超中心对等离子脂蛋白的分离和分析
Summary
用于分析等离子脂蛋白的几种方法：然而，超中心化仍然是最流行和最可靠的方法之一。在这里，我们描述了如何使用顺序密度超中心化将脂蛋白从血浆中分离的方法，以及如何分析用于诊断和研究目的的非脊髓灰质素。
Abstract
血浆脂蛋白和非政治蛋白的分析是血脂血症诊断和脂质代谢和动脉粥样硬化研究的重要组成部分。虽然有几种方法分析等离子脂蛋白，超中心化仍然是最流行和最可靠的方法之一。由于其完整的分离程序，通过这种方法分离的脂蛋白分数可用于分析脂蛋白，阿波利波蛋白，蛋白质组，和功能研究的脂蛋白与培养细胞体外。在这里，我们提供了隔离七个脂蛋白分数的详细协议，包括 VLDL（d=lt;1.006 g/mL）、IDL（d=1.02 g/mL）、LDL（d=1.04 和 1.06 g/mL）、硬盘（d=1.08， 1.10 和 1.21 克/mL） 从兔子血浆使用顺序浮动超中心。此外，我们向读者介绍如何分析阿波利波蛋白，如apoA-I，apoB，和apoE由SDS-PAGE和西方印迹，并显示具有代表性的结果脂蛋白和非波利波蛋白配置文件使用高脂兔模型。这种方法可以成为临床医生和基础科学家分析脂蛋白功能的标准方案。
Introduction
血脂血症是世界上动脉粥样硬化的主要危险因素。高浓度的低密度脂蛋白（LDLs）和低水平的高密度脂蛋白（HDL）与冠心病的高风险（CHD）1，2密切相关。在临床环境中，低密度脂蛋白胆固醇（LDL-C）和高密度脂蛋白胆固醇（HDL-C）都是在临床实验室3、4中使用自动分析仪进行常规测量的。尽管如此，还是有必要详细分析脂蛋白特征，以诊断脂质血症，研究人类和实验动物的脂质代谢和动脉粥样硬化。据报道，分析血浆脂蛋白的几种方法，如超中心化、大小排除色谱[快速蛋白质液体色谱（FPLC）和高性能液体色谱（HPLC）]、阿加罗斯和多丙烯酰胺凝胶的电磷化、核磁共振以及使用多面体和二价沉淀或其他化学品的选择性化学沉淀。20世纪50年代，Havel的研究小组首次提出了由密度定义的利波蛋白的概念，并将其分为低密度脂蛋白（CM）、低密度脂蛋白（VLDL）、中密度脂蛋白（IDL）、LDL和HDL5等，后来，其他组6、7进一步修改了该方法。到目前为止，超中心化是最流行和最可靠的方法，而实用的协议仍然不可用。在本文中，我们试图描述一个易于使用的方案，用于隔离小比例的等离子体使用顺序密度浮动超中心最初描述以前8。隔离七个等离子脂蛋白分数 [VLDL（d=lt;1.006 g/mL）、IDL（d=1.02 g/mL）、LDL（d=1.04 和 1.06 g/mL）、高密度脂蛋白（d=1.08， 1.10 和 1.21 克/mL）]使研究人员能够对脂蛋白及其组成非脊蛋白9、10、11进行广泛分析。完整的连续七密度脂蛋白可用于分析脂蛋白功能，也可用于基于细胞的体外策略。此协议应可用于临床诊断和基础研究。在这里，我们以兔子等离子体为例来证明这项技术，而来自其他物种的等离子体也可以以同样的方式应用。
Protocol
兔子研究的所有程序均经山梨大学机构动物护理和使用委员会批准（批准编号：A28-39）。 1. 血浆与兔血分离 准备 1.5 mL 微管，其中含有 15μL 的 0.5 M EDTA （pH 8.0）， 用于采血。 将兔子放入约束器中，用22量针刺穿动脉中间动脉，并将血液收集到管子中。轻轻地将血液与EDTA混合，放在冰上。 在4°C下将血管在1，500 x g 上集中20分钟，并将血浆收集到?…
Representative Results
使用此协议，我们使用 1 mL 的血浆分离兔子脂蛋白，并获得了 7 个密度分数。对于大多数研究目的，孤立的密度分数足以测量上文所述的脂质和非政治蛋白。同样的程序也可用于将等离子脂蛋白与人类和其他物种分离。对于小动物，如小鼠，需要汇集血浆。图 3显示了野生类型 （WT） 兔子喂养正常标准 （NS） 饮食或高胆固醇 （HC） 饮食的脂蛋白特征。兔子是食草动物，因?…
Discussion
高脂血症是动脉粥样硬化疾病最重要的危险因素之一。因此，对血浆脂蛋白的分析不仅对血脂血症患者的诊断至关重要，而且对研究脂蛋白代谢和动脉粥样硬化的分子机制也具有重要意义。在这项研究中，我们描述了等离子脂蛋白的分离和分析方案，这些协议可以应用于有超中心化的实验室。通过这种方法获得的信息是全面和直接的，因此建议临床和基础研究科学家。分离脂蛋白也可用于研究脂?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了JSPS KAKENHI赠款编号JP 20K08858、中国国家自然科学基金（第81941001号和81770457号）的研究资助。
Materials
| 22-gauge needle | Terumo | NN-2232S | For blood collection |
| 96-well microplate | greiner bio-one | 655101 | For lipids measurment |
| Anti-apolipoprotein A-I antibody | LifeSpan BioSciences | LS-C314186 | For Western blottng, use 1:1,000 |
| Anti-apolipoprotein B antibody | ROCKLAND | 600-101-111 | For Western blottng, use 1:1,000 |
| Anti-apolipoprotein E antibody | Merck Millipore | AB947 | For Western blottng, use 1:1,000 |
| CBB staining kit | FUJIFILM Wako Pure Chemical | 299-50101 | For apolipoprotein analysis |
| Centrifuge | HITACHI | himac CF15RN | |
| Closure | Spectrum | 132736 | For lipoprotein dialysis |
| Dialysis tubing | FUJIFILM Wako Pure Chemical | 043-30921 | For lipoprotein dialysis, MWCO 14,000 |
| Dry heat block | Major Science | MD-01N | For SDS-PAGE sample preparation |
