高含量<em>体外</em>胰岛β细胞复制发现平台
Summary
对于糖尿病研究领域的关键挑战是要明白，调节胰岛β细胞的复制，并制定刺激β细胞再生方法的分子机制。在此高内涵筛选的方法来识别和评估小分子的β细胞复制促进活性表示。
Abstract
Loss of insulin-producing β-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet β-cells remains an attractive strategy. β-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain β-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify β-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a β-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on β-cells and the molecular mechanisms that regulate β-cell growth.
Introduction
糖尿病包括病症共享打乱葡萄糖稳态的共同终点的集合。虽然糖尿病亚型的致病机理是不同的，它们共享降低β细胞量， 即 ，胰岛素生产能力1,2-损失的后果。目前，糖尿病的治疗策略依赖外源性胰岛素，胰岛素的产生或增强胰岛素敏感性的药理学刺激的长期管理，也很少，胰岛或全胰腺3,4移植。令人遗憾的是，这些策略的成功是短命的和/或不能充分概括内源性胰岛素产生的作用。尽管显影刺激β细胞再生的方法的实用性，没有这样的方法存在。因此，一个重要的糖尿病研究的目标是开发的方法来产生新的β细胞或展开内源性β细胞量5 </sup>。虽然来自可再生资源，如胚胎干细胞β细胞再生正在推进，安全性和效率的关注使得替代策略，包括膨胀成熟β细胞，优先级6,7的追求。重要的是，新的β细胞在体内的主要来源是预先存在的β细胞，而不是专门祖细胞8,9。虽然β细胞看起来具有有限的复制能力，在β细胞量略有增加（〜30％）可足以在许多糖尿病患者恢复葡萄糖动态平衡。另外， 在 β细胞量的原位药物刺激是一种潜在的廉价的和可扩展的治疗策略。这里提出了识别和表征刺激β细胞生长的小分子的高含量筛选方法。
各种体外实验方法可以用于鉴定基因产物和/或分子塔ŧ促进初级β细胞复制。用于测量β细胞复制诱导早期的努力用于胎儿啮齿动物的胰腺文化或完整隔离胰岛文化来衡量响应特定的处理条件10 [3 H]胸苷掺入，BrdU的醛，硫堇或胰岛素染色人群中掺入或有丝分裂体， 11。 这些体外的方法和密切变体具有一些局限性。突出的缺陷包括：（1）使用胎儿细胞，不同于成熟β细胞，显示高基底β细胞复制速率和在明显的方式12调节生长; （2）β细胞复制事件实验者相关裁决的主观性; （3）β细胞复制事件实验者依赖计数的劳动和时间密集性质阻碍了实验的吞吐量; （4）利用核掺入/染色/外观识别复制甚至TS和一个非重叠的胞质染色，以确定β细胞导致邻近非β细胞复制事件对β细胞的错误认定。
最近成熟原β细胞已被用于评估转基因过度表达对β细胞复制13-16的影响，以及基因产物或化合物处理。然而，这些研究还依赖于复制事件，对β细胞鉴定和/或限制通过劳动密集型的步骤， 例如 ，细胞或完整胰岛个体滑动孔电镀细胞质staining-或非特异性方法的主观计数石蜡包埋和处理17。值得注意的是，基于图像的人β细胞复制的筛选方法，类似于本文中所呈现的人，已发表18;然而，成功地利用该测定的尚未得到证实和利用对于初筛人胰岛的可能不是大致FEAsible。
识别复制促进物质的另一种策略是评估的β细胞系的生长诱导。最初的努力用于转化的β细胞，系如MIN6细胞或INS一十三分之八百三十二细胞14,19-21。然而，这些细胞系中表现出奔放增长，没有什么相似之处分化良好的β细胞22。因此，增长的感应能力是最小的，目前还不清楚相关的，有时难以概括。基于细胞系的筛选改进策略采用“可逆转变”是生长在没有四环素（强力霉素）的逮捕依赖SV40 T抗原表达23,24细胞。然而，目前还不清楚这些细胞是否恢复到在强力霉素除去一个“正常”β细胞样状态。不幸的是，使用这些细胞已经产生，不会出现有直接效用广义生长促进化合物24。总体而言，使用的细胞系来研究细胞类型中显示最小的自发复制活动的生长调控可能具有有限的适用性。
本文中所呈现的β细胞复制筛选平台利用成熟原代大鼠β细胞在体内的生长调节以保持在可能的范围内，混合细胞型组合物，以便能够谱系限制性生长促进性活动的胰岛细胞培养物，多-Well格式以最大化吞吐量和自动化的分析，以消除偏见和促进吞吐量。成功地利用这个平台，使促进胰岛β细胞的复制25,26几种化合物的鉴定。此外，该测定已被用于结构 – 活性关系的研究和化学上位实验以提供机械见解β细胞复制的分子调控。所提出的平台成功地适应FOβ细胞复制的RNA干扰基于-R的慢病毒调查通路25。该测定的局限性包括限制可扩展性（使用原代细胞），利用啮齿动物，而不是基于抗体的成像和初级胰岛使用，使用相关联的人胰岛细胞（尽管该测定可适于人胰岛研究），费用的的分散的胰岛（胰岛破坏架构），以方便自动图像采集和依赖性在与图像采集和分析能力的自动显微镜的可用性。虽然用于鉴定基因产物或刺激原位 β细胞再生化合物的简便体内筛选方法将是理想的，这样的平台尚不可用27。因此，所描述的平台是适合研究者感兴趣的研究β细胞复制的大多数方面。
Protocol
Representative Results
Discussion
为研究控制β细胞的生长和再生的分子通路的实验方法对于糖尿病研究的重要工具。在此，基于大鼠胰岛筛选平台，以确定和描述β细胞复制的小分子刺激呈现。
虽然该协议的大部分内容很容易被经验丰富的研究人员进行了几步需要特别的技术。首先，胰岛分离过程中，而不破坏其完整性胆管插管需要实践。一个有用的策略是最低限度地膨胀胆管，以确保充分配药胰腺消化液?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by NIDDK grants DK098143 and DK101530 from the NIH (JPA), Stanford’s Spectrum Child Health Research Institute (CHRI) and SPARK (UL1 TR001085, JPA).
