl溶液(见食谱)
  24. PBMC培养基(见食谱)

设备

  1. 移液器,Eppendorf研究®加10μl,20μl,200μl,1,000μl(Eppendorf,目录号:3123000020,3123000039,3123000055,3123000063)
  2. Stratalinker ® UV交联剂(Stratagene,目录号:400072,型号:Model 1800)
  3. 带旋转斗转子的离心机(Eppendorf,目录号:5805000010,5804709004)
  4. Neubauer计数室改进(Carl Roth,目录号:PC72.1)
  5. 具有电容扩展器和脉冲控制器的Gene Pulser II(Bio-Rad Laboratories,Inc。,目录号:165-2106,165-2108,165-2110)
  6. CO 2 培养箱Hera细胞150(Kendro Laboratory Products,目录号:51013568)
  7. BD FACSCalibur流式细胞仪系统采用488 nm激光(Becton,Dickinson and Company)
  8. -80°C冰箱HFU600TV(赛默飞世尔科技 TM ,目录号:11670823)

软件

  1. CellQuest Pro(Becton,Dickinson and Company,版本4.0.2)
  2. Prism(GraphPad Software,Inc。)

程序

第1天

  1. 准备质粒溶液
    1. pDsRedExpress-N1:使用EB缓冲液将浓度调节至最大900 ng /μl。
      注意:质粒浓度越高,质粒在照射过程中越可能位于彼此之上,并且照射效果越差。这导致最终的DNA修复能力更高。通过将40μl的液滴吸移到培养皿(直径10cm)中照射一半体积。使用Stratalinker 紫外线,用5 kJ / m 2 UVC(254 nm)照射培养皿盖子交联剂。照射后,将液滴收集在1.5ml反应管中。另一半质粒溶液保持未照射。
    2. pEGFP-N1不需要进一步处理,因为它可用作转染对照。质粒溶液的浓度应> 600 ng /μl以避免体积过大。 260nm和280nm处的吸光度比应为~1.8。
    3. 对于DNA修复的测量,混合10μg的pEGFP-N1和30μg的经照射的pDsRedExpress-N1。为了计算校正因子,每次电穿孔混合10μg的pEGFP-N1和30μg的未照射的pDsRedExpress-N1。注意,所需的体积取决于质粒溶液的浓度。使用EB缓冲液将混合物的体积调节至100μl。为整个计划的研究准备足够的质粒-DNA混合物。将混合物储存在4°C下短期储存或在-80°C下储存较长时间。
  2. 根据制造商的说明,使用Leucosep TM 管从12 ml人血液中分离PBMC,如下所示:
    1. 使用0.9%NaCl溶液(24ml终体积)以1:2稀释12ml抗凝血,并转移至Leucosep TM 管中。
    2. 在摇摆式转子转子中以1,000 x g 离心10分钟并关闭制动器。图1A显示离心前的Leucosep TM 管,图1B显示离心后的管。


      图1.离心前后的Leucosep TM 管。 A. Leucosep TM 管充满抗凝血液,之前用1:2稀释0.9%NaCl溶液。 B.密度梯度离心后的Leucosep TM 管。从上到下可以清楚地看到不同的层:血浆,富集细胞,分离介质,多孔屏障,再次分离培养基,最后是红细胞和粒细胞。

