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Tissue culture studies on immortalized Schwann cells.

永生化雪旺细胞的组织培养研究。

基本信息

批准号：
04670218
负责人：
WATABE Kazuhiko
金额：
$ 1.22万
依托单位：
The Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670218/
关键词：
Schwann cell Tissue culture Mouse Immortalization Transfection Heparin-binding protein Dorsalrootgenglia Sphingomyelinosis PDGF FGF TGF-β

项目摘要

1. Cultured Schwann cells derived from adult mouse dorsal rootganglia (DRG) and peripheral nerves were transfected with a plasmid containing SV-40 large Tantigen gene One of transfected cell lines, designated MS1, had distinct Schwann cell phenotypes such as S100 protein, laminin, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and Poprotein, as shown by imuunofluorescence. The growth of MS1 cells was partially suppressed by incubation with dibutyryl cyclic adenosine-3', 5'-monophosphate (dbc AMP), forskolin and retinoic acid.2. Mitogenic effects of fetal calf serum (FCS), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta) and forskolin to adult mouse Schwann cells were examined by bromodeoxy uridine (BrdU) incorporation and double immunofluorescence for S100 and BudU.PDGF-BB, basic FGF, TGF-beta 1 and beta 2 were all mitogenic for Schowann cells in media containing FOS.Forsk olin suppressed the mitogenic activity of th … More ese factors. In serum-free media, PDGF-BB and bFGF were also mitogenic, but TGF-beta1 and beta2 were not. Heparin-binding fractions of FCS obtained by heparin-Sepharose chromatography synergized with TGF-beta1 and beta2 to produce a mitogenic response. Since PDGF-BB, acidic FGF, basic FGF were not detected in these fractions by immunoabsorption and immunoblot assays, the presence of unidentified heparin-binding molecules in FCS bioactive for adult mouse Schwann cells is suggested.3. We obtained several of spotaneously immortalized celllines from long-term cultures of adult mouse Schwann cells. Conditioned media derived from one of the celllines had a mitogenic activity to original Schwann cell cultures. These cultures can be useful in studies on autocrine growth mechanism of Schwann cells in vitro.4. We established Schwann cell cultures of sphingomyelinosis (spm) mouse, which develops hepatosplenomegaly and ataxic movements with tremors. Primary cultures of DRG and peripheral nerves derived from affected mice were treated with anti-Thy1.2.and rabbit complement, which resulted purified Schwann cell cultures. Electron-microscopically, characteristic lamellar inclusions were demonstrated in these cells. We also obtained spontaneously immortalized Schwann cell colonies from long-term cultures. These Schwann cell cultures can be useful in studies on the nervous system involvement of spm mouse. Less

1.用含SV-40大T抗原基因的质粒转染成年小鼠背根神经节（DRG）和周围神经的雪旺细胞。免疫荧光显示，转染的细胞系MS 1具有不同的雪旺细胞表型，如S 100蛋白、层粘连蛋白、2 ′，3 ′-环核苷酸3 ′-磷酸二酯酶（CNP）和Poprotein。二丁酰环腺苷-3 '，5'-一磷酸（dbc AMP）、毛喉素和维甲酸孵育MS 1细胞，可部分抑制MS 1细胞的生长。胎牛血清（FCS）、血小板衍生生长因子（PDGF）、成纤维细胞生长因子（FGF）、转化生长因子-β的促有丝分裂作用通过溴脱氧尿苷（BrdU）掺入和对S100和BudU的双重免疫荧光检测TGF-β和毛喉素对成年小鼠雪旺细胞的作用。TGF-β 1和TGF-β 2在含FOS的培养基中对Schowann细胞都有促有丝分裂作用，Forsk olin抑制TGF-β 1和TGF-β 2的促有丝分裂活性。 ...更多信息这些因素。在无血清培养基中，PDGF-BB和bFGF也有促有丝分裂作用，但TGF-β 1和β 2没有。通过肝素-琼脂糖色谱法获得的FCS的肝素结合组分与TGF-β 1和β 2协同产生促有丝分裂反应。由于免疫吸附和免疫印迹分析在这些组分中未检测到PDGF-BB、酸性FGF、碱性FGF，因此证实FCS中存在对成年小鼠雪旺细胞具有生物活性的未知肝素结合分子.我们从成年小鼠雪旺细胞的长期培养中获得了几个孢子虫永生化细胞系。来自其中一种细胞系的条件培养基对原始许旺细胞培养物具有促有丝分裂活性。这些培养物可用于体外研究雪旺细胞的自分泌生长机制.我们建立了神经鞘磷脂病（spm）小鼠的雪旺细胞培养，这种小鼠出现肝脾肿大和震颤性共济失调运动。用抗Thy1.2和兔补体处理源自受影响小鼠的背根神经节和外周神经的原代培养物，得到纯化的许旺细胞培养物。电子显微镜下，这些细胞中显示出特征性的层状包涵体。我们还从长期培养中获得了自发永生化的雪旺细胞集落。这些雪旺细胞培养物可用于spm小鼠神经系统受累的研究。少