이식된 췌장소도의 면역보호 효과를 위한 헴산소화효소-1를 암호화한 아데노바이러스 형질도입 최적화 연구

Abstract

Diabetes mellitus is regarded one of the leading causes of death in the world. As a treatment of type-1 diabetes mellitus, pancreatic islet transplantation is one of the most promising strategies for diabetic patients. However, graft rejection severely limits its clinical application. In the graft rejection of transplanted islets, the host’s immune cells recognize the islets as antigens, which then stimulate the immune cells to begin the cytokine secretion and proliferation of immune cells. Many physicians are using immunosuppressive medication continuously after the transplantation. These immunosuppressive drugs have significant side effects and their long-term effects are still not fully known. Researchers are also trying to find new approaches that will allow successful islet transplantation without the use of immunosuppressive drugs. Heme oxygenase-1 (HO-1) enzyme shows the rate-limiting step in the conversion of heme and it shows most critical cytoprotective mechanisms activated during cellular stress. Overexpression of HO-1 exerts beneficial effects in various transplantation models including xenotransplantation. The HO-1 system exerts four major functions- (1) antioxidant function; (2) anti-inflammatory function; (3) maintenance of microcirculation; (4) modulatory function upon the cell cycle. Therefore, this study proposes a newly designed hemeoxygenase-1encoded adenovirus (AV-HO1) gene delivery for effective immunoprotection on the xenogeneic islet transplantation. A replication defective adenoviral vector was used for successfully transfer of HO-1 gene to pancreatic islets. The capacity of AV-HO1 to infect non-dividing cells allows insertion of cDNA into pancreatic islets with potential application. Optimization process of AVHO1 gene delivery into pancreatic islet is a very important issue. Transduction into pancreatic islet is difficult because islets have an extracellular matrix. Moreover, pancreatic islet is a non-dividing cell and cultured islets exist as spheroidal clusters of about 1,000 single cells. For optimization of transduction process, intact islet was transduced with different MOI values of AV-HO1 gene. After that, adverse effects of AV-HO1 gene delivery were evaluated by conducting glucose-stimulated insulin secretion. Results showed that, transduction of AV-HO1 gene did not affect the viability and insulin secretion. When the transduced islets were transplanted into streptozotocin induced diabetic balb/c mice, the survival rate of the mice was 17.0 ± 1.31 days; however, when the untransduced islets were transplanted, they were rejected within 5.0 ± 0.48 days. Histological analysis additionally revealed the presence of viable islets after transplantation. As a result, this study demonstrated AV-HO1 gene can successfully transduced pancreatic islet. Moreover, in-vivo results showed that treatment of AV-HO1 gene delivery could significantly increase the survival time without using any immunosuppressive drugs. This ongoing study will examine the synergism effect of HO-1 gene therapy and PEG-conjugation on immune reaction after pancreatic islet xenotransplantation, without using any immunosuppressive drugs. In conclusion, the overexpression of HO-1 could enhance the beta cell function of transplanted islets and following protocol of AV-HO1 gene delivery to pancreatic islet cell will certainly be an effective therapy for the treatment of type-1 diabetes mellitus.