β细胞的有丝分裂原持续管理局完整小鼠胰岛<em>离体</em>使用可生物降解的聚（乳酸 - 共 - 乙醇酸）微球
Summary
Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens.
Abstract
生物材料的发展已显著增加用于靶向药物递送的潜在的各种细胞和组织类型，包括在胰岛β细胞。此外，生物材料颗粒，水凝胶，和支架还提供了一个独特的机会来管理持续的，可控的药物输送到β细胞的培养和移植的组织模型。这些技术允许使用完整的小岛和一个翻译相关制度的候选β细胞增殖因子的研究。此外，确定用于在培养系统刺激β细胞增殖的候选因素的有效性和可行性是向前移动到体内模型之前的关键。这里，我们描述的方法共培养完整小鼠胰岛与感兴趣的可生物降解化合物（COI）-loaded聚（乳酸-共-乙醇酸）（PLGA）微球，以评估原位释放ö持续的影响的目的˚F促有丝分裂因子对β细胞的增殖。这种技术详细描述了如何生成包含使用市售的试剂所需的货物PLGA微球。而所描述的技术使用的重组人结缔组织生长因子（rhCTGF）作为一个例子，可以很容易地用于各种COI的。此外，此方法利用的96孔板，以尽量减少必要评估β细胞增殖的试剂的量。该协议可以很容易地适合于使用替代生物材料和其它内分泌细胞特征，例如细胞存活和分化的状态。
Introduction
胰岛β细胞是在体内仅产生胰岛素的细胞，并维持血糖稳态的关键。而健康个体具有足够的β细胞量和功能适当地调节血糖，糖尿病的个体被不足β细胞量和/或功能1,2-特征。已经提出了诱导β细胞增殖可以最终增加β细胞量和个体的糖尿病3恢复葡萄糖动态平衡。然而，评价和完整的胰岛β潜能细胞增殖的化合物的验证是必要的，可以制定有效的治疗之前。尸体人胰岛与糖尿病患者的移植恢复血液葡萄糖稳态了一段时间，但是可用性和此实验过程的成功是由可供移植人胰岛的缺乏和由β细胞死亡中的胰岛船尾受阻呃移植4。即使使用的诱导胰岛素产生细胞的倍增因子的发现，一个重大的挑战仍然存在于提供这些因素的体内相关网站。对于持续局部递送β细胞增殖的化合物的一个策略是聚（乳酸 – 共 – 乙醇酸）（PLGA）。 PLGA在FDA的使用的历史认可药物递送制品，由于其高的安全性，生物降解性，并且延长释放动力学5。具体地，PLGA是通过水解与水在体内或在培养成乳酸和乙醇酸，其天然存在于体内存在的代谢产物降解的丙交酯和乙交酯的共聚物。包封的药物化合物可以通过扩散和/或降解受控释放机构周围环境释放。 COI的封装提供了保护，防止酶降解，提高试剂的生物利用度相比unencapsula特德COI 5。我们建议，PLGA微球能够最终在体内用于施用在培养候选化合物对完整的胰岛和。测试PLGA的功效来管理β细胞有丝分裂原到胰岛离体进行了探索移植协议之前是至关重要的。
目前，没有任何技术来测量在活的动物的β细胞增殖。实验评估体内潜在的增殖的化合物的有效性，因此需要这些化合物的行政生活的动物，为胰腺免疫标记随后的解剖和处理。这些协议是昂贵和费力的，并且所需要的化合物要全身施用，不作任何保证，他们将到达小岛。相反地，几种永生β细胞系可用于在培养产生胰岛素的细胞的研究，但这些细胞系缺乏胰岛架构和环境下吨生物6中。永生化β细胞系也被表征为具有高得多的程度比内源性β细胞在体内的复制，从而变得复杂诱导增殖的化合物的分析。在这项研究中，我们描述了使用从成年小鼠中分离的完整的胰岛的协议。与β细胞系，完整保留胰岛胰岛的正常结构。同样，在对比体内进行的试验中，给予增殖性化合物直接向培养的完整胰岛显著减少了必须精确地测量β细胞增殖的试剂的量。
目前的研究利用PLGA来管理COI，在这个例子中，重组人结缔组织生长因子（rhCTGF）。此处所描述的方法赋予通过原始化合物培养的胰岛给药的显著优势，因为它允许对一个持续释放化合物的成第e媒体。值得注意的是，该测定可以被修饰以管理各种蛋白质和感兴趣的完整胰岛抗体。在其他内分泌细胞类型，包括α-细胞的作用，也可以被分析。
Protocol
Representative Results
Discussion
β细胞增殖的培养物研究通常是由几个困难阻碍。首先，永生化β细胞系由较高程度的增生比什么是内源性β细胞活胰岛发现的特点。此外，这些永生化细胞系缺乏正常结构正常β细胞功能的关键。这两个事实使得难以确定是否使用永生化的β细胞系在体内或整个胰岛进行测试时将保持真获得的结果。我们描述的方案，它使用新鲜分离完整小鼠胰岛，如胰岛架构被保持和β细胞增殖相当于?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Bethany Carboneau (Vanderbilt University) for critical reading of this manuscript. We also thank Anastasia Coldren (Vanderbilt University Medical Center Islet Procurement and Analysis Core) for islet isolations, and Dr. Alvin C. Powers (Vanderbilt University Medical Center) and Dr. David Jacobson (Vanderbilt University) for use of their centrifuge and tissue culture facility. This research involved use of the Islet Procurement and Analysis Core of the Vanderbilt Diabetes Research and Training Center supported by NIH grant DK20593. This work was supported by an American Heart Association Postdoctoral Fellowship (14POST20380262) to R.C.P., and grants from the Juvenile Diabetes Research Foundation (1-2011-592), and Department of Veterans Affairs (1BX00090-01A1) to M.A.G.
