IGF-I triple helix gene therapy of rat and human gliomas.
L. TrojanPiotr KopiñskiAndrzej MazurekL ChyczewskiA. LyPiotr JarockiJacek NiklińskiAlexander ShevelevR.J. TrzosYinghong PanD. J. GitisMaciej BierwagenJoanna CzapiewskaMing WeiJacek MichałkiewiczDominique HéninT PopielaF. EvrardH KasprzakDonald D. AnthonyJerzy Trojan
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IGF-I anti-gene technology was applied in treatment of rat and human gliomas using IGF-I triple helix approach.CNS-1 rat glioma cell and primary human glioblastoma cell lines established from surgically removed glioblastomas multiforme were transfected in vitro with IGF-I antisense (pMT-Anti-IGF-I) or IGF-I triple helix (pMT-AG-TH) expression vectors. The transfected cells were examined for immunogenicity (immunocytochemistry and flow cytometry analysis) and apoptosis phenomena (electron microscopy). 3 x 10(6) transfected cells were inoculated subcutaneously either into transgenic Lewis rats or in patients with glioblastoma. The peripheral blood lymphocytes (PBL) derived from "vaccinated" patients were immunophenotyped for the set of CD antigens (CD4, CD8 etc).Using immunocytochemistry and Northern blot techniques, the transfected "antisense" and "triple-helix" cells showed total inhibition of IGF. Transfected cultures were positively stained either for both MHC-I and B7 antigens--60% of cloned lines, or for MHC-I only--40% of cloned lines. Moreover "triple helix" cells as compared to "antisense" cells showed slightly higher expression of MHC-I or B7. Transfected cells also showed the feature of apoptosis in 60%-70% of cells. In in vivo experiments with rats bearing tumors, the injection of "triple helix" cells expressing both MHC-I and B7 interrupted tumor growth in 80% of cases. In contrast, transfected cells expressing only MHC-I stopped development in 30% of tumors. In five patients with surgically resected glioblastoma who were inoculated with "triple helix" cells, PBL showed an increased percentage of CD4 + CD25+ and CD8 + CD11b-cells, following two vaccinations.The anti-tumor effectiveness of IGF-I anti-gene technology may be related to both MHC-I and B7 expression in cells used for therapy. The IGF-I antigene therapy of human glioblastoma multiforme increases immune response of treated patients.Cite
Objective To observe the inhibitory effects of c-myc antisense oligonucleotides on the proliferation of MKN-45 cells.Methods Cell proliferation was measured by MTT assay.Expres- sion of c-myc protein was measured by immunocytochemistry after transfection.Apoptosis of cells was detected by flow cytometry and fluorescence microscope.Results After sealing c-myc gene with ASODN,the proliferation of MKN-45 cells was inhibited markedly.FCM analysis showed that an obvious apoptosis peak appeared after transfection.The immunocytochemistry staining showed that c-myc protein expression was suppressed significantly 64.9%±5.68%.Conclusion Liposome-mediated c-myc ASODN can evidently inhibit cell proliferation,down-regulate c-myc protein expression,and induce apoptosis of MKN-45 cells.
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To construct eukaryotic expression vector of human T-cell immunoglobulin mucin 3(TIM-3) and transfect mammalian cells to establish stable cell line.The whole coding region of TIM-3 was amplified by PCR and inserted into eukaryotic expression vector pIRES2EGFP. The recombinant plasmid was transfected into mammalian cells by Lipofectamine. The expression product was analyzed by flow cytometry and Western blot. The stable transfectant was screened and established by flow cytometry and selective medium.COS-7 and CHO cells were transfected with recombinant plasmid by Lipofectamine. The expression speciality was identified by Flow cytometry and Western blot. The stable transfectant of CHO cell line was established.The whole coding region of TIM-3 was successfully subcloned into eukaryotic expression vector and expressed on the surface of mammalian cells. The stable transfectant of CHO cell line was established.
