The Therapeutic Effect Of Intrasplenic Transplantation Of STGF-βRⅡ And IFN-γ Gene Combined Modified Hepatocytes On Mice Transplanted Liver Tumor

Tumor immunity treatment and gene treatment are major focus of current tumor research, including tumor cell gene treatment which is a hopeful approach to tumor treatment. It transfers cell gene to tumor organization and expresses it, thus the anti-tumor ability of immunity system is developed and strengthened. Experiments show that transplanted liver cells in the spleen are directionally distributed in the liver, which effectively expresses exogenous gene.Research shows that many malignant tumor cells secrete TGF-β through the form of paracrine and have an effect on stroma cells, thus helping their growth and escape from immunity monitoring system. They make tumor cells more aggressive and transferable through inducing formation of blood vessel, regulating production of ECM and proteolytic ferment, and expressing adhesion molecule. As the natural antagonist of active TGF-β, TGF-β type Ⅱ receptor ectodomain (sTGFβRⅡ) interdicts the reciprocity of TGF-β and membrane Ⅱ receptor by competitive combination with TGF-β, thus specifically interdicting biologic effect of TGF-β. Besides, IFN-γ is a cell gene which plays an important role in immune response. It can directly restrain tumor cell and induce activity of NK cell and CTL cell, so as toenhance adhesion molecule and MHC I antigen expression on the surface of tumor cells and antigen presenting cells (APCs), and make active the body's cell immunity. Considering the fact that promoting endogenous anti-tumor effect is a major part of sTGFPRII anti-tumor effect, its combination with IFN-y will be more effective than single gene method.Liver targeted gene treatment is one of the major focus in today's gene treatment. In order to discuss the potential synergistic effect of sTGFPRII and IFN-y in anti-fibrosis of liver, we will build up liver cell line BNL-sTR/ IFN-y which can steadily co-express sTGFPRII protein and IFN-y. Anti-tumor experiment in vivo shows that co-expression of sTGFpR II protein and IFN-y has synergistic effect on anti-tumor immunity, which provides an experimental gist for clinical treatment of malignant tumor.Part I Construction of sTGFpRlIand IFN-y gene combinedmodified hepatocytessTGFpR II gene were amplified from mouse liver tissur with specific primers and the sTGFPRII fragment was cloned into pcDNA3.1 eukaryotic expression vector . The recombinant, having been screened by amplicillin and identified by double digestion and sequencing , was transfected into BNL cells. The cell lysis was examined by Western Blotting for sTGFPRII expression. This assay revealed in the lysis a 20kDa(approx.) target protein which reacting with sTGFpR II specific antibodies. Later the BNL-sTR cells were infected by IFN-y/ Ad adenovirus. The supematants of infected BNL-sTR were examined by ELISA for IFN-y expression.Conclusion1. We successfully constructed a eukaryotic expression vector of sTGF(5R II;2. We successfully constructed a BNL-sTR cell line which can steadily express sTGFPRII;3. By infected with IFN-y/ Ad adenovirus, we successfully constructed a BNL-sTR/ IFN-y cell line which can steadily combined express sTGFpR II and IFN-y.Part II A preliminary study of BNL-sTR/ IFN-y cells in genetherapy of mice transplanted liver tumorTo investigate the cooperative therapeutic effect of sTGF0R II and IFN-y on tumor therapy in vivo, we introduced this cell line into transplanted liver tumor model mice via inrtasplenic transplantation.A mice persistent transplanted liver tumor model was induced by intrasplenic transplantation of CT26 cells. In the preliminary experiment, mice were transplanted with 105 CT26 cells/per mouse. After 2 weeks, some mice were sacrificed. Tumor tissue on the liver were macroscopic. Animals died in 4 to 5 weeks. After the model was esdablished, we divided the mice randomly into four groups, which were later intrasplenic transplanted with 2><106cell/mouse BNL-sTR/ IFN-y, BNL-sTR, BNL- IFN-y and BNL respectively. Untreated group and normal mice were used as controls. The resolution of liver tumor was expected through in vivo expression of the corresponding protein.ELISA of sera collected 2 weeks and 4 weeks after cell transplantation revealed that the serum sTGFpR II level increased notably in BNL-sTR/ IFN-y cells treated group and BNL- IFN-y cells treated group, compared with the other groups. The CTL activity and NK cell activity increased the most in the BNL-sTR/ IFN-y cells treated group, compared with the other groups. And the BNL-sTR/ IFN-y cells treated grouphad the longest survival period and the shortest tumor volume in all the groups. Taken the results together, we can conclude that combined expression of sTGFpR II and IFN-y reveals a cooperative anti-tumor effect in vivo.Conclusion1. We successfully established a persistent mice transplanted liver tumor mode;l2. Gene therapy by intrasplenic transplantation of BNL-sTR/ IFN-y cells can significantly reduce tumor development. This effect is more potent than those of individual sTGFpR II and IFN-y expressed cells. All the evidence above indicated that combined express of sTGFpRHand IFN-y may exhibits a combined anti-tumor effect.