复制和β细胞功能评估Adenovirally转分离的啮齿动物胰岛
Summary
该协议允许一个确定调节功能的β细胞质量的因素，找到治疗糖尿病的潜在的治疗靶点。该协议包括一个精简的方法来评估胰岛细胞复制和β细胞功能，在离体大鼠胰岛细胞与腺病毒基因表达操纵。
Abstract
主要是葡萄糖稳态控制的内分泌激素的胰岛素和胰高血糖素，胰岛β和α细胞分泌，分别。功能的β细胞质量是决定解剖β细胞质量以及β细胞的能力，以应对营养负荷。一个丧失功能的β细胞质量是中心1-3糖尿病的两种主要形式。而下降功能的β细胞自身免疫性1型糖尿病发作的质量结果，在2型糖尿病，这一递减的发展都无法适当分泌胰岛素的β细胞和β细胞的破坏从干部机制。因此，努力恢复功能的β细胞质量更好的治疗和糖尿病的潜在治疗至关重要。
正在努力找出刺激的复制和增强β细胞的功能，可以利用的分子途径。理想的情况下，治疗的目标，同时提高β细胞的生长和功能。也许更重要的虽然是一种策略，刺激β细胞的生长，以确定是否损害的β细胞功能（如某些癌基因），反之亦然成本。
由系统抑制或过度在离体大鼠胰岛靶基因的表达，可以增加功能的β细胞质量4-6找出潜在的治疗靶点。腺病毒载体可有效地过度或击倒蛋白在离体大鼠胰岛4,7-15就业。在这里，我们提出了一种方法来操纵利用腺病毒转导的基因表达和评估胰岛复制，并在离体大鼠胰岛β细胞功能（ 图1）。这种方法已经被以前使用，以确定新的目标，调节β细胞复制或功能5,6,8,9,16,17。
Protocol
1。腺病毒介导和培养大鼠胰岛准备加入所需数量的2毫升（载8毫米葡萄糖，10％胎牛血清，50个单位/ ml青霉素，50μg/ mL链霉素的RPMI 1640培养媒体）媒体6以及非组织文化的涂层板井。例如，一个典型的实验可能需要三口井 – 一个无病毒控制，病毒控制（例如，表达GFP的腺病毒），实验组。 暖板放置到组织文化的孵化器，为至少30分钟至37°C。 紧随6以及非组织文化的涂层板个别?…
Discussion
建立通路，可调制刺激的复制和增强β细胞的功能有关糖尿病的两个主要形式。因为功能的β细胞质量是依赖胰岛素分泌细胞的存在和功能上，评估这些因素，同时有它的优势。这个协议描述为确定一种蛋白质的过度表达或抑制是否会导致功能的β细胞质量的变化， 在体外培养 ，然后可以测试体内疗效的精简协议。
这个协议的一个限制是，胰岛是一种微型器…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作是格兰特DK078732支持由美国国立卫生研究院（的PTF）。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| RPMI 1640 media | Gibco | 11879 | |
| Penicillin/streptomycin | Gibco | 15140 | |
| 6-well plate | BD-Falcon | 35-1146 | Non-TC treated |
| [methyl-3H]-thymidine | Perkin Elmer | NET027Z001MC | 1 mCi/ml |
| Micro-centrifuge tubes | Denville | C2170 | 1.7 ml |
| NaCl | Sigma | 59888 | |
| KCl | Acros | 42409 | |
| KH2PO4 | Acros | 20592 | |
| MgSO4 | Acros | 41348 | |
| CaCl2 | Acros | 34961 | |
| HEPES | Sigma | H0887 | 1 M solution |
| 35% BSA | Sigma | A7979 | |
| NaHCO3 | Acros | 42427 | |
| d-glucose | Sigma | G8769 | |
| TCA | Fisher Scientific | SA9410-1 | 10% w/v |
| NaOH | Acros | 12426 | |
| Scintillation counting tube | Sarstedt | 58.536 | 7 ml, PP |
| Scintillation counting tube cap | Sarstedt | 65.816 | |
| Econo-Safe counting cocktail | RPI | 111175 | |
| Insulin RIA | Siemens | TKIN2 | |
| BCA Assay Kit | Thermo Scientific | 23250 | |
| Equipment | |||
| Centrifuge | Eppendorf | 5415R | |
| Scintillation counting tube rack | Sarstedt | 93.1431.001 | |
| Liquid scintillation counter | Perkin Elmer | Tri-Carb 2910TR |
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Tags
ReplicationBeta Cell FunctionAdenovirally-transduced Isolated Rodent IsletsGlucose HomeostasisInsulinGlucagonPancreatic Beta CellsAlpha CellsFunctional Beta Cell MassNutrient LoadDiabetesAutoimmune AttackType 1 DiabetesType 2 DiabetesInsulin SecretionMolecular PathwaysTherapeutic TargetsBeta Cell GrowthImpairing Beta Cell FunctionOncogenesTarget Genes
Citar este artigo
Fueger, P. T., Hernandez, A. M., Chen, Y., Colvin, E. S. Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets. J. Vis. Exp. (64), e4080, doi:10.3791/4080 (2012).