Establishment of a serum free medium for human muscle cells and its application of studyefor muscle disease

1.人软肌化学成分确定培养基的建立。我们为人体肌肉制备了一种化学成分确定的培养基。简言之，培养基由用胰岛素、转铁蛋白等强化的F12培养基组成。该培养基允许人肌细胞生长并产生具有多核素的肌管。该培养液也可用于细胞色素c氧化酶缺乏症患者肌肉的体外研究和未分化恶性纤维组织细胞瘤肌细胞的选择性诱导.用酶免疫法检测培养的肌肉细胞表达Thy-1抗原，并对活检肌肉组织冰冻切片进行免疫组化定量研究。从组织化学定性研究中，Thy-1抗原被认为是肌肉分化的表面标志。我们开发了一种酶免疫测定法，用于定量在96孔微孔板中培养的肌细胞表达的细胞外和细胞内Thy-1抗原。CK和LDH活性也被测量作为肌肉分化标志物。Thy-1抗原可以检测到早在24小时后的肌细胞胰蛋白酶消化我们的检测方法，但不能通过免疫荧光染色。细胞外Thy-1抗原的减少，但细胞内Thy-1抗原的增加与肌管的发育平行。当这个程序被应用于活检肌肉组织的冷冻切片，几种肌肉酶成功地定量。

1. Establishment of a chemically defined medium for humuan muscles. We prepared a chemically defined medium for human muscles. Briefly, the medium consisted of F12 medium intensified with insulin, transferin and etc.The medium allowed to grow human muscle cells and to generate myotubes with poly nucliei. These myotubes were able to contract spontaneously.This medium was also useful for in vitro studies of muscles from patients with cytochrome c oxidase difficiency and for selective induction of muscle cells from undifferentiated malignant fibrous histiocytoma.2. Enzyme immune assay for Thy-1 antigens expressed on cultured muscle cells and quantative immunohistchemical study on frozen sections of biopsied muscle tissues.From the qualitative studies of histochemistry, Thy-1 antigens have been suggested to be a surface marker of muscle differentiation.In the present study, we developed an enzyme immuno assay for quantitation of extracellular and intracellular Thy-1 antigens expressed by muscle cells cultured in 96 well-microplates. CK and LDH activities were also measured as muscle differentiation markers. Thy-1 antigens could be detected as early as 24 hours after trypsinization of muscle cells by our assay method but not by the immunofluorescence staining. The decrease of extracellular Thy-1 antigens but the increase of intracellular Thy-1 antigens were observed in parallel with the development of myotubes. When this procedure was applied for frozen sections of biopsied muscle tissues, several muscle enzymes were successfully quantitated.