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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，我们构建了热休克蛋白70（hsp 70）启动子调控下的LacZ基因表达载体（hsp 70/LacZ）。用脂质体法将hsp 70/LacZ载体转染人胶质瘤细胞系U251-MG、T98 G、NP 2和小鼠胶质瘤细胞系203。经G418筛选，获得稳定的转化子。使用这些转染子，测量加热后β-半乳糖苷酶的表达。加热条件为41℃ 2 h、42℃ 1h、43℃ 1h。结果表明，β-半乳糖苷酶的表达高峰在加热后3 ~ 24 h之间，尽管其表达程度因细胞系而异。(In与其他细胞系相比，U251-MG细胞即使在37℃加热作为阴性对照时也呈阳性表达。2)下一步，我们构建了hsp 70启动子调控下的CD 40配体（CD 40 L）基因表达载体（hsp 70/CD 40 L）。将该载体转染小鼠203胶质瘤细胞， ...更多信息或人NP 2胶质瘤细胞。经G418筛选，获得了稳定的转染细胞hsp 70/CD 40 L-NP 2和hsp 70/CD 40 L-NP 2。使用每种转染子，我们使用ELISA测量了加热后培养基上清液中CD 40 L蛋白的表达。结果表明，CD 40 L蛋白表达的高峰期在热处理后3 ~ 48 h之间。3）构建了hsp 70调控的IFN-γ基因表达载体（hsp 70/IFN-γ）。经G418筛选后，建立了稳定的hsp 70/IFN-γ-203转染子。用此转染子，我们用ELISA法检测了42℃加热1h后培养上清中IFN-γ蛋白的表达。结果表明，加热后24小时表达达到峰值。4）除U251-MG细胞外，大部分胶质瘤细胞系中，插入基因在加热后3 ~ 48 h表达最高，但不同细胞系的表达模式不同。因此，该基因表达系统使用热休克蛋白70启动子可以是一个很好的候选热诱导基因治疗。少