Experimental Studies On Immune Response To Recombined Plasmid Inserted MIP-3α Gene Driven By Irradiation

BackgroundMobidity and mortality of lung cancer, one of the most common malignant diseases,is still increasing in many countries. Although surgery, radiotherapy and chemotherapy have been developed for several decades, the prognosis for patients with lung cancer remains poor. The great developments of molecular biology and immunology provide some new methods to the treatment for lung cancer, e.g.immunotherapy and gene therapy. Up to now, gene therapy cannot be used extensively in clinic because of some crucial problems such as absence of efficient and specific method on gene transfer, and difficulty of regulating target genes. Traditional scheme of immunotherapy such as utilizing of cytokines have no good effects on lung cancer because specific anti-tumor immune response cannot be induced. American radiotherapist Weichselbaum RR suggested that radio-genetic therapy might have great anti-tumor effects. It means the combination of radiotherapy with gene therapy and is aimed at overcoming the problems and side effects, such as radiation resistance of some tumor and the toxicities to normal tissues, from the use of either radiation or gene therapy alone. In their research, TNF cDNA was linked with Egr-1 promotor. The results indicated that Egr-1 promotor could successfully induce TNF expression after the transfected cells exposed to irradiation. Other authors subsequently studied on radio-genetic therapy with Egr-1 promotor in fibrosarcoma, glioma, hepatocarcinoma and malignant melanoma and also showed promising outlook. However, radio-genetic therapy is not mature. At present, most of the target genes used in these studies were tumoricidal such as TNFα and OSM. The expression levels of these genes driven by Egr-1 were low and transient after routine radiation, and even lower in vivo than that in vitro. Target gene is the key of radio-genetic therapy since that the expression levels directly influenced the anti-tumor effects. If a gene, which can activate anti-tumor immune response, is linked to Egr-1 as target gene instead of the tumoricidal genes, the subsequent anti-tumor effect may be increased. Radiation can mediate modulation of tumor antigen-specific immunity by upregulating expression of immunomodulatory surface molecules (such as MHC, costimulatory molecules, adhesion molecules, death receptors, heat shock proteins) and secretory molecules (cytokines, inflammatory mediators) in tumor, stromal, and vascular endothelial cells. DCs play pivotal roles in the procedures of T-cell-mediated antitumor immune response. They process and present antigen to T cells and mediate antigen-specific immune responses. Macrophage inflammatory protein-3α (MIP-3α), a CC chemokine, is a potent chemoattractant for unmatrure DCs and, plays a role in the modulating function of DCs. We hypothesized that MIP-3α might be strongly induced by irradiation if MIP-3α gene could be linked to Egr-1 promotor. The consequences should be accumulation of DCs within the tumor, and the in vivo interaction of DCs with the apoptosis tumor cells/tumor antigens induced by radiation, and DC mature in the presence of cytokines such as TNF-α,and then DCs should induce immunity against the tumor with consequent suppression of tumor growth. To evaluate this hypothesis, expression vectors of MIP-3α gene driven by Egr-1 promotor were constructed in this study. The vectors were transferred into Lewis lung cancer cells and systemic administrated into implanted murine subcutaneous tumors. The expressions of MIP-3α were assessed both in vitro and in vivo. The effect of radio-genetic therapy using the vectors has been studied. Part 1 The constructions and identification of the MIP-3α expression vectors driven by Egr-1 promotorMaterial and methods: P-Egr plasmids carrying Egr-1 promotor were digested by XbaⅠand NcoⅠ. The CAT gene was cut away and the line vectors were isolated and purified from 0.6% agarose gel. The mouse MIP-3α cDNA fragments were amplified from pCMV-MIP3α plasmids by polymerase chain reaction (PCR). The products of PCR wer...