A novel model for studying fetal skeletal muscle development

描述（由申请方提供）：育龄期女性超重和肥胖的患病率日益增加，这是一个日益严重的公共卫生问题，可能对后代造成有害的持续影响，包括肥胖和糖尿病的易感性。母亲肥胖和营养过剩与后代肥胖和糖尿病之间的联系机制仍不清楚。骨骼肌是负责葡萄糖和脂肪酸利用的主要外周组织。由于出生后肌纤维数量没有增加，胎儿阶段对骨骼肌发育至关重要。胎儿肌肉直到妊娠晚期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定型直接影响发育的肌纤维的数量和大小。多能细胞致力于脂肪生成而不是肌生成将增加肌内脂肪细胞的数量，这是与骨骼肌中的胰岛素抵抗相关的事件。在我们的初步研究中，我们观察到母亲肥胖和营养过剩下调AMP激活蛋白激酶（AMPK）活性和胰岛素信号，并促进脂肪生成，这应该与多能祖细胞增殖和分化的改变有关。AMPK可能在体内调控胎儿肌肉多能细胞的增殖和分化中起重要作用。为了证实这一点，需要一个良好的体内实验模型。目的建立一种能在体内跟踪胎儿肌肉多能细胞增殖分化的实验模型，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到胎儿肌肉中，在胎儿肌肉中增殖并分化为成肌细胞、成脂细胞和其他细胞。将分别用携带AMPK恒定活性构建体的pDsRed载体和携带显性阴性AMPK构建体的pAcGFP载体转染间充质干细胞C3 H10 T1/2。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。将允许携带这些胎儿的妊娠绵羊产羔，然后将其安乐死以采集肌肉。将对肌肉进行切片，并测量骨骼肌中肌纤维、肌内脂肪细胞和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。该模型对于理解AMPK在体内多能细胞增殖和分化中的作用至关重要。此模型也将是有用的研究细胞内信号通路，影响胎儿肌肉中的多能细胞的分化。这些研究将具有巨大的生物医学意义，并将大大促进有关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.