体外生物荧光法对乳腺上皮细胞昼夜节律的表征
Summary
一个体外生物荧光法测定乳腺上皮细胞的细胞昼夜节律。该方法利用哺乳动物细胞报告质粒表达不稳定的荧光在周期 2基因启动子的控制下。它可以适应其他细胞类型, 以评估器官特定的影响昼夜节律。
Abstract
昼夜节律是所有生物体中的基本生理过程, 它们调节着从基因表达到睡眠行为等生物过程。在脊椎动物中, 昼夜节律由一个分子振荡器控制, 它在交叉核 (SCN; 中央起搏器) 和包括大多数外围组织的单个细胞中起作用。更重要的是, 暴露于 light-at 夜间、环境压力源和/或毒物的昼夜节律的破坏与慢性疾病和衰老的风险增加有关。能够识别能扰乱中枢和/或周边生物生物钟的药物, 以及能预防或减轻昼夜节律紊乱影响的药物, 对预防慢性病具有重要意义。虽然啮齿类动物模型可以用来识别诱发或预防/减轻昼夜节律紊乱的暴露和药物, 但这些实验需要大量的生物。在体内研究还需要大量的资源和基础设施, 并要求研究人员整夜工作。因此, 迫切需要一个细胞类型的适当的体外系统, 以筛选环境昼夜干扰和细胞类型的促进剂从不同的器官和疾病状态。我们构建了一个向量, 驱动转录的不稳定的荧光在真核细胞的控制下的人的周期 2基因启动子。这种昼夜节律的报告结构被稳定地转染为人乳腺上皮细胞, 并选择了昼夜节律性的记者细胞来发展体外生物荧光检测。在这里, 我们提出了一个详细的协议, 建立和验证的化验。我们进一步提供详细的概念实验证明的能力, 我们的体外化验, 以重述的体内的影响, 各种化学品的细胞生物钟。结果表明, 该方法可以适应各种细胞类型, 以筛选的环境干扰和化学促进剂的昼夜节律时钟。
Introduction
生物钟昼夜节律调节着一系列的生物学过程, 从昼夜的基因表达到睡眠行为, 周期约24小时. 流行病学研究强烈表明慢性扰乱昼夜节律节奏增加了轮班工作人员的乳腺癌和前列腺癌的风险, 包括护士和空勤人员1,2,3。这些发现得到了啮齿动物研究的证实, 证明暴露在恒定的光照、light-at 夜间或模拟时差的光周期会增加肿瘤发病率并加速肿瘤的生长4,5。根据人类和啮齿类研究的数据, 国际癌症研究机构在 2010年6将轮班工作归类为可能的人类致癌物 (类型 2A)。
此前, 我们证明, 一个单一的致癌剂量的乳腺肿瘤特异性致癌物, n-硝基-n-脲 (NMU), 扰乱了主要昼夜节律基因 (厘米) 的昼夜表达 (如,期间2, Per2) 和几个昼夜节律控制的基因 (CCGs), 包括靶乳腺 (但不是肝脏) 中的关键 DNA 损伤应答和修复 (复员) 基因。此外, 通过化学饮食的膳食L-甲基胱氨酸 (MSC), 将Per2和复员基因的昼夜节律表达复位为正常, 将肿瘤发病率降低63%。这些发现首次显示了昼夜节律, 化学致癌和化学7,8之间的机械联系。暴露于其他环境毒物显示扰乱昼夜基因表达在体内也与增加环境疾病的风险相关9,10。了解环境毒物和致病机制将昼夜节律紊乱联系起来的机理, 可能导致 mechanistically-based 的疾病预防方法。然而, 旨在确定暴露和昼夜节律之间相互作用的研究通常在体内进行.一个典型的体内实验研究对昼夜节律的影响需要大量的动物, 因为至少有三控制的组织和三暴露的动物必须在24或48小时内每3-4 小时收集一次。开发一个验证的体外系统, 重述在体内的观察和机制, 因此不仅减少了所需的动物数量, 而且大大降低了实验成本, 并要求研究人员在24-48 小时内连续工作。此外, 经验证的体外系统可用于高吞吐量的化合物筛选和/或影响昼夜节律的基因改变, 或对环境压力源或毒物的反应。因此, 需要对体外和体内模型和实验的策略性结合, 以获得不同焦点的不同见解。
在哺乳动物中, 昼夜节律振荡器不仅存在于 SCN 的专门神经元中, 而且还出现在大多数外围细胞类型中。这些分子钟类似于已建立成纤维细胞系和从胚胎或成年动物的原代成细胞;但是, 需要组织类型特定的蜂窝模型11。因此, 传统的运动活动的研究在体内, SCN外植体的ex 体内, 和细胞的在永生化成纤维细胞中的检测被广泛应用于研究自主性的昼夜生理缺陷。然而, 没有证据表明, 一个体外成纤维细胞的检测可以重述其他周边器官的细胞中的昼夜节律机制和反应,体内。不同的细胞类型可以有不同的基因表达模式, 异代谢, 和复员, 以及毒性和昼夜基因表达之间的联系可能是细胞类型的具体和/或不同的生理参数调制。此外, fibroblast-based 系统中的昼夜振荡器尚未得到充分评估, 以应对环境毒物、压力源和预防性药物, 它们将暴露与疾病发展和预防机制联系起来。因此, 需要简便、有效的细胞类型特异性、体外生物荧光检测来研究器官特定的环境昼夜干扰物。虽然各种细胞时钟模型 (例如, 在肝脏, 角质形成细胞, 和脂肪细胞, 以及骨肉瘤干细胞线) 已发展在最近几年12,13,14,15, 这里所描述的是乳腺上皮细胞中的第一个细胞时钟模型, 也是第一次在体内对环境压力源、毒物、药物和化学剂的反应进行复述的示范。
