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

在正常的人类怀孕期间，母亲对胰岛素的需求突然增加了一倍。为了满足这一需求，她的身体产生了额外的胰岛素分泌细胞--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？了解人体产生新的β细胞本身就是一个重要的生物学问题。这种理解对于糖尿病的治疗也很重要，因为对疾病生理学的深入理解建立在对正常生理学的理解之上。不能在人类受试者身上做实验。我们使用的是老鼠，幸运的是，它们对怀孕的生理反应与人类相似。例如，在怀孕期间，小鼠体内的β细胞质量增加了一倍以上，人类也是如此。这种显著的增加只发生在小鼠的几天内，这对我们的研究是方便的。这个项目的问题可以重塑：在怀孕期间，哪些类型的细胞会自我转化为β细胞？直到最近，关于这个问题的公认智慧是由Melton和Dor进行的一项奇妙的实验提供的。他们从转基因小鼠开始，也就是DNA以某种方式改变的小鼠。这种改变对小鼠的生理影响很小，直到小鼠被注射某种激素(他莫昔芬)，在这一点上，改变的DNA开始产生，但只在一种特定类型的细胞中--在Dor-Melton案例中，仅在β细胞中--染色后在显微镜下可见的某种蛋白质(HPAP)。该细胞据说是“被HPAP标记的”。这项实验之所以如此丰富，是因为当HPAP标记的细胞分裂(复制)时，分裂产生的细胞也被标记。标记的细胞必须具有标记的祖先，而未标记的细胞必须具有未标记的祖先。这为跟踪细胞的祖先提供了一种机制，无论细胞是否更改为不同类型的细胞，该机制都有效。Melton和Dor证明，在自然界中不存在的情况下(手术切除部分胰腺)，新的β细胞仅来自现有的β细胞，通过通常的细胞分裂过程。我们小组最近重复了Melton-Dor实验，使用正常妊娠来产生大量但正常的额外胰岛素需求。在这些条件下，同样的标记技术导致了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贝塔细胞确实来自哪里，尽管我们可以确定一些来自现有贝塔细胞的分裂。我们现在的目标是精确地确定怀孕期间新的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。在我们的项目中发现的祖先细胞和祖先细胞产生的β细胞，可能会在体外大量培养。将道路交通事故受害者的β细胞移植到糖尿病患者体内已经是1型和2型糖尿病的标准治疗方法。然而，这样的来源提供的β细胞只能满足需求的一小部分。华威大学的机器人控制的TIS显微镜是德国以外唯一的同类显微镜，它允许我们从不同时间点不同蛋白质的表达中推断细胞在成为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到40种蛋白质，而不是传统显微镜可以检测到的2到3种蛋白质。TIS的这种强大功能将使我们能够识别关于β细胞祖先身份的许多建议中的哪些是正确的。

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.