| ECL Western blotting detection reagents | GE Healthcare | RPN2209 | For Western blotting |
| Electrophoresis Chamber | BIO-RAD | Mini-PROTEAN Tetra Cell | |
| Ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) disodium salt dihydrate | FUJIFILM Wako Pure Chemical | 345-01865 | For anticoagulant (0.5 M), for dialysis (1 mM) |
| Filter paper | ADVANTEC | 590 | For Western blotting |
| Fixed angle ultracentrifuge rotor | BECKMAN COULTER | 357656 | TLA-120.2 |
| Fixing solution | For SDS-PAGE (50% Methanol/ 10% Acetic acid) | ||
| Immun-Blot PVDF menbrane | BIO-RAD | 1620177 | For Western blotting |
| Lumino image analyzer | GE Healthcare | For Western blotting, ImageQuant LAS 4000 | |
| Magnetic stirrer | ADVANTEC | SR-304 | For lipoprotein dialysis |
| Microplate reader iMARK | BIO-RAD | For lipids measurment | |
| Microtube | INA-OPTIKA | SC-0150 | |
| Orbital agitator USBDbo | Stovall Life Science | ||
| Peroxidase congugated anti goat IgG antibody | Jackson ImmunoResearch | 705-035-003 | For Western blotting, use 1:2,000 |
| Peroxidase congugated anti mouse IgG antibody | Jackson ImmunoResearch | 715-035-150 | For Western blotting, use 1:2,000 |
| Phospholipids assay kit | FUJIFILM Wako Pure Chemical | 433-36201 | For lipids measurment |
| Polycarbonate ultracentrifuge Tubes | BECKMAN COULTER | 343778 | |
| Potassium Bromide | FUJIFILM Wako Pure Chemical | 168-03475 | For density solution |
| Power Supply | BIO-RAD | For SDD-PAGE and Western blotting, PowerPac 300, PowerPac HC | |
| Protein standards Precidion Plus Protein Dual Xtra | BIO-RAD | 161-0377 | For SDS-PAGE and Western blotting |
| Rabbit restrainer | Natsume Seisakusho | KN-318 | For blood collection |
| Rotor | HITACHI | T15A43 | |
| SDS-PAGE running buffer | 25 mM Tris/ 192 mM Glycine/ 0.1% SDS | ||
| SDS-PAGE sample buffer (2x) | 0.1M Tris-HCl (pH 6.8)/ 4% SDS/ 20% glycerol/ 0.01% BPB/12% 2-merpaptoethanol | ||
| SDS-polyacrylamide gel | 4-20% gradient polyacrylamide gel | ||
| Skim milk powder | FUJIFILM Wako Pure Chemical | 190-12865 | For Western blotting blocking buffer (5% skim milk/ 0.1% Tween 20/ PBS) |
| Total cholesterol assay kit | FUJIFILM Wako Pure Chemical | 439-17501 | For lipids measurment |
| Triglyicerides assay kit | FUJIFILM Wako Pure Chemical | 432-40201 | For lipids measurment |
| Tube slicer for thick-walled tube | BECKMAN COULTER | 347960 | For lipoprotein isolation |
| Tween 20 | SIGMA-ALDRICH | P1379 | For Western blotting washing buffer (0.1% Tween 20/ PBS) |
| Ultracentrifuge | BECKMAN COULTER | A95761 | Optima MAX-TL |
| Western blotting wet transfer system | BIO-RAD | Mini Trans-Blot Cell |
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Citazione di questo articolo
Niimi, M., Yan, H., Chen, Y., Wang, Y., Fan, J. Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation. J. Vis. Exp. (167), e61790, doi:10.3791/61790 (2021).