Renilla 荧光 (rLuc) 和萤火虫荧光是 30-61 kDa 单体蛋白, 不需要后处理酶活性, 并可以作为一个遗传学记者后立即翻译。一旦基体与荧光酶相关联, 生物化学反应就会产生闪光。因此, 荧光结构被广泛应用于基因表达报告系统体外和体内。然而, 在昼夜节律研究中, 荧光记者的效用受荧光蛋白 (T1/2 = 3.68 h) 相对较长的半衰期的限制, 因为它相对于昼夜节律基因的变化 (特别是短周期)。表达;然而, 多年来许多研究成功地使用了荧光基因在 pGL3 传染媒介, 表明快速地可降解的荧光可能不是必要的为报告昼夜节律, 特别是为节奏以较长的期间, 例如24小时因此, 一个记者质粒使用不稳定的荧光载体, pGL [Luc2P/Neo], 其中包含 hPEST (蛋白质失稳序列) 已经开发, 使我们可以使用它作为一个昼夜报告矢量我们目前的体外生物荧光测定法。由Luc2P编码的蛋白质具有更短的半衰期 (T1/2 = 0.84 h), 因此, 对转录活性的变化比对野生类型的反应更快和更大的程度, 我ndicating 可用于监视PER2启动器在 real-time16中精确调节的荧光的节律表达式。
Protocol
Representative Results
Discussion
在哺乳动物细胞中, 昼夜节律时钟的周期性由相互关联的转录/平移反馈回路调节。Heterodimers 的 Bmal1 和时钟或 Npas2 调节昼夜转录通过绑定到电子盒元素的推动者的核心厘米, 包括Per2和哭, 和许多 CCGs4。当它们在细胞中堆积时, heterodimers 的每一个: 哭 post-translationally 被修饰并传送到细胞核以抑制 Clock:Bmal1 的转录活性。这个负反馈回路允许核心厘米调节自己的转录, 并?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了2012毒理学学会的支持–高露洁榄替代研究基金 (m) 和大韩民国动植物检疫机构国际合作研究基金会 (m. 方) 和 NIEHS 赠款P30ES005022 (h. Zarbl)。我们要感谢 Dr. (在休斯敦德克萨斯健康科学中心的麦戈文医学院) 为他的有益的讨论, Mr. 绍-一个胡安为他的实验协助, 和 Ms. 基米中田为她的校对阅读。
Materials
| pLS[hPER2P/rLuc/Puro] vector | SwitchGear Genomics | S700000 | customized vector |
| pGL4.18[Luc2P/Neo] vector | Promega | 9PIE673 | destabilized luciferase expression vector |
| T4 DNA ligase | Invitrogen | 15224041 | For subcloning |
| TOPO TA cloning kit | Invitrogen | K4500-02 | with One Shot TOP10 Chemically Competent E. coli |
| Sac I | New England BioLabs | R0156S | Restriction enzyme |
| Hind III-HF | New England BioLabs | R3104S | Restriction enzyme |
| CutSmart buffer | New England BioLabs | B7204S | Restriction enzyme buffer |
| DNA gel extraction kit | Qiagen | 28704 | Purify DNA fragments from agarose gel |
| PCR Purification Kit | Qiagen | 28104 | DNA clean up |
| LB Miller's modification | TEKNOVA | L8600 | For transformed E. Coli culture |
| LB Agar Plate | TEKNOVA | L1902 | For white/blue selection |
| QIAprep spin miniprep Kit | Qiagen | 27104 | For extraction of plasmide DNA |
| EndoFree plasmid maxi prep Kit | Qiagen | 12362 | For extraction of plasmide DNA |
| FuGene HD | Promega | E2311 | Transfection reagent |
| Opti-MEM reduced serum medium | Invitrogen | 31985-062 | For transfection |
| MCF10A cell line | American Type Culture Collection | CRL-10317 | Mammary Epithelial Cells |
| MEGM BulletKit | Lonza | CC-3150 | Mammary Epithelial Growth Medium |
