Preclinical studies for measuring beta cell mass in vivo

测量体内β细胞质量的临床前研究

• 批准号: 7734215
• 负责人: David Harlan
• 金额: $ 24.85万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734215
• 关键词: Adrenal Medulla; Adult; Animal Model; Animals; Antibodies; Attention; Autoimmune Process; Basal Ganglia; Beta Cell; Binding; Cell Proliferation; Cells; Clinical; Count; Data; Data Reporting; Diabetes Mellitus; Disease; Endocrine; Enrollment; Eye; Flow Cytometry; Functional disorder; Glucose; Goals; Hormones; Human; Image; Individual; Injection of therapeutic agent; Insulin; Insulin-Dependent Diabetes Mellitus; Label; Ligands; Lung; Magnetic Resonance Imaging; Manuscripts; Measures; Mediating; Modeling; Mus; Names; Numbers; Pancreas; Patient currently pregnant; Positron-Emission Tomography; Pregnancy; Primates; Process; Protocols documentation; Publications; Radioactive; Radioactivity; Radiolabeled; Rate; Rattus; Reporting; Research Personnel; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Risk; Rodent; Role playing therapy; Secondary to; Signal Transduction; Staining method; Stains; Structure of beta Cell of islet; T-Lymphocyte; Techniques; Testing; Thymidine; Tissue-Specific Gene Expression; Tissues; Universities; Week; Work; analog; brain tissue; cell type; design; dihydrotetrabenazine; enantiomer; falls; improved; in vivo; islet; mouse model; nonhuman primate; preclinical study; radiotracer; receptor; response; species difference; vesicular monoamine transporter 2

Much of the work described in this report emanated from observations made by Dr. Paul Harris at Columbia University who, using differential gene expression studies, noted that beta cells (to a much greater degree than other pancreatic cells) express the vesicular monoamine transporter-2 (VMAT-2). Dr. Harris and colleagues went on to perform studies using a VMAT-2 radiolabeled ligand named 11C-dihydrotetrabenazine (11C-DTBZ) as an imaging agent for positron-emission tomography (PET) studies in a rat model for T1DM with results suggesting that as beta cell mass fell during the autoimmune process, so did the 11C-DTBZ-PET pancreatic signal. Indeed, in that model the PET signal seemed able to predict diabetes onset. With an eye toward developing the technique for application in clinical interventional trials enrolling humans at risk for T1DM, we initiated several studies to assess 11C-DTBZ-PET using islets from human cadaveric donors and various mouse and non-human primate models at our disposal. Consistent with the techniques promise, we found that: 1. Human islets immunostained with anti-VMAT-2 and with anti-insulin revealed nearly coincident staining, while other islet endocrine cells did not stain for VMAT-2. 2. Mixing variable human islet numbers with 3H-DTBZ generated radioactive counts directly correlated with the islet number. 3. Cold DTBZ could complete away 3H-DTBZ bound to human islets suggesting binding to specific receptors. 4. Labeling human islets with 3H-DTBZ, then injecting the labeled islets intravenously into mice promptly euthanized after that injection, generated radioactivity counts in the animals lungs consistent with the islet number injected. 5. Non-human primates injected with 11C-DTBZ then studied using PET revealed bright signals that co-registered with magnetic-resonance imaging (MRI) of the animals pancreas. Unfortunately, with further testing we found: 1. In our mouse models, native pancreatic DTBZ-PET signals did not reliably correlate with predicted beta cell mass. 2. Immunostaining of both mouse and non-human primate pancreas with anti-VMAT2 and anti-insulin (or other endocrine hormone-specific antibodies) was not consistent with beta cell specific VMAT2 expression. To test whether the apparent absence of beta cell VMAT2 expression might be secondary to species differences (i.e. the staining antibody was raised against human VMAT2), we immunostained other primate tissue (brain basal ganglia and adrenal medulla) known to express VMAT2 and found clear expression in those positive control tissues, but absent staining in appropriate negative control tissues. 3. Individual cells from isolated non-human primate islets studied using both flow cytometry and reverse-transcriptase-polymerase chain reaction (RT-PCR) revealed no evidence for beta cell specific VMAT2 expression. 4. 11C-DTBZ exists as both a positive enantiomer, and a mirror image negative enantiomer. Only the positive 11C-DTBZ enantiomer binds to VMAT2. We performed PET scans using both forms in the same primate and found that both generated similar PET signals. Taken together (in conjunction with data reported in work unit number DK055110-01), we concluded that while it showed considerable promise, 11C-DTBZ PET pancreatic signals are not sufficiently specific for pancreatic beta cells to serve as a useful technique for measuring beta cell mass in vivo and we have turned our attention to other techniques. A manuscript describing these results has been prepared for publication. We have moved on to assess, using the non-human primate model, whether pregnancy promotes beta cell proliferation as is reported in rodents. Under an approved animal use protocol, we have administered thymidine analogs to a pregnant non-human primate, then examined the animal's pancreas to assess rates of beta cell turnover. Another such study is planned in the next few weeks. Preliminary data suggests that as opposed to responses observed in rodents, adult primate beta cells display little if any capacity for proliferation, even during pregancy.

