Characterisation of skeletal development and the use of anabolic agents in murine models of Duchenne muscular dystrophy

杜氏肌营养不良症（DMD）是一种严重且最终致命的疾病。每4000名男性中就有1人患此病，而且无法治愈。类固醇是唯一可以帮助减缓肌肉无力的治疗方法，但它们有许多副作用，包括生长衰竭和骨质疏松症。具体原因尚不清楚，但即使是没有接受类固醇治疗的男孩也往往身材矮小，骨骼脆弱。我们不知道骨骼和生长问题是否仅仅是由肌肉无力引起的（我们的骨骼需要肌肉活动“负荷”才能加强和有效地生长），或者是否有其他主要问题。这可能是因为DMD是一种慢性炎症过程，它破坏了生长激素(GH)/胰岛素样生长因子（IGF-1）途径。生长激素是由脑下垂体分泌的。它可以直接作用于骨骼，但主要是通过IGF-1间接作用。总之，GH/IGF-1通路在儿童时期正常骨生长和骨量的调节中起着至关重要的作用，GH或IGF-1缺乏的儿童身材矮小，患有骨质疏松症。目前还没有有效的治疗方法来帮助DMD患者的骨骼生长。我们将探讨生长激素和IGF-1在DMD小鼠模型骨和生长中的作用。DMD常用的小鼠模型是x连锁肌营养不良（mdx）小鼠，但它有明显的局限性，因为疾病过程不像Duchenne患者那样严重，mdx小鼠发生肌肉再生。很少有在mdx小鼠中有效的药物被证明对患者有用。因此，我们将使用一种新的小鼠模型，Cmah-/-mdx小鼠。这只小鼠是从mdx小鼠培育出来的，但也有一个额外的基因缺失，使它更“人性化”，因此我们希望它的行为方式与杜氏症患者更相似。我将第一次观察Cmah-/-mdx的骨骼和生长情况，并将其与mdx和健康的“野生型”小鼠进行比较。然后我会给老鼠注射类固醇以确定对它们生长和骨骼的影响。我还将观察Cmah-/mdx小鼠是否对GH和IGF-1有反应，无论是单独的还是联合的。为了做到这一点，我将使用一系列技术，包括微型CT和标记骨骼和生长板来监测生长速度。我还将从这些小鼠模型的胚胎中培养跖骨（脚），看看它们是否也表现出生长迟缓和骨骼脆弱。如果他们这样做，这将向我们表明除了类固醇和肌肉无力引起的问题外，还有一个与骨骼有关的主要问题。这项工作非常重要，因为生长激素和IGF-1都很容易获得，并且已经在儿童中使用。如果在小鼠模型中发现它们单独或共同作用有助于骨骼发育和促进生长，那么这项工作就可以作为DMD患者的临床试验进行。进一步描述新的Cmah-/-mdx小鼠模型也很重要，可以用于其他DMD实验。我们收集的数据也可能有助于设计新的治疗方法，专门针对导致骨骼健康状况不佳和DMD生长的问题的机制。

Duchenne muscular dystrophy (DMD) is a severe and ultimately fatal disease. It affects up to 1 in 4000 males and there is no cure. Steroids are the only treatment that can help slow down muscle weakness but they have many side effects including growth failure and osteoporosis. It is unclear exactly why, but even boys who are not treated with steroids tend to be short and have weak bones. We do not know whether the bone and growth problems are just caused by the muscle weakness (our bones need to be 'loaded' by muscle activity in order to strengthen and grow efficiently), or whether there is another primary problem. It may be that because DMD is a chronic inflammatory process, this disrupts the growth hormone (GH)/insulin-like growth factor (IGF-1) pathway. Growth hormone is produced by the anterior pituitary gland. It can act directly on the skeleton, but mainly acts indirectly, through IGF-1. Together the GH/IGF-1 pathway plays a crucial role in regulating normal bone growth and bone mass during childhood, and children with either GH or IGF-1 deficiency are short and have osteoporosis. There are no treatments available to help bone growth in DMD. We will investigate the role of GH and IGF-1 in bone and growth of mice models of DMD. The mouse model commonly used in DMD is the X-linked Muscular Dystrophy (mdx) mouse, but it has significant limitations because the disease process is not as severe and muscle regeneration occurs in the mdx mice, unlike in patients with Duchenne. Very few medications that are effective in the mdx mouse have shown to be useful in patients. Therefore we will use a new mouse model, the Cmah-/-mdx mouse. This mouse is bred from the mdx mouse, but also has an additional gene deletion to make it more 'human' and therefore we hope will behave in more similar way to patients with Duchenne. I will look, for the first time, at bone and growth in the Cmah-/-mdx and compare it to the mdx and a healthy 'wildtype' mouse. I will then give the mice steroids to determine the effects on their growth and skeleton. I will also see if the Cmah-/mdx mouse responds to GH and IGF-1, both alone and in combination. To do this I will use a range of techniques, including micro CT and labeling of the bones and growth plate to monitor the rate of growth. I will also culture metatarsals (feet) bones from the embryos of these mice models to see if they also display reduced growth and bone fragility. If they do this will show us that there is a primary problem relating to the skeleton in addition to the problems caused by steroids and muscle weakness. This work is very important because both GH and IGF-1 are readily available and are already used in children. If they are found to help bone development and encourage growth, either alone or acting together, in mice models, then this work could be carried out as a clinical trial in patients with DMD. It is also important to further describe the new Cmah-/-mdx mouse model, so that it can be used in other DMD experiments. The data that we collect may also help in the design of new therapies that are directed specifically at the mechanism of the problem that leads to poor bone health and growth in DMD.