Construction And Therapeutic Effects Of The Attenuated Salmonella Typhimurium Strain Carrying KGF Gene (SPK)

Keratinocyte growth factor (KGF) could mediate proliferation, migration and differentiation of various kinds of epithelial cells. As a result, KGF has been used to treat many diseases. However, the recombinant protein of KGF is limited for clinical application because of its short half-life, high cost and adverse side effects resulting from large dose administration. Therefore, we focused on the gene therapy of KGF mediated by the attenuated Salmonella typhimurium.In this study, KGF gene was cloned into a eukaryotic expression vector and then the recombinant vector was transducted into attenuated Salmonella typhimurium by electrotansfection method. The positive strain carrying KGF gene was identified and was designated to Salmonella-plasmid-KGF strain, abbreviated to SPK strain. Then the therapeutic effects of SPK were evaluated in three intractable disease modelsCombined radiation-wound injuries occurring in clinic, are the chronic and refractory wound, and there are still no effective treatments at present. The biological function of SPK strain was verified firstly in vitro by transfection of the HaCaT cells originating the human keratinocytes. The results demonstrated that KGF gene could be expressed effectively and the proliferation of HaCaT cells was significantly promoted. Then combined radiation-wound injuries were treated with SPK stain by local subcutaneous injection around the wounds in rats. The inflammatory response in the wound was inhibited obviously by SPK strain administration that accelerated the wound healing rate resuling from angiogenesis and formation of granulation tissues.Radiation therapy of lung cancer may induce serious side effects including radiative pneumonia and pulmonary fibrosis that not only interfere in curative effect but also endanger the life of patient. The treatment of radioactive pneumonia with administration of SPK strain by intratracheal instillation was evaluated in rats. The results showed that administration of SPK strain could significantly ameliorate X-ray induced lung injury characterized by inhibitition of inflammatory response and decrease of the total number of cells in lung lavage fluid. Furthermore, the accumulation of reactive oxygen species (ROS), such as malondialdehyde (MDA), inducing lung injury was suppressed by the increase of superoxide dismutase (SOD) activity. SPK strain could mediate the expression of some cytokines to maintain the normal physiological function of lung.Ulcerative colitis is an inflammatory bowel disease with high morbidity and difficult cure. There are still no effective drugs for the desease at present since the exact pathogeny remains elusive. The effects of oral administration of SPK stain on acetic acid induced ulcerative colitis in rats were investigated. The results demonstrated that SPK strain treatment could significantly ameliorate symptoms of ulcerative colitis. The inflammatory response and colonic injury were obviously inhibited in the rats administrated with SPK strain with the decrease of tumor necrosis factor-α(TNF-α) expression and ROS induced injury in damaged colonic tissues. Moreover, the proliferation of intestinal epithelial cells and healing process of injured colonic tissues were attributed to the increase of KGF and its receptor (KGFR) expression.Results of the three animal models demonstrated that SPK could transfect epithelial cells effectively, and promote proliferation and migration, antioxidation and anti-inflammatory response of epithelial cells; mediate pathological process and repair damaged tissues. SPK may become a novel and effective therapy of the damage of epithelial tissues.