Recombinant Adenovirus Carrying Mouse CCR7 Gene Transfect Immature Dendritic Cells Derived from Mouse Bone Marrow
|School||Third Military Medical University|
|Keywords||Immature dendritic cells Immune tolerance Recombinant adenovirus CCR7|
Research background and purpose of clinical skin allograft large area deep burn patients with early wound coverage is the most direct and effective method of treatment, but due to strong antigenic properties of the skin, resulting in about 3 weeks after transplantation of exogenous skin occurs irreversible rejection greatly inhibit a the autologous MICROSKIN mixed-allogeneic skin transplantation effect. How successful immune tolerance induced skin grafts clinically so far found no effective measures. Dendritic cells (dendritic cell, DC) is known most powerful professional antigen-presenting cells, play an extremely important role in the immune response. DC differentiation and development process is accompanied by the DC by the differentiation of immature precursor cells to develop into mature cells, and cell morphology, surface markers, and biological functions have undergone a corresponding change. As DC immature form --- imDC (immature dendritic cell) because the most significant features in its function is can induce T lymphocyte specificity lower response current study immune tolerance strategy, which in our last also been confirmed by the National Natural Science Foundation of China (No.30271341). The imDC or genetically modified DC joint transplantation experiments have been a more satisfactory effects in organs such as the kidney, liver, heart, pancreas and small intestine, which provides a very favorable basis for skin transplantation experiments. DC maturation process associated with changes in phenotype, including reduced uptake and MHC class I molecules, costimulatory molecule upregulation, and ultimately become a powerful antigen-presenting cells. Study found imDC gradual migration of inflammatory mediators mature, its surface chemotactic factor receptor (Chemokine receptor-7, CCR7) were up-regulated. The the CCR7 ligand with MIP3β (ie CCL19) and SLC (CCL21) interactions, guided DC chemotactic migration to the lymph nodes, thereby CCR7 is considered mature antigen-presenting DC (mature DC, mDC) migration homing completed which one of the main receptor activation of naive T cells. The ability to migrate in the CCR7 chemokine been confirmed, the transfer of some malignant showed CCR7 strict regulation of tumor cell metastasis. Of this non-random, organized organ selective transfer process is similar to the dendritic cell immunostimulatory process directed migration of tumor cells. Based CCR7 has a very important chemotactic migration feature, if the higher the degree of DC maturation, CCR7 expression stronger immune system is rendered more robust immune response, whereas the induction of immune tolerance, however, imDC lack of CCR7 expression, chemotactic migration characteristics to the lymph nodes is weak, a long time in a non-T cell zones vulnerable to a variety of inflammatory cytokines or foreign antigens stimulate growth of mDC serious impact on the effect of inducing immune tolerance. Based on the above analysis, the research is intended to be funded by the National Natural Science Foundation of China, by constructing a recombinant adenovirus containing mouse CCR7, CCR7 expression try to establish in imDC make imDC efficient migration has only obtained originally mDC avoid imDC peripheral tissue differentiation to a mature state, to ensure its play to induce immune tolerance to provide new solutions and ideas for large area deep burn patients with early clinical wound coverage caused by the immune rejection. Method 1, extract total RNA from mouse thymus application of reverse transcriptase PCR (RT-PCR) method, in order to design primers with restriction sites CCR7 gene sequence after TA cloning, enzyme Qieya clone BJ5183 bacteria into the shuttle plasmid pAdTrack-CMV pAdeasy-1 homologous recombination screening positive clones, restriction enzyme digestion, linearized liposome transfected HEK293 cells for packaging, PCR and amplification to obtain containing the the CCR7 gene of recombinant adenovirus and viral titer was determined according to the reporter gene GFP. The small dose rmGM-CSF and rmIL-4 induced by cultured BALB / c mice bone marrow-derived imDC light microscope and harvested in six days; the constructed recombinant adenovirus transfection imDC According to the reporter gene green fluorescent protein (green fluorescence protein, GFP), detection of recombinant adenovirus infection efficiency; empty virus control, application of RT-PCR and immunoblotting (western blot) method to detect transfected 3 days after imDC CCR7 gene mRNA and Protein level expression changes. Results 1, was successfully constructed mouse CCR7 recombinant adenovirus vector, recombinant adenovirus titer of 1 × 109U / ml. 2, the culture of imDC loose adherent growth visible on the surface of irregular dendritic processes, the volume increase over the previous; visible adenoviral transfection cell surface stained with green fluorescence under a fluorescence microscope, was irregular burr-like protrusions . 3 recombinant adenovirus transfection imDC, the infection efficiency up to 70%, significantly higher CCR7 mRNA and protein expression compared with empty virus group. Conclusion 1 mouse CCR7 recombinant adenovirus vector was constructed successfully. Obtain a high titer of recombinant adenovirus. 2 mouse bone marrow-derived mononuclear cells obtained after the application of low-dose rmGM-CSF and rmIL-4 co-stimulation typical morphological characteristics imDC. 3, the build recombinant adenovirus infection imDC that after CCR7 mRNA and protein expression was significantly higher in imDC CCR7 expression, and once again proved correct constructed an adenovirus vector transfection efficiency, can effectively expression of the target gene, has a higher appeal. The chemotactic migration feature for further research transfection of CCR7 imDC change its skin transplantation induced immune tolerance provided the experimental basis and foundation.