A novel model for studying fetal skeletal muscle development

描述(由申请人提供)：超重和肥胖育龄妇女的日益普遍是一个日益严重的公共卫生问题，这可能会对后代造成有害的、持久的影响，包括肥胖和糖尿病的易感性。母体肥胖和营养过剩与子代肥胖和糖尿病之间的联系机制仍不明确。骨骼肌是负责葡萄糖和脂肪酸利用的主要外周组织。由于出生后肌肉纤维数量不会增加，因此胎儿期对骨骼肌的发育至关重要。直到妊娠晚期的胎儿肌肉中含有大量的间充质干细胞(多能祖细胞)。多能细胞的增殖和谱系承诺直接影响肌肉纤维的数量和大小。多能细胞致力于脂肪生成而不是肌肉生成将增加肌肉内脂肪细胞的数量，这一事件与骨骼肌中的胰岛素抵抗有关。在我们的初步研究中，我们观察到母亲肥胖和过度营养下调了AMP激活的蛋白激酶(AMPK)活性和胰岛素信号转导，并促进了脂肪生成，这可能与多能祖细胞的增殖和分化改变有关。AMPK可能在体内调控胎肌多能细胞的增殖和分化中发挥重要作用。为了证实这一点，需要一个良好的体内实验模型。建立一种可在体内追踪胎儿肌肉多能细胞增殖和分化的实验模型，并利用该模型检测AMPK在胎儿肌肉发育中的作用。骨髓间充质干细胞注射入胎儿肌肉后，可在胎儿肌肉中增殖分化为肌源性、成脂性等细胞。将携带AMPK持续活性结构的pDsRed载体和携带显性负AMPK结构的pAcGFP载体分别导入骨髓间充质干细胞C3H10T1/2。PDsRed和pAcGFP载体是哺乳动物表达载体，分别表达红色和绿色荧光蛋白。稳定表达红色或绿色荧光蛋白的细胞将被克隆，然后注射到胎儿的骨骼肌中。怀有这些胎儿的怀孕绵羊将被允许产下羔羊，然后对羔羊进行安乐死以采集肌肉。将对肌肉进行切片，并测量肌肉纤维、肌肉内脂肪细胞和骨骼肌结缔组织中是否存在荧光细胞。使用携带两种不同荧光蛋白的细胞将允许直接比较具有不同AMPK活性的细胞的目的地。这一模型对于理解AMPK在体内多能细胞增殖和分化中的作用至关重要。该模型也将有助于研究影响胎儿肌肉多能细胞分化的细胞内信号通路。这样的研究将具有巨大的生物医学意义，并将极大地促进关于胎儿发育规划的研究。

DESCRIPTION (provided by applicant): The increasing prevalence of overweight and obese women of childbearing age is a growing public health concern, which can result in harmful, persistent effects in offspring, including pre-disposition to obesity and diabetes. Mechanisms linking maternal obesity and over nutrition to obesity and diabetes of offspring remain poorly defined. Skeletal muscle is the main periphery tissue responsible for glucose and fatty acid utilization. Since there is no increase in muscle fiber number after birth, fetal stage is crucial for skeletal muscle development. Fetal muscle up to late gestation contains a large number of mesenchymal stem cells (pluripotent progenitor cells). The proliferation and lineage commitment of pluripotent cells directly affect the number and size of muscle fibers developed. The commitment of pluripotent cells to adipogenesis instead of myogenesis will increase the number of intramuscular adipocytes, an event associated with insulin resistance in skeletal muscle. In our preliminary studies, we observed that maternal obesity and over-nutrition down-regulated AMP-activated protein kinase (AMPK) activity and insulin signaling, and promoted adipogenesis which should be associated with altered proliferation and differentiation of pluripotent progenitor cells. AMPK may play an important role in the control of proliferation and differentiation of pluripotent cells in fetal muscle in vivo. To confirm it, a good in vivo experimental model is needed. To establish an experimental model which can track the proliferation and differentiation of pluripotent cells in fetal muscle in vivo and to use this model to test the role of AMPK in fetal muscle development. Mesenchymal stem cells injected into fetal muscle will proliferate and differentiate into myogenic, adipogenic and other cells in fetal muscle. Mesenchymal stem cell C3H10T1/2 will be transfected with a pDsRed vector carrying AMPK constant active construct and a pAcGFP vector carrying dominant negative AMPK constructs respectively. pDsRed and pAcGFP vectors are mammalian expression vectors and express red and green fluorescent proteins respectively. Cells stably expressing red or green fluorescent proteins will be cloned and then injected into the skeletal muscle of fetuses. Pregnant sheep carrying these fetuses will be allowed to lamb which will then be euthanized for muscle collection. Muscle will be sectioned and the existence of fluorescent cells in muscle fibers, intramuscular adipocytes and connective tissues in skeletal muscle will be measured. The use of cells carrying two different fluorescent proteins will allow a direct comparison for the destination of cells with different AMPK activities. This model will be crucial for understanding the role of AMPK in the proliferation and differentiation of pluripotent cells in vivo. This model will also be useful for studying intracellular signaling pathways which affect differentiation of pluripotent cells in fetal muscle. Such studies will have enormous biomedical implications and will greatly facilitate studies regarding fetal developmental programming.