Development of novel heat-induced cytokine gene therapy against malignant gliomas

针对恶性胶质瘤的新型热诱导细胞因子基因疗法的开发

基本信息

批准号：
12470286
负责人：
TANAKA Ryuichi
金额：
$ 2.82万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470286/
关键词：
heat shock promoter heat shock protein 70 gene therapy hyperthermia glioma interferon-gamma interferon-pamma heat shock protein gene terapy

项目摘要

1) First, we constructed LacZ gene expression vector under the control of heat shock protein 70 (hsp70) promoter (hsp70/LacZ). Then three human glioma cell lines (U251-MG, T98G, NP2) and one mouse glioma cell line (203) were transfected with hsp70/LacZ vector using lipofection method. After selection with G418, the stable transfectants were obtained. Using these transfectants, the expression of beta-galactosidase after heating was measured. The condition of heating was 41℃ for 2h, 42℃ for 1h, or 43℃ for 1h. The results showed that the peak of the beta-galactosidase expression was between 3h and 24h after heating, even though the degree of its expression varied among the cell lines. (In contrast to other cell lines, the expression was positive in U251-MG cells even with 37℃ heating as a negative control.)2) As a next step, we constructed CD40 ligand (CD40L) gene expression vector under the control of hsp70 promoter (hsp70/CD40L). Then we transfected the vector into mouse 203 glioma cell … More or human NP2 glioma cell by lipofection method. After the selection with G418, we obtained stable transfectants, hsp70/CD40L-203 or hsp70/CD40L-NP2. Using each transfectants, we measured the expression of CD40L protein in the supernatant of the culture medium after heating using ELISA. The results revealed that the peak expression of CD40L protein was between 3h and 48h after heating.3) Furthermore, we constructed IFN-gamma gene expression vector under the control of hsp70 (hsp70/IFN-gamma). The stable transfectant with this vector (hsp70/IFN-gamma-203) was established after the selection with G418. Using this transfectant, we measured the expression of IFN-gamma protein in the supernatant of the culture medium after heating at 42℃ for 1h using ELISA. The result showed that the expression peaked at 24h after the heating. There was no expression of IFN-gamma observed with the treatment of 37℃ heating as a negative control.4) Taken together, in most of the glioma cell lines except for U251-MG cells, the peak expression of the inserted gene by heating was observed between 3h and 48h after heating, even though the expression pattern varied depending on each cell line. Therefore, this gene expression system using hsp70 promoter can be a good candidate for heat-inducible gene treatments. Less

1）首先，我们在控制热休克蛋白70（HSP70）启动子（HSP70/LACZ）的控制下构建了LACZ基因表达载体。然后使用LiPofection方法将三种人神经瘤细胞系（U251-MG，T98G，NP2）和一只小鼠胶质瘤细胞系（203）翻译。选择G418后，获得了稳定的转化体。使用这些转化体，测量加热后β-半乳糖苷酶的表达。加热状况为41℃，为2H，42℃为1H，或43°℃，为1H。结果表明，加热后β-半乳糖苷酶表达的峰值在3h至24h之间，即使其表达程度在细胞系之间有所不同。（与其他细胞系相反，即使以37°加热为阴性对照，在U251-mg细胞中的表达也是阳性的。）2）作为下一步，我们在HSP70启动子（HSP70/CD40L）的对照下构建了CD40配体（CD40L）基因表达载体。然后，我们将载体转化为小鼠203神经胶质瘤细胞…通过脂肪触发方法，更多或人类NP2神经胶质瘤细胞。选择G418后，我们获得了稳定的转化体HSP70/CD40L-203或HSP70/CD40L-NP2。使用每个转化体，我们在使用ELISA加热后测量了培养基上清液中CD40L蛋白的表达。结果表明，加热后CD40L蛋白的峰值表达在3H和48H之间。3）此外，我们在HSP70（HSP70/IFN-GAMMA）的控制下构建了IFN-GAMMA基因表达载体。使用G418选择后，建立了使用该载体（HSP70/IFN-GAMMA-203）的稳定变换剂（HSP70/IFN-GAMMA-203）。使用了这一翻译，我们使用ELISA在42℃加热后，在42℃加热后，测量了IFN-GAMMA蛋白在培养基的上清液中的表达。结果表明，加热后的表达在24小时处达到峰值。通过处理37℃加热作为阴性对照的IFN-gamma没有观察到的IFN-gamma。因此，使用HSP70启动子的这种基因表达系统可以成为热诱导基因处理的良好候选者。较少的