    3. 从顶部吸取约10ml血浆以更容易地到达富集的细胞部分。
    4. 使用1,000μlEppendorf移液管收集富集的细胞级分,并将细胞转移到50ml离心管中。
    5. 用10ml PBS洗涤细胞,并在250μL离心下离心10分钟。
    6. 用5ml PBS重复洗涤步骤两次。当第二次加入5ml时,使用Neubauer计数室计数细胞。
  3. 对于每次电穿孔,将每孔3ml PBMC培养基填充到6孔细胞培养板中。
  4. 对于每次电穿孔,在15ml离心管中以250×10μg/ ml离心2×10 6个细胞10分钟。准备两个管子,以便能够正确计算一个样品的DNA修复能力。一个管将包含经照射的pDsRedExpress-N1和pEGFP-N1,另一个管包含未经照射的pDsRedExpress-N1和pEGFP-N1。在不含酚红的150μlRPMI培养基中重悬沉淀。
  5. 向细胞悬浮液中加入100μl先前制备的质粒混合物,使悬浮液含有10μgpEGFP-N1和30μgpDsRedExpress-N1(照射或未照射)。
  6. 将250μl细胞悬浮液转移至具有4mm间隙的电穿孔比色皿,将其置于Gene Pulser II的冲击室中,并在400V和500μF下开始电穿孔。
    注意:始终一个接一个地对一个辐照批次和一个未辐照的批次进行电镀。这两个将用于计算一个样本的DNA修复能力。
  7. 将电穿孔顺序转移到含有RPMI培养基的制备的6孔细胞培养板中。
  8. 在37°C,5%CO 2 和95%的相对湿度下孵育24小时。

第2天
  1. 为每种电穿孔方法准备一个1.5 ml反应管。
  2. 使用1,000μlEppendorf移液管将细胞混合在细胞培养板的孔中,然后将1.5ml细胞悬浮液转移到先前制备的1.5ml反应管中。 
  3. 在250 x g 下离心10分钟。 
  4. 除去1.4ml上清液,在剩余的100μl中重悬细胞,并转移到0.5ml FACS管中。
  5. 将0.5ml管置于5ml管中,并使用488nm激光进行FACS分析。

数据分析

使用FACSCalibur,CellQuest Pro软件和488nm激光进行FACS分析。用对照样品进行目标细胞(淋巴细胞)的门控。为了分析含有经照射的pDsRedExpress-N1的样品,使用相同的门。转染的对照样品和含有经照射的pDsRedExpress-N1的样品的门控分别显示在图2和图3中。


图2.对照样品的FACS分析。 A.对照样品含有未经辐照的pDsRedExpress-N1和门控淋巴细胞。 B.用门R1门控的转染细胞。右下区域(R2)显示仅表达GFP的细胞,左上区域(R3)显示仅表达DsRed的细胞,右上区域(R4)显示表达GFP和DsRed的细胞。


图3.含有经辐照的pDsRedExpress-N1的样品的FACS分析。 A.含有经过门控的淋巴细胞的经照射的pDsRedExpress-N1的样品。 B.用门R1门控的转染细胞。右下区域(R2)显示仅表达GFP的细胞,左上区域(R3)显示仅表达DsRed的细胞,右上区域(R4)显示表达GFP和DsRed的细胞。

为了计算DNA修复能力,将表达GFP和DsRed的细胞的百分比加起来如下。所有表达GFP的细胞是门R2和R4中的细胞。所有表达DsRed的细胞是门R3和R4中的细胞。
 为了校正关于转染功效的修复能力,需要校正因子F. F总是根据对照样品的GFP和DsRed的百分比计算。因此,对于经辐照的pDsRedExpress-N1的每次转染,需要对照。 F是表达GFP的细胞与对照样品的表达DsRed的细胞的百分比之比。 DNA修复能力根据Matt 等人(2016)计算如下:



对于统计分析,可以使用任何版本的Prism(GraphPad Software,Inc。)。我们建议一式三份来计算DNA修复能力。

笔记

  1. 注意电穿孔过程中的时间常数。在我们的实验中,时间常数通常在20到25毫秒之间。此外,太高的时间常数导致低转染效率或细胞死亡。
  2. 为了让激光器预热,在FACS分析前大约30分钟打开流式细胞仪。
  3. 为了补偿488 nm激光对DsRed的非最佳激发,与GFP相比,DsRed的三倍用于电穿孔。 
  4. 我们建议每个样品一式三份(三种控制方法和三种含有辐照pDsRedExpress-N1的匹配方法)。

