Origin of new beta cells during pregnancy using PCLT and TIS microscopy

在人类正常怀孕期间，母亲对胰岛素的需求突然增加了一倍。为了满足这种需求，她的身体产生了额外的胰岛素生产细胞——这些细胞被称为β细胞。这个项目的问题是：在正常妊娠中，额外的细胞来自哪里？了解人体产生新β细胞的过程本身就是一个重要的生物学问题。这种认识对糖尿病的治疗也很重要，因为对病变生理学的深刻理解是建立在对正常生理学的理解之上的。实验不能在人体上进行。我们使用了老鼠，幸运的是，老鼠对怀孕的生理反应与人类相似。例如，在怀孕期间，老鼠体内的β细胞数量增加了一倍以上，人类也是如此。这种显著的增长只发生在老鼠身上几天，这对我们的研究很方便。这个项目的问题可以重新定义：在怀孕期间，哪些类型的细胞将自己转化为β细胞？直到最近，关于这个问题的公认的智慧是由梅尔顿和多尔进行的一个精彩的实验提供的。他们从转基因老鼠开始，也就是DNA以某种方式被改变的老鼠。这种改变对小鼠的生理几乎没有影响，直到给小鼠注射某种激素（他莫西芬），此时改变的DNA开始产生，但只在一种特定类型的细胞中——在多尔-梅尔顿的情况下，只在β细胞中——在显微镜下染色后可以看到某种蛋白质（HPAP）。细胞被称为“HPAP标记”。这个实验之所以能提供如此丰富的信息，是因为当一个hpap标记的细胞分裂（复制）时，分裂产生的细胞也会被标记。标记的细胞一定有标记的祖先，而未标记的细胞一定有未标记的祖先。这提供了一种追踪细胞祖先的机制，无论细胞是否转变为不同类型的细胞，这种机制都有效。Melton和Dor表明，在自然界中不会发生的情况下（手术切除部分胰腺），新的β细胞只能通过通常的细胞分裂过程从现有的β细胞中产生。我们的研究小组最近重复了梅尔顿-多尔实验，用正常妊娠来制造大量但正常的额外胰岛素需求。在这些条件下，同样的标记技术会得出一个非常不同的结论——大约一半的新β细胞不是通过复制现有的β细胞产生的，而是来自其他地方。我们的实验并不是为了显示新的β细胞从何而来，尽管我们可以确定一些来自现有β细胞的分裂。我们现在的目标是精确地确定怀孕期间新β细胞的各种类型的祖先。这将是了解糖尿病中胰岛素缺乏的重要一步，并可能导致有效的治疗。在体外大量培养我们项目中发现的祖细胞和祖细胞产生的β细胞可能成为可能。将从道路交通事故受害者身上采集的糖尿病β细胞移植到患者体内，已经成为1型和2型糖尿病的标准治疗方法。然而，来自这种来源的β细胞只能满足需求的一小部分。华威大学（University of Warwick）的机器人控制TIS显微镜是德国以外唯一的同类显微镜，它使我们能够从不同时间点不同蛋白质的表达推断出细胞在变成β细胞时是如何变化的。TIS在同一组织切片的同一位置检测40种蛋白质，而不是通过传统显微镜检测到的2或3种蛋白质。TIS的强大功能将使我们能够确定关于β细胞祖先身份的许多建议中哪一个是正确的。

During normal human pregnancy, the mother's need for insulin is suddenly doubled. To fulfil this need, her body creates additional insulin-producing cells--these are called beta cells. This project's question is: Where do the additional cells come from in normal pregnancy? Understanding the body's production of new beta cell is an important biological problem in its own right. This understanding is also important for the treatment of diabetes, because a deep understanding of diseased physiology rests on understanding normal physiology. Experiments cannot be done on human subjects. We use mice, whose physiological response to pregnancy is fortunately similar to that of humans. For example, during pregnancy, the mass of beta cells more than doubles in mice, and the same is true for humans. This remarkable increase takes place over only a few days in a mouse, which is convenient for our study. This project's question can be recast: During pregnancy, which types of cells transform themselves into beta cells? Until recently, the accepted wisdom on this question was provided by a wonderful experiment carried out by Melton and Dor. They start with transgenic mice, that is, mice whose DNA has been altered in a certain way. This alteration has little effect on the physiology of the mouse until the mouse is injected with a certain hormone (tamoxifen), at which point the altered DNA starts to produce, but only in one particular type of cell--in the Dor-Melton case, only in beta cells--a certain protein (HPAP) visible under the microscope after staining. The cell is said to be "labelled by HPAP". What makes the experiment so informative is that, when an HPAP-labelled cell divides (replicates), then the cells resulting from division are also labelled. A labelled cell must have had labelled ancestors, and an unlabelled cell must have had unlabelled ancestors. This gives a mechanism for tracking the ancestry of a cell, which works whether or not the cell changes to a cell of different type. Melton and Dor showed that, under circumstances that do not occur in nature (surgical removal of part of the pancreas), new beta cells come only from existing beta cells, via the usual process of cell division. Our group recently repeated the Melton-Dor experiment using normal pregnancy to create a large but normal demand for additional insulin. Under these conditions, the same labelling technique leads to a very different conclusion--about half of the new beta cells do NOT arise through replication of existing beta cells, but come from somewhere else. Our experiment was not designed to show where the new beta cells did come from, even though we could be sure that some came from division of existing beta cells. Our objective now is to determine with precision the various types of ancestors of new beta cells in pregnancy. This would be a major step towards understanding insulin-deficiency in diabetes, and may lead to effective treatment. It may become possible to cultivate outside the body, in large numbers, ancestor cells discovered during our project and beta cells that the ancestor cells generate. It is already standard treatment in both type 1 and type 2 diabetes to transplant into the body of a diabetic beta cells that are harvested from road traffic accident victims. However, a supply of beta cells from such a source can supply only a tiny fraction of the need. The University of Warwick's robotically controlled TIS microscope, the only one of its kind outside Germany, allows us to deduce, from the expression of different proteins at different time points, how a cell changes while becoming a beta cell. TIS detects 40 proteins at the same place in the same tissue section, instead of the 2 or 3 detectable through conventional microscopy. This great power of TIS will enable us to identify which of many suggestions are correct as to the identity of ancestors of beta cells.