Study On The Amelioration Of Type 1 Diabetes Mellitus By A Naked DNA Plasmid Encoding Proinsulin And Pancreatic RegeneratingⅢProtein

Type 1 diabetes mellitus (TIDM), which also be named as insulin dependence diabetes, is an autoimmune disease mediated by auto-reactive T cells and characterized by selective destructions of insulin-producingβcells together with absolute insulin defency. At the begnning of T1DM, the activated auto-reactive T lymphocytes and macrophages infiltrated into the pancreatic islets. They released the inflammatory mediators like Th1 cytokines and NO which induced the apoptosis and necrosis ofβcells, and damaged the insulin-secreting cells, then caused the hyperglycemia and other complications of T1DM. Currently, the therapeutic options used for treating T1DM have several limitations and alternative therapies for this devastating disease are urgently needed to be explored. With the continous development of molecular biology techniques, gene therapy becomes a very promising approach for the treatment of diseases, and a new field has been opend up for the treating of T1DM.Proinsulin has been identified as a unique auto-antigen which triggers autoimmune diabetes based on its highly restricted expression in the pancreaticβcells. It plays a very important role in the process ofβcell damages and acts as a vital antigen to induce immunological tolerance. Pancreatic regeneratingⅢ(RegⅢ) gene is an important gene involved'n the regeneration or growth ofβcells.In this study, we chosed these two vital genes (proinsulin and RegⅢgene) which play important role in the different stages of T1DM as target gene, and inserted the cDNAs of them into the multiple cloning sites of pBudCE4.1, thus, a plasmid encoding RegⅢprotein and proinsulin was constructed. Then, we observed the expressions of RegⅢprotein and proinuslin in the cos-7 or NIT-1 cells transfected with pReg/PI, and its effects on NIT-1 cells were studied by series in vitro experiments. Type 1 diabetic mice model was established by multiple intraperitoneal injection of low dose streptozocin (STZ). pReg/PI plasmid was intramuscular injected into the mice, its therapertic effects and underlying mechanisms were investigated in this study.Firstly, we isolated the cDNA of proinsulin from the genome of Balb/c mice by RT-PCR, and acquired the RegⅢcDNA by the digestion of pCMV-RegⅢplasmid with two restriction enzymes. The two cDNAs were inserted into the multiple cloning sites of pBudCE4.1, thus, a recombination plasmid encoding RegⅢand proinsulin (pReg/PI) was constructed. This plasmid was identified by restriction enzyme digestion and gene sequencing, the results showed that the orientation of the inserted cDNAs was correct and no point mutation was found in the cDNA sequences.Secondly, we transfected cos-7 cells or NIT-1 cells with pReg/PI plasmid by Lipofectamine 2000, and assessed the expressions of RegⅢprotein and proinsulin in the cells by western blot. The results showed that pReg/PI plasmid could be expressed effeciently in these cells. Additionally, we investigated the effects of pReg/PI on the proliferation of NIT-1 cells, and apoptosis induced by high glucose or cytotoxic T lymphocytes. We found that transfection with pReg/PI could promote the proliferation of NIT-1 cells, and inhibit the apoptosis induced by cytotoxic T lymphocytes. However, pReg/PI plasmid exhibited no significant amelioration of the apotosis induced by high glucose in NIT-1 cells.Finally, we established the animal model of T1DM by multiple intraperitoneal injections of low dose STZ and the pReg/PI plasmid was given to the T1DM mice by intramuscular injection for 5 weeks. We monitored the blood glucose levels of the mice and observed that treatment with pReg/PI could ameliorate the symptoms of T1DM and delayed the onset of T1DM. To investigated the underlying mechanisms of pReg/PI, we assessed the insulitis in the pancreas of the mice by histopathology analysis, apoptosis ofβcells in the pancreatic islets with Tunel assay, the proliferation of splenic lymphocytes by MTT, the differentiation of CD4+CD25+Foxp3+ T regulatory cells by FACS, the contents of insulin and Thl or Th2 cytokines in the serum by ELISA assay. We found that intramuscular delivery of pReg/PI could increase insulin contents in the serum of T1DM mice model induced by STZ, restore the balance of Thl/Th2 cytokines and expanded CD4+CD25+Foxp3+ T regulatory cells, which may attribute to the establishment of self-immune tolerance. Additionally, in comparison to the mice treated with empty vector pBudCE4.1 (pBud), attenuated insulitis and apoptosis achieved by inhibiting activation of NF-κB in the pancreas of pReg/PI treated mice were observed.In summary, pReg/PI plasmid could be efficiently expressed in the mammaliam cells, promote the proliferation of NIT-1 cells and inhibit the apoptosis induced by immunological related factors in vitro. It also can delay the onset of T1DM induced by multiple low doses of STZ, which was associated with not only the reconstruction of immunological self-tolerance but also regeneration ofβcells in diabetic mice.This study is the first time to combine the treatments aimed at suppression of autoimmunity and expansion ofβcells by gene recombinant technology, and it provids theoretical basis for the exploring of new theapy approaches for T1DM.