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Functional analysis of p130 oncogene in malignant glioma

恶性胶质瘤p130癌基因的功能分析

基本信息

批准号：
13671475
负责人：
KAWAMOTO Keiji
金额：
$ 2.05万
依托单位：
Kansai Medical 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-13671475/
关键词：
glioma p130 transfection cyclin B1 cyclin D1 cyclinB1 cyclinD1

项目摘要

I. Analysis of cell cycle and cyclin with use of FCMThree kinds of glioma cell lines were analyzed the cell cycle and expression of cyclins from the DNA histogram stained with FTTC laelled cyclin A, B1, D1, E and propidium iode. These cell lines experssed all of cyclin A, B1, D1. Cyclin B1 was prooved to be expressed on the S, G2, M phases and cyclin D1 was expressed on the phase of G0, G1 phases.II. Analysis of cell cycle and cyclin with use of LSCSame results were obtained by LSC as well as FCM.III. Effect of As2O3 on cell cycle progression and cyclin expression in glioblastoma cell linesAs2O3 induced an increase in p53 level and a decrease in level of cyclin B1, combined with cell arrest at G2/M in cell lines. Cell arrest in G1, however, was associated with a decline in cyclin D1 expression in the U87 MG cells.IV. Rb2/p130 expression on the surgical specimen of gliomasBenign gliomas grade I and II were expressed strongly, but malignant glioma grade IV showed negative stain. On the other hand, p53 showed high positive rates in the cases of grade IV.V. Functional analysis of p130 induced gliomap130 cDNA induced subclone to BCMGSneo and this p130 was transfected into U87 MG with use of Lipofact AMINE. After transfected cells were sellected by G418, U87 MG expressed of p130 oncogene was success to be produced. Plasmid from the oncogene of p130 was obtained over 6 months and p130 was possible to be transofected into U87MG cell line. We are planning now how to controll the proliferation of malignant gloma, especially mutant glioma (N-319) or express of the cyclins.

I.流式细胞术分析细胞周期及cyclin的表达用FTTC标记cyclin A、B1、D1、E和碘化丙啶染色的DNA直方图分析三种胶质瘤细胞系的细胞周期及cyclin的表达。细胞周期蛋白A、B1、D1均表达。Cyclin B1表达于S、G2、M期，cyclin D1表达于G 0、G1期。用LSC分析细胞周期和细胞周期蛋白用LSC和FCM获得相同的结果。III. As_2O_3对胶质母细胞瘤细胞周期进程和cyclin表达的影响As_2O_3可诱导胶质母细胞瘤细胞株p53水平升高，cyclin B_1水平降低，并使细胞阻滞于G_2/M期。然而，细胞停滞在G1期与U87 MG细胞中细胞周期蛋白D1表达的下降相关。Rb 2/p130在胶质瘤手术标本中的表达：Ⅰ、Ⅱ级良性胶质瘤Rb 2/p130表达强，Ⅳ级恶性胶质瘤Rb 2/p130表达阴性。另一方面，p53在IV. V级病例中显示高阳性率。p130诱导的胶质瘤蛋白130 cDNA诱导的亚克隆至BCMGSneo的功能分析，并使用Lipofact AMINE将该p130转染至U87 MG中。转染细胞经G418筛选后，成功产生表达p130癌基因的U87 MG。经6个月以上的时间获得了p130癌基因的质粒，并有可能将p130转染到U87 MG细胞系中。我们正在研究如何控制恶性胶质瘤的增殖，特别是突变型胶质瘤（N-319）的增殖或细胞周期蛋白的表达。