Materials
| 250g male Male Sprague Dawly Rat | Charles River | Stain # 400 | |
| 12 cm teeth tisuue forceps | Fine Science Tools | 11021-12 | |
| 11.5 cm fine scissors | Fine Science Tools | 14058-11 | |
| 14.5 cm surgical scissors | Fine Science Tools | 14001-14 | |
| 16 cm curved forceps | Fine Science Tools | 11003-16 | |
| 12 cm curved hepostat | Fine Science Tools | 13011-12 | |
| 12 cm scalpel handle | Fine Science Tools | 10003-12 | |
| Tissue sieve-30 mesh | Bellco Glass | 1985-85000 | |
| Cizyme RI, 375,000 CDA units | VitaCyte | 005-1030 | |
| Hanks' Balanced Salt solution (Ca++ and Mg++) | Gibco | 24020-117 | |
| Ketamine HCl (200 mg/20 ml) | JHP Pharmaceuticals | NDC# 42023-113-10 | to make anesthetic cocktail |
| Xylazine (5 g/50 ml) | LLOYD | NADA# 139-236 | to make anesthetic cocktail |
| Histopaque 1077 | Sigma | H-1077 | to make histopaque 1100 |
| Histopaque 1119 | Sigma | H-1119 | to make histopaque 1100 |
| Newborn Calf Serum 500 ml | Hyclone | SH30118.03 | |
| Hanks' Balanced Salt solution | Hyclone | SH30268.01 | |
| Dulbecco's Modified Eagle Medium/Low Glucose | Hyclone | SH30021.01 | |
| Functionality/Viability Solution | Mediatech | 99-768-CV | |
| RPMI1640 media | Hyclone | SH30096.01 | to make conditioned medium |
| 804G rat bladder carcinoma cell-line | Available upon request | to make conditioned medium | |
| Fetal Bovine Serum, Qualified | Gibco | 26160 | |
| GlutaMax-I | Gibco | 35050-061 | |
| Penicillin (5,000 IU/ml/Strptomycin (5 mg/ml) | MP Biomedicals | 1670049 | |
| Formamide 500 mL | Fisher BioReagents | BP227-500 | |
| Antigen Unmasking Solution 250 mL (PH 6.0) | Vector Laboratories | H-3300 | to make 0.15 M Sodium Sitrate solution |
| Dextrose, Anhydrous | EMD Chemicals | DX0145-1 | to make 1 M glucose solution |
| Nomal Donkey Serum (Powder) | Jackson ImmunoResearch | 017-000-121 | |
| Triton X-100 | Sigma | T8787-100ML | |
| Mouse anti-human Ki67 antibody | BD Biosciences | 556003 | |
| Goat anti-human PDX-1 antibody | R&D Systems | AF2419 | |
| Polyclonal Guinea Pig anti-insulin antibody | Dako | 2016-08 | |
| Polyclonal Rabbit anti-glucagon antibody | Dako | 2014-06 | |
| Polyclonal Rabbit anti-somatostatin antibody | Dako | 2011-08 | |
| Polyclonal chicken anti-vimentin antibody | abcam | ab24525 | |
| Biotin-SP-conjugated, Donkey Anti-Mouse IgG | Jackson ImmunoResearch | 715-065-150 | |
| StreptAvidin, Alex Flour 488 conjugated | Invitrogen | S32354 | |
| Rhodamine-conjugated Donkey Anti-Goat IgG | Jackson ImmunoResearch | 705-025-147 | |
| Rhodamine-conjugated Donkey Anti-Guinea Pig IgG | Jackson ImmunoResearch | 706-025-148 | |
| Rhodamine-conjugated Donkey Anti-Rabbit IgG | Jackson ImmunoResearch | 711-025-152 | |
| Cy 5-conjugated Donkey Anti-Guinea Pig IgG | Jackson ImmunoResearch | 706-175-148 | |
| Cy 5-conjugated Donkey Anti-Goat IgG | Jackson ImmunoResearch | 705-175-147 | |
| Cy 5-conjugated Donkey Anti-Rabbit IgG | Jackson ImmunoResearch | 711-175-152 | |
| Cy 5-conjugated Donkey Anti-Chicken IgG | Jackson ImmunoResearch | 703-175-155 | |
| DAPI | Millipore | S7113 | |
| Disposable Reagent Reservoir 25 mL | Sorenson BioScience | 39900 | |
| 384 well, black/clear, tissue culture treated plate | BD Falcon | 353962 | |
| 96 well, black/clear, tissue culture treated plate | Costar | 3603 | |
| Multi-channel pipettor | Costar | 4880 | |
| 12-channel vaccume aspirator | Drummond | 3-000-096 | |
| Cell Scraper | Falcon | 353085 | |
| Isotemp Water Bath Model 2223 | Fisher Scientific | ||
| High-content screening instrument: ArrayScan VTI | Thermo Scientific |
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