| MEBM | Lonza | CC-3151 | Mammary Epithelial Basal Medium |
| MEGM SingleQuot Kit | Lonza | CC-4136 | Suppliments & Growth Factors |
| ReagentPack | Lonza | CC-5034 | Reagent for subculture |
| D-PBS (10X) | Sigma | D1408 | Wash cells in culture dishes |
| UltraPure Distilled Water | Invitrogen | 10977 | Dilute 10X D-PBS |
| Tissue culture dish (35 mm) | BD/Falcon | 353001 | cell culture dish suitable to LumiCycle |
| Silicon grease | Fisher | NC9044707 | For sealing dish with recording medium |
| Round cover glass | Harvard Bioscience | 64-1500 (CS-40R) | For sealing dish with recording medium |
| Cholera toxin | Sigma | C8052 | Supplement for growth medium |
| G418 sulfate (Geniticin) | Invitrogen | 10131035 | Antibiotic for selection of stabliy transfected cells |
| Ampicillin | Sigma | A9393 | For colony selection |
| Forskolin | Sigma | F6886 | Synchronization agent |
| Melatonin | Sigma | M5250 | Synchronization agent |
| Dexamethasone | Sigma | D4902 | Synchronization agent |
| Horse serum | Sigma | H1138 | Synchronization agent |
| d-Luciferin, sodium salt | Invitrogen | L2912 | Luciferase substrate |
| IC261 | Sigma | 10658 | Positive control for circadian disruptor |
| Methylnitrosourea (NMU) | Sigma | N1517 | Mammary specific carcinogen |
| Methylselenocysteine (MSC) | Sigma | M6680 | Organic selenium (chemopreventive agent) |
| EX527 | Sigma | E7034 | SIRT1 specific inhibitor |
| Cambinol | Sigma | C0494 | SIRT1 & SIRT2 inhibitor |
| NanoDrop Spectrophotometer | Thermo Scientific | NanoDrop 8000 | Quantify nucleotide |
| GeneAmp PCR System 9700 | Applied Biosystems | N805-0200 | For molecular biology experiment |
| CO2 Incubator | NAPCO | Series 8000 DH | For cell culture at 5% CO2 at 37 °C |
| Desktop centrifuge with refrezerator | Eppendorf | 5430R | For molecular biology experiment |
| Centrifuge with swing bucket | Eppendorf | 5810 R | For cell culture |
| Inverted microscope | Nikon | 80124 | Phase contrast optional |
| Tissue culture hood | Labconco | Class II A2 | BSL-2 certified |
| LumiCycle 32 | Actimetrics | Not Available | Luminoscence detector |
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