AIM: Cancer gene therapy has received more and moreattentions in the recent decade. Various systems of genetherapy for cancer have been developed. One of the mostpromising choices is the suicide gene. The product ofth...AIM: Cancer gene therapy has received more and moreattentions in the recent decade. Various systems of genetherapy for cancer have been developed. One of the mostpromising choices is the suicide gene. The product ofthymidine kinase (TK) gene can convert ganciclovir (GCV)to phosphorylated GCV, which inhibits the synthesis of cellDNA, and then induces the cells to death. Cytolines play animportant role in anti-tumor immunity. This experiment wasdesigned to combine theTK gene and mIL-2/mGM-CSFgenes to treat gastric cancer, and was expected to producea marked anti-tumor effect.METHODS: TK gene was constructed into the retroviralvector pLxSN, and the mIL-2 and mGM-CSF genes wereinserted into the eukaryotic expressing vector pIRES. Thegastric cancer cells were transfected by retroviral serum thatwas harvested from the package cells. In vitro study, thetransfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect andbystander effect. In vivo experiment, retroviral serum andcytokines plasmid were transfected into tumor-bearing mice,to observe the changes of tumor volumes and survival ofthe mice.RESULTS: In vitro experiment, 20 % TK gene transducedcells could cause 70-80 % of total cells to death. In vivoresults showed that there was no treatment effect in controlgroup and TK/GCV could inhibit the tumor growth. Thestrongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis ofthe cancer in the treated groups.CONCLUSION: TK/GCV can kill tumor cells and inhibit thetumor growth in vivo IL-2 and GM-CSF strongly enhancethe anti-tumor effect. Through the retrovirus and liposomemethods, the suicide gene and cytokine genes are allexpressed in the tissues.展开更多
Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for high...Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.展开更多
基金Health Ministry Scientific and Research Foundation of China,No.98-1-312
文摘AIM: Cancer gene therapy has received more and moreattentions in the recent decade. Various systems of genetherapy for cancer have been developed. One of the mostpromising choices is the suicide gene. The product ofthymidine kinase (TK) gene can convert ganciclovir (GCV)to phosphorylated GCV, which inhibits the synthesis of cellDNA, and then induces the cells to death. Cytolines play animportant role in anti-tumor immunity. This experiment wasdesigned to combine theTK gene and mIL-2/mGM-CSFgenes to treat gastric cancer, and was expected to producea marked anti-tumor effect.METHODS: TK gene was constructed into the retroviralvector pLxSN, and the mIL-2 and mGM-CSF genes wereinserted into the eukaryotic expressing vector pIRES. Thegastric cancer cells were transfected by retroviral serum thatwas harvested from the package cells. In vitro study, thetransfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect andbystander effect. In vivo experiment, retroviral serum andcytokines plasmid were transfected into tumor-bearing mice,to observe the changes of tumor volumes and survival ofthe mice.RESULTS: In vitro experiment, 20 % TK gene transducedcells could cause 70-80 % of total cells to death. In vivoresults showed that there was no treatment effect in controlgroup and TK/GCV could inhibit the tumor growth. Thestrongest anti-tumor effect was shown in TK+mIL-2+mGM-CSF group. The pathologic examination showed necrosis ofthe cancer in the treated groups.CONCLUSION: TK/GCV can kill tumor cells and inhibit thetumor growth in vivo IL-2 and GM-CSF strongly enhancethe anti-tumor effect. Through the retrovirus and liposomemethods, the suicide gene and cytokine genes are allexpressed in the tissues.
基金supported by the National Natural Science Foundation of China (30771952)the National Natural Science Foundation of Guangdong Province (07117783)NSFC and the Research Grants Council of Hong Kong Joint Research Scheme (30418003).
文摘Objective To study the activities of interleukin （IL）-2 and granulocyte-macrophage colony-stimulating factor （GM-CSF） （hlL-2/mGM-CSF）. Methods SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli （E. coil） BL21 （DE3） in inclusion body （IB） form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay. Results The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein/L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4×10^6 IU/mg and 7.1×10^6 IU/mg, respectively. Conclusion This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hlL-2/mGM-CSF used in immune therapy.
