权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

An attempt of application Myo D transfected skin fibroblasts of patients to enhance gene expression and to analyze muscle specific Na channel proteins.

尝试应用Myo D转染患者的皮肤成纤维细胞来增强基因表达并分析肌肉特异性Na通道蛋白。

基本信息

批准号：
07457129
负责人：
SHISHIBA Yoshimasa
金额：
$ 3.01万
依托单位：
OKINAKA MEMORIAL INSTITUTE FOR MEDICAL RESEARCH
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457129/
关键词：
Hypocalcemia Hypercalcemia Ion channel Myo D gene Myosin Fibroblast Na channel 皮膚線維芽細胞

项目摘要

Purpose : Our goal of the research is to transform human fibroblasts to myoblasts by transfecting MyoD gene. Such transfected and successfully transformed cells would give us a very handy tool to examine genetic abnormality of muscle-specific praoteins such as ryanodine-receptor protein or TTX-sensitive Na channel. Such tranformed cells would also allow us to examine physiological lproperties of those channel proteins.Materials and methods : As our rigorous attempts to transfect MyoD gene into human fibfoblasts employing Cap, lipofectine, or lipofectamine, we adopted animal cell-line which has higher rate of transfection efficiency and examined basic requirements to induce TTX-sensitive Na channel employing a sensitive method to detect the channel mRNA.Results : One of the collaborator, Dr.Okamura succeeded to modify RT-PCR to be sensitive nough to detect changes in TTX-insensitive Na channel expression according to the development of motor neuron in isolated blastomeres of Halocynthia roretzi. Drs.Takahashi and Tanaka have been engaged in developing a new method to detect transfection efficiency directly under the microscope. They arae trying to establish a Myo-DGFP fusion protein and examine the expression in an in-vitro culture system. Although the attempts are currently under the progress, they have already successfully praoduced a fusion protein which connects functional channel protein and also succeeded in expressing the fusion protein in an in-vitro system. By employing these methods, it is feasible to establish a clinically important in vitro system to study transformed skin fibroblasts as a surrogate of muscle biopsy specimen.

目的：本研究的目的是通过转染MyoD基因，将人成纤维细胞转化为成肌细胞。这种转染和成功转化的细胞将为我们提供一个非常方便的工具来检查肌肉特异性前蛋白，如ryanodine受体蛋白或TTX敏感性Na通道的遗传异常。材料与方法：我们采用Cap、Lipofectine、Lipofectamine等方法将MyoD基因导入人成纤维细胞，采用转染率较高的动物细胞系，采用灵敏的方法检测通道mRNA，考察了诱导TTX敏感性Na通道的基本条件。合作者之一Okamura博士成功地根据Halocynthia roretzi分离卵裂球中运动神经元的发育将RT-PCR修改为灵敏的，足以检测TTX不敏感Na通道表达的变化。Takahashi和Tanaka博士一直致力于开发一种在显微镜下直接检测转染效率的新方法。他们试图构建Myo-DGFP融合蛋白，并在体外培养系统中检测其表达。虽然目前的尝试还在进行中，但他们已经成功地制备了连接功能性通道蛋白的融合蛋白，并成功地在体外系统中表达了融合蛋白。通过这些方法，可以建立一个临床上重要的体外系统来研究转化的皮肤成纤维细胞作为肌肉活检标本的替代物。