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Objective: To construct cation liposome vector and transfect interferon-β(IFN-β) gene into human glioma cell line with it,and to observe the inhibitory effect of IFN-β on the proliferation of the cell line.Methods: The cation liposome vector containing IFN-β was constructed by reverse-phase evaporation and was used to transfect human glioma cell line SK-MG-1.The inhibitory effect of IFN-β on the proliferation of the cell line was measured by MTT method,and the apoptosis of glioma cells was determined by flow cytometry.Results: IFN-β gene transfection inhibited the proliferation of human glioma cell line SK-MG-1 and induced the apoptosis of glioma cells.Conclusion: Cation liposome is an effective vector used to transfect IFN-β gene into human glioma cell line.IFN-β can inhibit the proliferation of human glioma cell line SK-MG-1 and induce the apoptosis of the glioma cell.
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Objective To establish a rat myoblast cell line(L6) with stable expression of human calcitonin(hCT) gene and observe hCT expression and secretion in L6 cells transfected by hCT gene.Methods We constructed a recombinant eukaryotic expression plasmid,pcDNA3.0-Igκ-hCT,which contains human calcitonin gene and Murine Igκ-chain leader sequence.The recombinant hCT pcDNA plasmid was transfected into L6 cells by cation liposomes and selected by G418.The control group transfected with plasmid pcDNA3.0.The mRNA and protein expression of hCT were detected by reverse transcription-PCR(RT-PCR) and immunocytochemistry staining respectively,and the hCT secretion in the culture supernatant of L6 cells were analyzed by RIA.Results The expressions of hCT mRNA and protein could be detected by RT-PCR and immunocytochemistry staining in the recombinant hCT gene transfected L6 cells,but not in pcDNA3.0 transfected cells.During serial passages,we had detected average concentration of hCT,which was 42.22ng/(106·24h) in the culture supernatant.Conclusion The pcDNA3.0-Igκ-hCT was successfully transfected into L6 myoblast;The vector could express hCT sustainedly.The stable system of hCT gene expression was constructed.
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OBJECTIVE:To observe the biological effect of c-myc antisense oligonucleotides(ASODN)on human gastric cancer MKN-45 cells.METHODS:The transfection on MKN-45 cells of c-myc antisense oligodeoxynucleotide was mediated by liposome.The cell proliferation was measured by MTT assay.The expression of c-myc protein was measured by immunocytochemistry after the transfection.The apoptosis of cells was detected by flow cytometry and fluorescence microscopr.The tumor models were constructed of nude mice transplanted with human gastric cancer MKN-45 cells and the observations were made on tumor suppression rate and tumor volume.RESULTS:After sealing c-myc gene with ASODN,the proliferation of MKN45 cells was inhibited markedly and the inhibition rates treated with 0.25-4 μmol/L ASODN were from 11.2% to 23.6%.FCM analysis showed that there appeared an obvious apoptosis peak after the transfection.The immunocytochemistry staining showed that c-myc protein expression was suppressed significantly(64.9±5.68)%.The tumor volume decreased obviously and the rate of tumor suppression reached 41.5% in vivo experiment.CONCLUSION:Liposome-mediated c-myc ASODN can evidently inhibit cell proliferation,down-regulate c-myc protein expression,and induce apoptosis of MKN45 cells.
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To investigate the effects of transfected exogenous p21 gene on the cells cycle of HLE-B3 cells line. The feasibility of prevention of secondary cataract by gene therapy was evaluated.Total length of human p21 gene cDNA was cloned on the parent's plasmid pcDNA3 to construct the recombinant plasmids of pcDNA3/p21, a large amount of pcDNA3/p21 plasmid DNA was prepared by QIAGEN endofree maxi kit. After harvest of the plasmid DNA, the HLE-B3 cells line was transfected. The cell growth was observed and the cells cycle was analyzed by flow cytometry. The expression of p21 mRNA was detected by RT-PCR and the expression of p21 protein was detected by immunohistochemistry and western blot analysis.Forty eight hours after transfection, the growth of transfected cells became slower, some cells floated and died; the control cells and blank plasmid (blank pcDNA3) transfected cells grew normally. Flow cytometry analysis revealed that the number of cells in G(1) phase increased markedly in transfected cells. The RT-PCR showed that the product of p21 in the transfected cells (dead or alive cells) was obviously higher than that of the controls. Immunohistochemical studies showed few positive cells in the controls, and very high positive signal was detected after transfection. Western blot showed a positive band at the level of 21 000 in transfected cells, no positive band could be found in the controls.Exogenous p21 gene can transfect the HLE-B3 cells and can be expressed. The transfection can affect the cells cycle by G(1) arrest and induces cells death through the apoptosis process. This result suggests that it is possible to prevent the occurrence of secondary cataract by gene therapy.
