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Médecine

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Published: October 23, 2018
doi:
10.3791/58323
, , , , , ,
1Department of Pharmacology and Systems Physiology, College of Medicine,University of Cincinnati, 2Division of Endocrinology,Cincinnati Children’s Hospital Medical Center, 3Division of Developmental Biology,Cincinnati Children’s Hospital Medical Center, 4Department of Pediatrics, College of Medicine,University of Cincinnati

Summary

在这里, 我们提出了一个分离健康和功能的原发性小鼠肝细胞的协议。通过非放射性标记基底检测肝新生蛋白合成的说明, 帮助了解肝脏能量代谢稳态背景下蛋白质合成的机理。

Abstract

肝细胞是肝脏的实质细胞, 参与多种代谢功能, 包括蛋白质的合成和分泌对系统能量稳态至关重要。从小鼠肝脏分离的原发性肝细胞构成了一个重要的生物工具来了解肝脏的功能特性或变化。本文介绍了一种通过两步胶原酶灌注技术分离和培养原发性小鼠肝细胞的方法, 并讨论了其在蛋白质代谢研究中的应用。成年小鼠肝脏依次用乙二醇-双四乙酸酸 (EGTA) 和胶原酶灌流, 其次是肝细胞与密度梯度缓冲液的分离。这些分离的肝细胞在培养板上是可行的, 并保持肝细胞的大部分赋存特征。这些肝细胞可用于评估蛋白质新陈代谢, 包括新生的蛋白质合成与非放射性试剂。我们表明, 分离的肝细胞是易于控制, 并包含更高的质量和体积稳定性与能量代谢相关的蛋白质合成, 利用化疗选择性结扎反应与四甲基罗丹明 (塔姆拉) 蛋白检测方法和西方印迹分析。因此, 该方法对研究与能量稳态有关的肝新生蛋白合成具有重要意义。下面的协议概述了分离高质量原代小鼠肝细胞和检测新生蛋白质合成的材料和方法。

Introduction

蛋白质是一个重要的营养元素, 人体干重的大约50% 是由具有多种生物学特性和功能的蛋白质组成的1。因此, 蛋白质合成是最消耗能量的事件之一, 蛋白质代谢的改变与疾病的发展密切相关, 包括代谢疾病2,3,4。在肝脏中, 蛋白质生物合成占总能量消耗的大约 20–30%5,6。此外, 蛋白质不仅作用于肝脏的被动或构建块, 而且还有主动信号调解因子潜伏或给予调节系统代谢7。例如, 血清白蛋白的水平降低, 由肝脏合成和分泌, 血浆中最丰富的蛋白质8, 增加2型糖尿病发展的风险9,10, 11, 而更高浓度的血清白蛋白是预防发展代谢综合征12。此外, 干扰或中断分泌或膜束缚肝蛋白, 调节胆固醇稳态, 包括脂蛋白, LDLR, 和 LRP1, 可能导致胰岛素抵抗, 高脂血症, 或动脉粥样硬化的发展13. 因此, 鉴定肝脏蛋白质代谢紊乱中涉及的分子病理生理学机制及其相关代谢并发症可能有助于发现新的药理学延缓发病或治疗代谢性疾病的方法, 如胰岛素抵抗、糖尿病和无酒精脂肪肝。

蛋白质合成与细胞能量状态密切相关 (例如, 在蛋白质合成的伸长步骤中形成一个肽键, 需要4磷酸二酯键14), 并通过分子通路来调节细胞间和内营养的可用性15,16。AMP 活化蛋白激酶 (AMPK) 是维持能量稳态17的细胞内能量传感器之一。一旦 AMPK 被激活时, 细胞能量水平变得较低, AMPK 及其靶向基底功能刺激分解代谢通路和抑制合成代谢过程, 包括蛋白质合成18,19。蛋白质合成的调节通过多种转化因子和核糖体蛋白的磷酸化介导20。值得注意的是, 雷帕霉素复合物 1 (mTORC1) 的哺乳动物靶点是蛋白质合成的主要驱动力, 是 AMPK21的主要靶点之一。激活 mTORC1 通路通过刺激蛋白质转化和自噬20,21提高细胞生长和增殖。因此, AMPK 活化能抑制 mTORC1-mediated 蛋白合成22是合乎逻辑的。事实上, 激活 AMPK 抵消和直接 phosphorylates mTORC1 对苏氨酸残留 2446 (2446) 导致其失活23和抑制蛋白质生物合成24。此外, AMPK 可以间接抑制 mTORC1 功能的磷酸化和活化的结节性硬化复合 2 (TSC2)25是 mTORC1 信号级联的上游调节器。简而言之, 这些途径在肝脏中的失调经常与代谢疾病的发展有关, 因此迫切需要建立有效的实验工具来研究这些途径在调节能源和肝细胞蛋白质代谢。