本报告中描述的大部分工作源自哥伦比亚大学的Paul Harris博士的观察，他使用差异基因表达研究指出，β细胞（比其他胰腺细胞更大程度地）表达囊泡单胺转运蛋白-2（VMAT-2）。 Harris博士及其同事继续使用VMAT-2放射性标记的配体11 C-DTBZ作为T1 DM大鼠模型正电子发射断层扫描（PET）研究的成像剂进行研究，结果表明，随着β细胞质量在自身免疫过程中下降，11 C-DTBZ-PET胰腺信号也是如此。 事实上，在该模型中，PET信号似乎能够预测糖尿病发作。 着眼于开发技术应用于临床干预试验，招募有T1 DM风险的人，我们启动了几项研究，以评估11 C-DTBZ-PET使用胰岛从人类尸体供体和各种小鼠和非人灵长类动物模型在我们的处置。 与技术承诺一致，我们发现： 1.用抗VMAT-2和抗胰岛素免疫染色的人胰岛显示出几乎一致的染色，而其他胰岛内分泌细胞不对VMAT-2染色。 2.将不同的人胰岛数量与3 H-DTBZ混合产生的放射性计数与胰岛数量直接相关。 3.冷DTBZ可完全消除3 H-DTBZ与人胰岛的结合，提示与特异性受体结合。 4.用3 H-DTBZ标记人胰岛，然后将标记的胰岛静脉注射到注射后立即安乐死的小鼠中，在动物肺中产生的放射性计数与注射的胰岛数量一致。 5.非人类灵长类动物注射11 C-DTBZ，然后使用PET进行研究，显示出与动物胰腺的磁共振成像（MRI）共同记录的明亮信号。 不幸的是，通过进一步的测试，我们发现： 1.在我们的小鼠模型中，天然胰腺DTBZ-PET信号与预测的β细胞质量不可靠相关。 2.用抗VMAT 2和抗胰岛素（或其他内分泌细胞特异性抗体）对小鼠和非人灵长类动物胰腺的免疫染色与β细胞特异性VMAT 2表达不一致。 为了测试β细胞VMAT 2表达的明显缺乏是否可能继发于种属差异（即，针对人VMAT 2产生染色抗体），我们对已知表达VMAT 2的其他灵长类动物组织（脑基底神经节和肾上腺髓质）进行免疫染色，并发现在那些阳性对照组织中明确表达，但在适当的阴性对照组织中不存在染色。 3.使用流式细胞术和逆转录酶-聚合酶链反应（RT-PCR）研究的来自分离的非人灵长类动物胰岛的单个细胞未显示β细胞特异性VMAT 2表达的证据。 4. 11 C-DTBZ以正对映体和镜像负对映体存在。 仅阳性11 C-DTBZ对映体与VMAT 2结合。 我们在同一灵长类动物中使用这两种形式进行PET扫描，发现两者产生相似的PET信号。 综上所述（结合工作单元编号DK 055110 -01中报告的数据），我们得出结论，尽管11 C-DTBZ PET胰腺信号显示出相当大的前景，但其对胰腺β细胞的特异性不足以作为体内测量β细胞质量的有用技术，我们已将注意力转向其他技术。 描述这些结果的手稿已经准备好出版。 我们已经开始使用非人类灵长类动物模型来评估怀孕是否会像啮齿类动物中报道的那样促进β细胞增殖。 根据批准的动物使用方案，我们已将胸苷类似物给予怀孕的非人灵长类动物，然后检查动物的胰腺以评估β细胞更新率。 另一项此类研究计划在未来几周内进行。 初步数据表明，与啮齿动物中观察到的反应相反，成年灵长类动物的β细胞即使在怀孕期间也几乎没有增殖能力。