Novel techniques for assessing beta cell turnover

评估 β 细胞更新的新技术

• 批准号: 7967592
• 负责人: David Harlan
• 金额: $ 22.06万
• 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/7967592
• 关键词: Adult; Age; Animals; Annual Reports; Autopsy; Beta Cell; Biopsy Specimen; Bromodeoxyuridine; C-Peptide; Carbon; Case Series; Cell Count; Cell Separation; Cells; Clinical Data; Clinical Protocols; Clinical Trials; Collaborations; DNA; DNA biosynthesis; Data; Diabetes Mellitus; Half-Life; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Islets of Langerhans; Journals; Label; Laboratories; Life; Manuscripts; Measures; Medicine; Natural regeneration; Neurons; Nuclear; Pancreas; Patients; Process; Proinsulin; Proteins; Publishing; Reagent; Recovery; Recovery of Function; Reporting; Research Personnel; Rodent; Rodent Model; Science; Scientist; Source; Specimen; Staining method; Stains; Stem cells; Structure of beta Cell of islet; T-Lymphocyte; Techniques; Testing; Therapeutic; Thymidine; Time; Transgenic Mice; Trees; USSR; Work; analog; base; cell injury; interest; islet; killings; man; non-diabetic; novel; novel strategies; regenerative; research study

Background: Considerable data from rodent models has shown that, given time, pancreatic beta cell numbers can return to near normal following an insult that has destroyed most of animals insulin-producing beta cells. Even so, considerable controversy exists as to whether that beta cell recovery results exclusively from the proliferation of surviving beta cells, or whether other pancreatic stem cells can also differentiate into mature insulin producing islet beta cells. The question is even murkier in man since we cannot employ techniques used to study the question in rodents (e.g. transgenic mice with labeled beta cells, bromodeoxyuridine incorporation into DNA, etc). Even so, evidence strongly suggests that pancreatic beta cells are being replenished throughout life. For instance, despite the fact that individuals with even long standing type 1 diabetes mellitus (T1DM) appear to harbor anti-beta cell specific T cells capable of aggressively killing beta cell targets; autopsy based studies find pancreatic beta cells in most subjects dying with T1DM. Further, large case series that have evaluated patients with long-standing T1DM find that many if not most continue to make small amounts of C-peptide. For instance, as cited in our annual report for work unit number DK055111-01, we can demonstrate that C-peptide is produced by the pancreas in subjects with long-standing T1DM. These observations, and others, have led several investigators to ask whether pancreatic beta cell regeneration might prove a useful therapeutic approach to treat diabetes. As critical scientists, however, we must consider other data suggesting that beta cell mass does not significantly change in adult life. For instance, classic studies by Ogilvie from 1937 (Quarterly Journal of Medicine. 23: p287-300) suggest that beta cell mass is stable beyond age 25, though it is possible that observation is explained by beta cell loss rates being exactly equaled by regeneration rates in adults. As a consequence of this ongoing discussion, much interest has been focused on measuring a pancreatic beta cells regenerative capacity. Some scientists have approached the question by analyzing pancreatic biopsy specimens using immunohistochemical techniques, while we (and others) have attempted to promote beta cell functional recovery in clinical trials (see annual reports for work units numbered DK055104-03 and DK055111-01). A manuscript published by Spalding et al in 2005 (Cell. 122: p133-143) suggested to one of us (SP) a novel approach to assess pancreatic beta cell turnover rates by measuring the cells DNA 14C content. The principle underlying these studies is that earth's atmospheric 14C content has been tracked and is precisely known since the early 1900s. Further, we know that atmospheric 14C content sharply peaked in the early 1960s due to atmospheric nuclear tests then being performed. Since 14C decays with a half-life of nearly 6000 years, by measuring a cell's DNA 14C content, one can assess when that cells DNA was synthesized. Spalding et al validated the technique using DNA isolated from tree rings, and further used the technique to measure neuronal cell turnover rates in man. During the past 2 years, we have attempted to adapt the 14C technique to study pancreatic beta cell turnover rates. Such studies are difficult however, and for several reasons. One, the technique requires quite pure beta cells and large numbers of them; this can be difficult since the cells must be derived from a single donor. Two, one must scrupulously avoid contamination with carbon from any source (proteins, reagents used in the beta cell isolation process, etc) since that carbons 14C content could confound the analysis. Many techniques for isolating pure beta cells were attempted before we settled on immunostaining the cells for proinsulin and gaiting on the positive cells using flourscence activated cell sorting. With that technique, weve prepared pure beta cell DNA from two donors (born in 1926 and 1958), and in collaboration with Dr. Bruce Buchholz at the Lawrence Livermore Laboratories, have estimated that their beta cell DNA dated approximately 25 years younger than each donor. Our preliminary data suggests that the pancreatic islet beta cells from non-diabetic individuals do not turnover at a very rapid rate once individuals reach adulthood, and the work was recently cited in Science (Sept 12, 2008, page 1437). We have also obtained data employing pancreatic sections from subject autopsied days to months after receiving thymidine analogs for other clinical trials. Those sections were stained to look for insulin positive cells that had incorporated the analog, suggesting new DNA synthesis. These studies too suggest very limited beta cell turnover occurs during adult life. A manuscript reporting these results has been submitted but remains in the review process