分离的原发性肝细胞和体内肝细胞的功能特性与体外肝细胞系26、27、28之间有较强的相似性。结果表明, 原代人肝细胞与肝活检有77% 相似之处, 而 HepG2 细胞是分化良好的肝癌细胞, 广泛用于研究肝功能, 在其背景下显示小于48%。基因表达谱29。因此, 利用原发性肝细胞, 而不是永生化培养细胞, 对肝脏功能和生理研究具有重要意义, 并可用于原代肝细胞的分离与培养。特别是从大鼠30,31。虽然大鼠肝细胞是有用的在一个相对较高的产量, 小鼠肝细胞在许多科学方面有更大的潜力, 因为转基因小鼠的广泛可用性。然而, 从小鼠分离健康和丰富的原发性肝细胞的细胞和分子的评估有几个技术挑战: 首先, 套管插入灌注肝用缓冲试剂是非常困难的处理由于小而薄小鼠门静脉或下腔静脉;其次, 在分离过程中, 细胞的操作时间越长, 细胞数量和质量就会降低;第三, 非酶机械分离方法可能导致严重的损伤, 并产生低产量的存活的分离原代肝细胞32,33。在二十世纪八十年代, 采用胶原酶灌注技术, 从动物肝脏中分离肝细胞34。这种方法是基于胶原酶灌注肝35、36、37、肝灌注钙螯合剂溶液38、39、酶消化和肝实质的机械解离35。在第一步, 小鼠肝脏灌流与钙 [ca2 +] 免费缓冲区包含 [ca2 +] 螯合剂 (乙二胺四乙酸酸, EDTA)。第二步, 小鼠肝脏灌入含有胶原酶的缓冲液, 以水解细胞外基质的相互作用。与第一步中使用的缓冲液不同, 在第二步的缓冲液中存在 [Ca2 +] 离子是有效的胶原酶活性所必需的, 之后消化的肝脏必须进一步轻柔地与机械分离, 使用钳肝囊和薄壁组织。最后, 通过过滤去除结缔组织, 随后离心分离非实质细胞和非活体肝细胞的可行肝细胞与使用密度梯度缓冲液40,41 ,42。在本研究中, 我们展示了一种改良的两步法胶原酶灌注技术, 用于分离小鼠肝脏的原代肝细胞, 用于蛋白质合成分析。

蛋白质的简単被广泛用于量化的表达水平, 更替率, 并确定蛋白质的生物分布43由于高灵敏度的放射性检测44。然而, 使用放射性同位素需要高度控制的研究环境和程序45。替代性的非放射性方法已得到发展, 越来越受到欢迎。四甲基罗丹明 (塔姆拉) 蛋白检测方法的化疗选择性结扎反应是其中之一, 是基于叠氮化物和炔烃基团之间的化学选择性反应46, 可用于分析细胞事件, 如检测新生蛋白合成和用叠氮化物组修饰的糖蛋白子类。对于新生的蛋白质合成, l-Azidohomoalanine (l-AHA, 一种叠氮化物改性氨基酸) 可以代谢纳入蛋白质和检测通过使用塔姆拉蛋白检测方法47。通过在原代小鼠肝细胞中使用这种检测方法, 我们表明, 新生的蛋白质合成速率与线粒体和 AMPK 活化 ATP 的可用性密切相关 (图 1)。

总之, 利用主鼠肝细胞是研究蛋白质和能量代谢和量化新生蛋白质合成的重要因素, 这对于深入了解与发展相关的通路的生理作用是有价值的。肝细胞相关疾病的治疗。

Protocol

本协议包含实验室小鼠的使用。动物护理和实验程序是按照辛辛那提儿童医院医疗中心动物保育委员会批准的程序进行的。 1. 原代小鼠肝细胞的分离 预隔离准备 如表 1所述, 准备450毫升40% 密度梯度缓冲器, 保持在4摄氏度 (15 mL/鼠标)。 准备500毫升威廉姆斯的培养基, 如表 1所述, 保持在4摄氏度。 准备500毫升的 DMEM 如…

Representative Results

主要的小鼠肝细胞隔离导致约 20 x 106总细胞/小鼠的产量。组织学, 活体和附着的原代肝细胞出现多边形或典型的六角形与明确概述的膜边界后24小时孵育 (图 2)。 为了证实孤立细胞是否为原代肝脏, 我们比较了分离的原代小鼠肝细胞 (PMHs)、小鼠胚胎成纤维细胞 (MEFs)、小鼠肝癌细胞系 (Hepa 1-6) 和小?…

