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

1)首先构建热休克蛋白70 （hsp70）启动子（hsp70/LacZ）调控的LacZ基因表达载体。然后用脂质转染hsp70/LacZ载体转染3株人胶质瘤细胞系U251-MG、T98G、NP2和1株小鼠胶质瘤细胞系203。用G418筛选后，获得了稳定的转染物。利用这些转染物，测定加热后β -半乳糖苷酶的表达。加热条件为41℃2h， 42℃1h, 43℃1h。结果表明，β -半乳糖苷酶的表达高峰出现在加热后的3h ~ 24h之间，尽管不同细胞系的表达程度不同。（与其他细胞系相比，U251-MG细胞即使在37℃加热作为阴性对照，表达也呈阳性。）2)下一步，构建hsp70启动子（hsp70/CD40L）调控的CD40配体（CD40L）基因表达载体。然后用脂质转染法转染小鼠203胶质瘤细胞和人NP2胶质瘤细胞。用G418筛选后，我们获得了稳定的转染物hsp70/CD40L-203或hsp70/CD40L-NP2。使用每种转染物，用ELISA法检测加热后培养基上清液中CD40L蛋白的表达。结果显示，CD40L蛋白在加热后3h ~ 48h达到表达高峰。3)进一步构建了hsp70调控下的IFN-gamma基因表达载体（hsp70/IFN-gamma）。用G418筛选后，与该载体（hsp70/ ifn - γ -203）建立稳定的转染载体。利用该转染物，在42℃加热1h后，用ELISA法检测培养基上清中ifn - γ蛋白的表达。结果表明，该表达在加热后24h达到峰值。在37℃的负对照处理下，ifn - γ没有表达。4)综上所述，除了U251-MG细胞外，在大多数胶质瘤细胞系中，加热插入基因的表达高峰出现在加热后的3h - 48h之间，尽管不同细胞系的表达模式不同。因此，利用hsp70启动子构建的基因表达体系可以作为热诱导基因处理的良好候选体系。少

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