食谱

  1. 1x PBS
    8克NaCl
    0.20克KCl
    2.88 g Na 2 HPO 4 ·12H 2 O
    1.24克KH 2 PO 4
    加入1L ddH 2 O,pH 7.4
  2. 0.9%NaCl溶液
    9克NaCl
    加入1L ddH 2 O,使用0.2μm无菌过滤器的无菌过滤溶液
  3. PBMC媒体
    RPMI 1640不含酚红
    20%胎牛血清
    储存在4°C

致谢

作者感谢医学博士。 Karin Rupprecht(中医中心 - TCM西格马林根)和医学博士。 Adrian Schulte(F. X. Mayr BodenseeCenterÜberlingen)用于抽取血液样本并通知志愿者。

利益争夺

其中一位作者与德国Bad Homburg的MSE Pharmazeutika GmbH签订了咨询合同。

伦理

所有实验均按照赫尔辛基宣言进行,并经德国LandesärztekammerBaden-Württemberg伦理委员会批准。在收集样本之前通知志愿者并且他们书面同意使用他们的血液样本。

参考

  1. Athas,W.F.,Hedayati,M.A.,Matanoski,G.M.,Farmer,E.R。和Grossman,L。(1991)。 开发和现场测试验证循环人淋巴细胞中DNA修复的分析。 Cancer Res 51(21):5786-5793。
  2. Burger,K.,Matt,K.,Kieser,N.,Gebhard,D。和Bergemann,J。(2010)。 改良的荧光测定宿主细胞再激活试验,以确定原代角质形成细胞,黑素细胞和成纤维细胞的修复能力。< / a> BMC Biotechnol 10:46。
  3. Matt,K.,Burger,K.,Gebhard,D。和Bergemann,J。(2016)。 减少卡路里对人类外周血单核细胞DNA修复能力的影响。 Mech Aging Dev 154:24-29。
  4. Mendez,P.,Taron,M.,Moran,T.,Fernandez,M。A.,Requena,G。和Rosell,R。(2011)。 经过修改的宿主细胞再激活试验,用于量化冷冻保存的外周淋巴细胞中的DNA修复能力。 DNA Repair(Amst) 10(6):603-610。
  5. Nouspikel,T。(2009)。 哺乳动物细胞中的DNA修复 - 核苷酸切除修复:多功能性的变异。 Cell Mol Life Sci 66(6):994-1009。
  6. Protić-Sabljić,M.,Whyte,D.,Fagan,J.,Howard,B.H.,Gorman,C.M.,Padmanabhan,R。和Kraemer,K.H。(1985)。 对猿猴病毒40转化的干皮病色素细胞系中连锁克隆基因的表达进行定量。 Mol Cell Biol 5(7):1685-1693。
  7. Qiao,Y.W.,Spitz,M.R.,Guo,Z.Z.,Hadeyati,M.,Grossman,L.,Kraemer,K。H. and Wei,Q.Y。(2002)。 使用荧光素酶报告基因通过改良的宿主细胞再激活试验快速评估紫外线DNA损伤的修复并且与正常人淋巴细胞中DNA修复基因的多态性相关。 Mutat Res 509(1-2):165-174。
  8. Roguev,A。和Russev,G。(2000)。 用于DNA修复的双波长荧光检测。 Anal Biochem 287(2):313-318。
  9. Shrinivas,S.A.,Shanta,S.H.,Prajakta,B.B。(2017)。 DNA:人体的损伤和修复机制。 Glob J Pharmaceu Sci 3(2):555613。
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引用:Matt, K. and Bergemann, J. (2019). Ex vivo Analysis of DNA Repair Capacity of Human Peripheral Blood Mononuclear Cells by a Modified Host Cell Reactivation Assay. Bio-protocol 9(15): e3325. DOI: 10.21769/BioProtoc.3325.
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