Discussion

虽然一些永生化的肝细胞系已经提出并用于调查肝功能49,50,51,52, 这些细胞一般缺乏重要的和基本的正常肝细胞的功能, 如白蛋白的表达 (图 3)。因此, 人们普遍认为, 利用原发性肝细胞是检查肝脏生理学和文化新陈代谢的一个有价值的选择, 尽管这些细胞的培养和维护的挑战。在?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢 Drs Joonbae Seo 和维薇安的科学投入和讨论。这项工作得到了国家卫生研究院 (NIH) (R01DK107530) 的支持。T.N. 得到日本科技署的支持。这项研究的一部分是由 NIH (P30DK078392) 为辛辛那提的消化系统疾病研究核心中心提供资助的。

Materials

HEPES buffer Fisher Scientific BP310-500
D-glucose Fisher Scientific D16-500
Ethylene glycol-bis(β-aminoethyl ether)-tetraacetic acid AmericanBio AB00505-00025
Antibiotic-Antimycotic (100X) Gibco 15240-062
HBSS (10X) no calcium, magnesium, phenol red Gibco 14185-052
Calcium Chloride Dihydrate (CaCl2.2H2O) Fisher Scientific C79-500
Density gradient buffer GE Healthcare 17-0891-02
DMEM (Dulbecco's Modified Eagle Medium) low glucose, pyruvate Gibco 11885-084
Fetal Bovine Serum Hyclone SH30910.03
Phosphate Buffered Saline (PBS) (1X) Gibco 1897141
Williams medium E, no glutamine Gibco 12551-032
L-alanyl-L-glutamine dipeptide supplement Gibco 35050-061
Collagenase Type X Wako Pure Chemical Industries 039-17864
Perfusion pump Cole-Parmer Masterflex L/S Equipment
IV administration set EXELINT 29081 Equipment
A water bath REVSCI RS-PB-200 Equipment
Tube heater Fisher Scientific Isotemp Equipment
Ethanol Decon Lab, Inc 0-39613
Isoflurane PHOENIX 10250
Autoclaved Cotton Tips Fisherbrand 23-400-124
100 mm Petri Dish TPP 93100
Connector (Male Luer Lock Ring) Cole-Parmer instrument EW-4551807
24G catheters TERUMO Surflo 24Gx3/4'
100 μm Filter (CELL STRAINERS) VWR 10199-658
15 ml conical-bottom centrifuge tubes VWR 89039-666
50 ml conical-bottom centrifuge tubes VWR 89039-658
Chemoselective ligation reaction PROTEIN ANALYSIS DETECTION KIT, TAMRA ALKYNE Invitrogen C33370
AHA (L-azidohomoalanine) Invitrogen C10102
DMEM (methionine free) Gibco 21013024
L-Cystine Dihydrochloride SIGMA C2526
Laemmli sample buffer BioRad 161-0737
Protease Inhibitor Cocktail SIGMA P9599
SDS solution (20%) BioRad 161-0418
Tris-HCL (1M) American Bioanalytical AB14044-01000
Phosphatase Inhibitor Cocktail SIGMA P5726
Protein concentration measuring Kit (Bovin Serum Albumin-BSA) BioRad 500-0207
6-well tissue culture plate TPP 92006
Digital Heatblock VWR 12621-092 Equipment
Multi-Rotator Grant-bio PTR-60 Equipment
Ultrasonic Sonicator Cole-Parmer GE130PB Equipment
Standard Heavy-Duty Vortex Mixer VWR 97043-566 Equipment
A variable mode laser scanner GE Healthcare Life Science FLA 9500 Equipment
Coomassie-dye reagent Thermo Scientific 24594
Inverted microscope Olympus CKX53 Equipment
Western Blotting apparatus BioRad 1658004 Equipment
Centrifuge Eppendorf 5424R Equipment
Automated cell counter BioRad TC20 Equipment
FluorChem R system proteinsimple – Equipment
p-Ampka (T172) antibody Cell signaling 2535
Total-AMPK antibody Cell signaling 5832
Albumin antibody Cell signaling 4929
beta actin antibody Santa Cruz sc-130656
Fine scissors and forceps
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Primary Mouse HepatocytesNascent Protein SynthesisL-azidohomoalanine LabelingNon-radioactiveLiver PerfusionCollagenaseDensity Gradient SeparationMetabolic Disease ResearchObesityNon-alcoholic Fatty LiverType Two Diabetes

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Salem, E. S., Murakami, K., Takahashi, T., Bernhard, E., Borra, V., Bethi, M., Nakamura, T. Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method. J. Vis. Exp. (140), e58323, doi:10.3791/58323 (2018).
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