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The de novo formation of intramuscular fat tissue by the insertion of myogenic and adipogenic cell lineage determination genes into satellite cell genome

通过将生肌细胞和脂肪细胞谱系决定基因插入卫星细胞基因组来从头形成肌内脂肪组织

基本信息

批准号：
13660291
负责人：
MURAYAMA Kimiaki
金额：
$ 2.24万
依托单位：
Meiji University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660291/
关键词：
satellite cells cell differentiation PPARγ myogenic cell adipose cell adult stem cell ウシ多能性骨格筋衛星細胞 CEB Pα

项目摘要

As the first stage of this investigation, the procedure for isolating, purifying, and culturing skeletal muscle satellite cells (satellite cells) from Japanese Black cattle and Holstein cattle was established. In this procedure, satellite cells were separated from cell debris and other cells by a series of differential centrifugation after minced muscle samples were digested by protease. The crude satellite cell preparation was further purified by removing fibroblasts with an aid of differential adhesion to the plastic bottom of culture dishes. By this procedure satellite cells were prepared at better than 95% purity, determined by the immunostaining with monoclonal antibody for desmin. For satellite cell culture, the DMEM/F-12 medium with 10% fetal bovine serum and antibiotics/antimycetics was found suitable for cell proliferation. In this medium, satellite cells attached to the culture dish bottom, took up the spindle-shape, and proliferated. After 3-5 days, cells reached confluence … More and became ready for the cell passage. When the culture medium was replaced with a serum-free medium for the confluent cells, satellite cells underwent differentiation in 1-2 days to form myotubules. In myotubules, the production of myosin was detected by the immunostaining with monoclonal antibody MF-20, proving that satellite cells completed the terminal differentiation as the myogenic cell. As described above, satellite cells spontaneously differentiated to myogenmic lineage under a normal condition. In the second stage of the investigation, the stability of the satellite cell lineage was put on a question and its plasticity was evaluated. With a hypothesis that satellite cells were able to differentiate to cell lineages other than myogenic cells, experiments were conducted. When spindle-shaped satellite cells were transfected with peroxisome proliferators-activated receptors γ (PPARγ)by the lipofection method and the cell culture was continued, satellite cells accumulated lipid droplets, detected by Oil-Red-O staining. This was the first report to demonstrate that satellite cells were capable of being directed the fat cell lineage. When PPARγ was inserted to satellite cells which initiated the terminal differentiation by fusion to form multinucleated cells, lipid accumulation, as well as myosin expression was detected in multinucleated cells. These observations were the direct evidence that satellite cells possessed pluripotency and functioned as adult stem cells. Furthermore, these observations provided a new insight to the concept of cell differentiation. Less

作为本研究的第一阶段，建立了日本黑牛和荷斯坦牛骨骼肌卫星细胞(卫星细胞)的分离、纯化和培养程序。在这个过程中，用一系列差速离心法从细胞碎片和其他细胞中分离出卫星细胞，然后将粉碎的肌肉样品用酶消化。粗制的卫星细胞制剂通过在培养皿塑料底部的差别化黏附去除成纤维细胞来进一步纯化。用结蛋白单抗进行免疫染色，可获得纯度在95%以上的卫星细胞。在卫星细胞培养中，添加10%胎牛血清和抗生素/抗真菌药的DMEM/F-12培养液适合于细胞增殖。在该培养液中，卫星细胞附着在培养皿底部，呈纺锤形，并增殖。3-5天后，细胞达到融合…更多，并为细胞通道做好准备。当融合细胞用无血清培养液代替培养液时，卫星细胞在1~2天内分化形成肌管。在肌小管中，用单抗MF-20免疫染色检测到肌球蛋白的产生，证明卫星细胞完成了作为肌源性细胞的终末分化。如上所述，卫星细胞在正常情况下会自发分化为生肌细胞系。在第二阶段的调查中，对卫星细胞谱系的稳定性提出了一个问题，并对其可塑性进行了评估。在假设卫星细胞能够分化为肌源性细胞以外的细胞系的情况下，进行了实验。用脂质体转染法将过氧化物酶体增殖物激活受体γ(PPARγ)基因导入梭形卫星细胞，继续培养，油红O染色检测到卫星细胞聚集脂滴。这是第一个证明卫星细胞能够引导脂肪细胞谱系的报告。将PPARγ插入卫星细胞，通过融合启动末端分化形成多核细胞，可在多核细胞中检测到脂质堆积和肌球蛋白表达。这些观察结果是卫星细胞具有多能性并具有成体干细胞功能的直接证据。此外，这些观察为细胞分化的概念提供了新的见解。较少