背景资料：来自啮齿动物模型的大量数据表明，在一定时间内，胰腺β细胞数量可以在破坏大多数动物产生胰岛素的β细胞的损伤后恢复到接近正常。 即便如此，关于β细胞的恢复是否完全来自存活β细胞的增殖，或者其他胰腺干细胞是否也可以分化成成熟的胰岛素产生胰岛β细胞，仍然存在相当大的争议。 这个问题在人类身上更加模糊，因为我们不能使用在啮齿动物身上研究这个问题的技术（例如，带有标记β细胞的转基因小鼠，溴脱氧尿苷掺入DNA等）。 即便如此，有证据强烈表明，胰腺β细胞在整个生命过程中都在不断补充。 例如，尽管患有甚至长期存在的1型糖尿病（T1 DM）的个体似乎具有能够积极地杀死β细胞靶标的抗β细胞特异性T细胞的事实;基于尸检的研究在大多数死于T1 DM的受试者中发现胰腺β细胞。 此外，评估长期T1 DM患者的大型病例系列发现，即使不是大多数，也有许多患者继续产生少量C肽。 例如，如我们工作单元编号DK 055111 -01的年度报告中所述，我们可以证明C肽是由长期T1 DM受试者的胰腺产生的。 这些观察结果以及其他一些观察结果导致一些研究人员询问胰腺β细胞再生是否可能被证明是治疗糖尿病的有用治疗方法。 然而，作为关键的科学家，我们必须考虑其他数据，这些数据表明β细胞的数量在成年后不会发生显着变化。 例如，Ogilvie从1937年开始的经典研究（Quarterly Journal of Medicine）。二十三日：p287-300）表明，β细胞群在25岁以后是稳定的，尽管观察结果可能是由β细胞损失率与成人再生率完全相等来解释的。 由于这种持续的讨论，许多兴趣集中在测量胰腺β细胞再生能力上。 一些科学家通过使用免疫组织化学技术分析胰腺活检标本来解决这个问题，而我们（和其他人）则试图在临床试验中促进β细胞功能恢复（参见编号为DK 055104 -03和DK 055111 -01的工作单元的年度报告）。 Spalding等人在2005年发表的手稿（Cell. 122：p133-143）向我们之一（SP）建议了一种通过测量细胞DNA 14 C含量来评估胰腺β细胞更新率的新方法。 这些研究的基本原理是，自20世纪初以来，地球大气中的14 C含量一直被跟踪并精确地知道。 此外，我们知道，由于当时进行的大气核试验，大气中的14 C含量在20世纪60年代初急剧达到峰值。 由于14 C衰变的半衰期接近6000年，通过测量细胞的DNA 14 C含量，可以评估细胞DNA合成的时间。 Spalding等人使用从树木年轮中分离的DNA验证了该技术，并进一步使用该技术测量人类神经细胞的更新率。 在过去的2年中，我们尝试采用14 C技术来研究胰腺β细胞更新率。 然而，这种研究是困难的，原因有几个。 首先，这项技术需要相当纯的β细胞和大量的β细胞;这可能很困难，因为细胞必须来自单一供体。 第二，必须严格避免任何来源的碳污染（蛋白质，β细胞分离过程中使用的试剂等），因为碳14 C含量可能会混淆分析。 在我们决定对细胞进行胰岛素原免疫染色并使用荧光激活细胞分选法对阳性细胞进行标记之前，尝试了许多分离纯β细胞的技术。 利用这种技术，我们从两个捐赠者（出生于1926年和1958年）身上制备了纯β细胞DNA，并与劳伦斯利弗莫尔实验室的布鲁斯布赫霍尔茨博士合作，估计他们的β细胞DNA比每个捐赠者年轻大约25岁。 我们的初步数据表明，非糖尿病个体的胰岛β细胞在成年后不会以非常快的速度周转，这项工作最近被《科学》杂志引用（2008年9月12日，第1437页）。 我们还获得了其他临床试验中使用接受胸苷类似物后几天至几个月尸检的胰腺切片的数据。 对这些切片进行染色，以寻找掺入类似物的胰岛素阳性细胞，这表明新的DNA合成。 这些研究也表明，在成年期，β细胞的更新非常有限。 报告这些结果的手稿已经提交，但仍在审查过程中