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AIM To study the effect of p38MAPK transfection on the biological characteristics of glioma cell C6. METHODS p38MAPK was transfected into glioma cell C6 by lipofectin. Expression of p38MAPK was detected by immunocytochemistry. HE staining and flow cytometry were adopted to measure the cell morphology, adhesion and cell cycle. RESULTS p38MAPK was expressed in transfected glioma cells, with cell biological characteristics changing and apoptotic cells emerging. CONCLUSION Apoptosis of glioma cell could be induced by p38MAPK transfection.
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Establishment of cell line transfected with human LIGHT gene and its co-stimulatory effect on T cell
To establish the human co-stimulatory molecule LIGHT gene transfected cell line and to investigate its co-stimulatory effect on T cell activation and proliferation in vitro,the full-length human LIGHT gene coding region was cloned from the activated T cells of human peripheral blood by RT-PCR and then inserted into the eukaryotic expression vector pIRES2-EGFP to construct the recombinant pIRES2-EGFP-LIGHT after double digestion with EcoR Ⅰ and BamH Ⅰ.The recombinant plasmid was transfected to murine L929 cells after induction with LipfectamineTM2000 and the cells were further selected with G418.The effect of the recombinant vector transfected into L929/LIGHT cells on T cells proliferation and cytokine production in vitro was studied by MTT,ELISA and flow cytometry.It was demonstrated that the stable expression of human LIGHT on the transfected cell line was identified by flow cytometry analysis.and the L929/LIGHT cells could promote obviously the proliferation of T cells in vitro and were stimulated by anti-CD3 mAb.The up-regulation on the cytokine production,such as IL-2,IFN-γ and IL-10 could be demonstrated by ELISA.It is evident that a cell line L929/LIGHT stably expressing the human LIGHT gene has been obtained and it triggers a signal which can prominently stimulate the proliferation and cytokine production of T cells in vitro.
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Objective:To investigate the effects of liposome (LipfectAMINE TM ,namely LR)-mediated c-myc antisense oligodeoxynucleotide(ASODN) on cell proliferation,c-myc protein expression,and apoptosis of MCF-7 cells. Methods:Cell proliferation was measured by MTT assay. Expression of c-myc protein was measured by immunocytochemistry before and after transfection. Apoptosis of cells was detected by flow cytometry. Results:Transfection of ASODN/LR markedly inhibited the proliferation of MCF-7 cells compared with sense and missense ODN( P 0.01). FCM analysis showed that there appeared an obvious apoptosis peak after transfection. The apoptosis rate was maximum (28.76±2.09)% at 72 h. The immunocytochemistry staining showed that c-myc protein expression was suppressed significantly(21.4±1.16)%. Conclusion:Liposome-mediated c-myc ASODN can evidently inhibit cell proliferation,down-regulate c-myc protein expression,and induce apoptosis of MCF-7 cells.
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To study the effects of alpha1,4Gal T antisense oligonucleotide mediated by lipofectin on human glioma cell line SWO-38.SWO38 glioma cells were exposed to 10 micromol/L alpha1,4Gal T antisense oligonucleotide for 72 h by means of lipofectin transfection, and the growth inhibition of the cells was detected by colony-forming unit assay. Analysis of DNA fragmentation was performed with flow cytometry (FCM) and agarose gel eletrophoresis with Fas protein expression determined by FCM.alpha1,4Gal T antisense oligonucleotide significantly inhibited the growth of glioma cells (P<0.01) and induced apoptosis in SWO-38 cells (P<0.05). Marked up-regulation of Fas protein expression was observed in response to the treatment (P<0.01).alpha1,4Gal T antisense oligonucleotide can significantly inhibit SWO-38 cell proliferation and induce cellular apoptosis, the mechanism of which may involve up-regulated Fas expression.
Agarose
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