Effect of Lipofectin on Antigen-presenting Function and Anti-tumor Activity of Dendritic Cells

BACKGROUND: Dendritic cells (DC) are professional antigen-presenting cells in the immune system and can induce T cell response against virus infections, microbial pathogens, and tumors. Therefore, immunization using DC loaded with tumor-associated antigens (TAAs) is a powerful method of inducing anti-tumor immunity. For induction of effective antitumor immunity, antigens should be efficiently introduced into DC and presented on MHC class I molecules at high levels to activate antigen-specific CD8+ T cells. We have been exploring methods for loading exogenous antigens into APC with high efficiency of Ag presentation. In this study, we tested the effect of the cationic liposome (Lipofectin) for transferring and loading exogenous model antigen (OVA protein) into BM-DC. METHODS: Bone marrow-derived DC (BM-DC) were incubated with OVA-Lipofectin complexes and then co-cultured with B3Z cells. B3Z activation, which is expressed as the amount of beta-galactosidase induced by TCR stimulation, was determined by an enzymatic assay using beta-gal assay system. C57BL/6 mice were immunized with OVA-pulsed DC to monitor the in vivo vaccination effect. After vaccination, mice were inoculated with EG7-OVA tumor cells. RESULTS: BM-DC pulsed with OVA-Lipofectin complexes showed more efficient presentation of OVA-peptide on MHC class I molecules than soluble OVA-pulsed DC. OVA-Lipofectin complexes-pulsed DC pretreated with an inhibitor of MHC class I-mediated antigen presentation, brefeldin A, showed reduced ability in presenting OVA peptide on their surface MHC class I molecules. Finally, immunization of OVA-Lipofectin complexes-pulsed DC protected mice against subsequent tumor challenge. CONCLUSION: Our data provide evidence that antigen-loading into DC using Lipofectin can promote MHC class I-restricted antigen presentation. Therefore, antigen-loading into DC using Lipofectin can be one of several useful tools for achieving efficient induction of antigen-specific immunity in DC-based immunotherapy.

Transfection and expressiOn of green fluorescent protein gene in prostate carcinoma PC-3M cell line / 吉林大学学报(医学版)

Objective：To reseach the expression of green fluorescent protein(GFP)gene in prostate car-cinoma PC-3M cell line and the efffect of cell cycle．Methods：GFP gene recombination DNA was construct-ed and PC-3M cells were transfected with lipofectin regant．The expression of GFP was observed under in-vert fluorescence microscope．Meanwhile，the cell cycle of positive cell colon was assayed by flOw cytome-ter．Resuits：After transfection，about 10％～15％cells expressed green fluorescent protein and there wasno significant change compared the cell cycle of positive cell colon with parents cells．Conclusion：GFP sup-plys a new way in reseaching metabolic phenomenon of live cells．

Optimizing condition for lipofectin-mediated NF-?B decoy oligodeoxynucleotides transfection into J774.1 cells / 第二军医大学学报

Objective: To search for the optimizing parameters and distribution pattern of NF ?B decoy oligodeoxynucleotides in transfecting J774.1 cells mediated by lipofectin. Methods: With the change of ODNs/lipofectin ratio and transfection time, the uptake rate and mean fluorescence intensity of NF ?B decoy oligodeoxynucleotides in J774.1 cells were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of NF ?B decoy oligodeoxynucleotides was determined with fluorescence microscopy. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity. Results: The uptake of NF ?B decoy oligodeoxynucleotides into J774.1 cells was significantly improved by lipofectin. In 24 well culture plate, 1 ?g ODNs with 5?g lipofectin ( W/W =1∶5) resulted in the highest transfection efficiency and lower cytotoxicity. The NF ?B decoy oligodeoxynucleotides localized in the nucleus as well as in the cytoplasm following an incubation of 6 h with lipofectin. While NF ?B decoy oligodeoxynucleotides had faint fluorescences in cytoplasm in the absence of lipofectin. Conclusion: lipofectin can enhance the cellular uptake of NF ?B decoy oligodeoxynucleotides in J774.1 cells and alter intracellular distribution of NF ?B decoy oligodeoxynucleotides. Efficiency of transfection is the highest when the ratio of ODNs/lipofectin is 1∶5 for incubation of 6 h.

Development of Gene Therapy Strategy Using Plasmid and Adenovirus in Cervical Cancer Treatment / 대한산부인과학회잡지

BACKGROUND: The basic treatment of malignant tumors is surgery, radiotherapy, chemotherapy. Even though, the object of these treatments is to kill cancer cells, they have limitations. So, in future studies of treatment of cancer, we should look into increasing human immune response using gene therapy in order to induce damage to tumor cells. OBJECTIVE: The cell growth inhibitory effect of cervical cancer cells was investigated by direct transfection using liposome(pRcCMVp53/lipofectin). and by indirect transfection using Adenovirus(AdCMVp53). METHODS: The cervical cancer cell lines we used in this study were HPV16 positive, having inhibitory gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3, LacZ gene was used as the marker gene for the transfection efficacy. Direct transfection was done by using lipofectin (pRcCMVp53/lipofectin) and indirect transfection was done by using virus, AdCMVp53. The effect of tumor cell growth inhibition was measured by cell counting assay. RESULT: Inhibition of growth of cervical cancer cells in cell counts of direct transfection was CaSki(88.5%), SiHa(59.1%), HeLa(86.0%), HeLaS3(78.0%), C33A(91.3%) and HT3(74.0%). Inhibition of growth of cervical cancer cells in cell counts of indirect transfection was CaSki(97.4%), SiHa(91.6%), HeLa(95.8%), HeLaS3(99.7%), C33A(97.3%) and HT3(87.4%). CONCLUSION: The inhibition of cell growth of cervical cancer cells by direct and indirect transfection was significantly reduced, and showed little differences depending on the type of cells. These results will have a great meaning in treating cervical cancer patients using gene therapy by direct or indirect transfection

The Construction of bFGF/Math1 Gene Vector and Its Expression in the Cochlea of Rat / 听力学及言语疾病杂志

Objective To construct the bFGF/Math1 f usion gene expression vector and to investigate its expression in the cochlea of rat. Methods Using the recombinant DNA technique to construct the bFGF/Math1 gene expression vector and the restriction enzyme analysis to ide ntify the correct construction. The mRNA expression was detected by the RT-PCR m ethod after being transfected into the cochlea of rat using lipofectin reagent. Results The recombinant was correct and the bFGF/Math1 wa s expressed in the cochlea of rat.Conclusion The PRK5-bFGF-Math1 eukaryotic expression plas mid was successfully constructed and the bFGF/Math1 was expressed in the mammali an cochlea.

Effect of c-erbB2 antisense oligodexynucleotides on radiosensitivity of human ovarian cancer cell line / 重庆医科大学学报

Objective:To explore the effect of lipofectin-c-erbB2 antisense oligodexynucleotides on radiosensitivity of human ovarian cancer cell line.Methods:The expression of c-erbB2 was detected by means of RT-PCR;cellular response to irradiation was evaluated by MTT test and the colony forming assay.Results:Lipofectin-c-erbB2 antisense oligodexynucleotides(AS-ODN) could suppress the expression of c-erbB2,and significantly decreased the survival fraction and colony forming rate of human ovarian cancer cells after ionizing irradiation( P 0.05).Conclusion:c-erbB2 antisense oligodexynucleotides sensitize the SKOV3 to ionizing irradiation through decreasing the expression of c-erbB2,which might be the result of the fact that c-erbB2 antisense oligodexynucleotides inhibit the cellular signal transduction pathway relating to the radiation-resistant phenotype.