Materials
| Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer | ThermoFisher Scientific | O6149 | For labeling COI with fluorophore |
| DMSO Dimethyl Sulfoxide | Fisher BioReagents | BP231-1 | For dissolving fluorophore in step 1 |
| Disposable PD-10 Desalting Columns | GE Healthcare | 17-0851-01 | Desalting column used in step 1 |
| Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000-69,000 | Sigma-Aldrich | 739960 | Used in generation of microspheres in step 2 |
| Poly(vinyl alcohol) molecular weight 89,000-98,000 | Sigma-Aldrich | 341584 | Used in generation of microspheres in step 2 |
| RPMI 1640 | Thermo Scientific | 11879-020 | For culturing islets |
| Dextrose Anhydrous | Fisher BioReagents | 200-075-1 | Supplement for islet media |
| Penicillin-Streptomycin | Sigma-Aldrich | P4333 | Antibiotics for islet media |
| Normal horse serum | Jackson ImmunoResearch | 008-000-121 | Supplement for islet media |
| 96-well tissue culture plate | Corning | 3603 | For culturing islets |
| Ethylene glyco-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid | Sigma-Aldrich | E4378 | Supplement for pre-assay islet media |
| Cytospin 4 Cytocentrifuge | Thermo Scientific | A78300003 | For spinning cells onto microscope slides |
| EZ Single Cytofunnel | Thermo Scientific | A78710020 | For spinning cells onto microscope slides |
| Ethylenediaminetetraacetic acid | Fisher BioReagents | BP118-500 | Used in dissociating islets |
| paraformaldehyde | Sigma-Aldrich | P6148 | For fixing cells |
| Triton X-100 | Fisher BioReagents | BP151 | For permeabilizing cells |
| Normal donkey serum | Jackson ImmunoResearch | 017-000-121 | Blocking reagents for immunofluorescence |
| Anti-Ki67 antibody | abcam | ab15580 | For Ki67 immunofluorescence |
| Polyclonal Guinea Pig Anti-Insulin | Dako | A0564 | For insulin immunofluorescence |
| Cy3 AffiniPure Donkey Anti-Rabbit | Jackson ImmunoResearch | 711-165-152 | For Ki67 immunofluorescence |
| Cy5 AffiniPure Donkey Anti-Guinea Pig | Jackson ImmunoResearch | 706-175-148 | For insulin immunofluorescence |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | ThermoFisher Scientific | D1306 | For nuclei visualization in immunofluorescence |
| Aqua-Mount | Lerner Laboratories | 13800 | Fast drying mounting media |
| FreeZone -105°C 4.5 Liter Cascade Benchtop Freeze Dry System | Labconco | 7382020 | For lyophilization of microspheres |
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