Antidiabetic Effects Of PBudCE4.1/Reg3 Gene Combined With Insulin On T1DM And Its Molecular Mechanism

Objective To construct Reg3 secretary eukaryotic expression vector and investigate the effects of PBudCE4.1/Reg3 gene combined with insulin on streptozotocin (STZ)-induced type 1 diabetic mice and explore a new way for diabetes gene therapy.Methods (1) Reg3 gene fragment was extracted for the further construction of plasmid. The full-length murine Reg3 cDNA was inserted into the restriction enzyme cutting site (Xho1, Kpn1) of plasmid PBudCE4.1. Positive clones were screened in order to obtain the recombinant vector PBudCE4.1/Reg3. Then Cos-7 cells were transfected with the recombinant vector and PBudCE4.1 vector, respectively, to establish Reg3-expression and empty vector Cos-7 cell line. The positive clones were selected withβ-galactosidase staining kit, and Reg3 protein in Cos-7 cells was detected by western blot.(2) The T1DM model was established in BALB/c mice by a daily intraperitoneal injection of 40 mg/kg of STZ per mouse for 5 consecutive days, PBudCE4.1/Reg3 gene and/or insulin was i.m. and s.c. injected, respectively, into the diabetic mice once a week for consecutive 4 weeks. At the end of experiment, pancreas tissues were taken for histologic examination with HE staining. Serum insulin was estimated according to the protocol described by the manufacturer of the mouse insulin ELISA kit; splenocytes were subjected to the determination of the ratio of the CD4+CD25+ Foxp3+ T cell subpopulations with FACS. Besides, splenocytes were also harvested for the assay of lymphocyte proliferation with MTT method.(3) Pancreas tissues were taken for isletsβcells apoptosis evaluation by TUNEL method. The levels of cytokines IL-2, IFN-γ, TGF-βand IL-10 in serum were estimated by ELISA kit. Pancreas tissues were also taken out to investigate the protein expression of Reg3, NF-κB, BcL-2 and p-Akt1 using wetern blot analysis. Results (1) It was shown that the recombinant vector contained Reg3 gene fragment by the agarose gel electrophoresis, and inserted into PBudCE4.1 without point and frameshift mutations by sequencing. PBudCE4.1/Reg3 gene was transfected into Cos-7 cell successfully. Western blot revealed Reg3 protein release into the culture medium, and expressed steadily in Cos-7 cell lines.(2) Multiple low dose of STZ in BALB/c mice could establish a stable model of T1DM. No lymphocyte infiltration in the islets of the pancreas and no obviousβcell destruction could be found in the pancreatic tissues from mice received the combination treatment.Moreover, administration of Reg3 combined with insulin tended to bring serum insulin to normal values compared with the diabetic control mice. In comparison to the diabetic model, the level of blood glucose in mice treated with Reg3 combined with insulin was significantly lower (P<0.01), and lasted stably after the treatment withdrawal. A depressed lymphocyte proliferation and higher ratio of the CD4+CD25+ Foxp3+ T cell subpopulations were demonstrated in the Reg3/insulin treated mice as compared with those in the diabetic control ones (P < 0.01 and P < 0.05, respectively).(3) TUNEL assay displayed that, no obviousβcell apoptosis in the pancreatic island were observed in the Reg3/ insulin treatment group. The increase of TGF-βand IL-10 extent as well as the depression of IL-2, IFN-γwas found in the STZ-diabetic mice. Reg3 combined with insulin treatment could improve the anomalous cytokines extent significantly. Meanwhile, western blot analysis was used to investigate the protein expression of Reg3, NF-κB, BcL-2 and p-Akt1. It was found that the expression of Reg3 protein increased significantly in Reg3/insulin treated mice (P<0.05). Reg3 could effectively activate the Akt pathway, in order to promote NF-κB nuclear translocation, and increase anti-apoptotic BcL-2 gene expression.Conclusions Reg3 gene combined with insulin prevents mice from developing hyperglycemia, possibly by inducing immunotolerance, which inhibiting immune response, reducing infiltration of inflammatory cells; and enhancing regeneration-related protein expression, improvingβcell function of the diabetic mice. They played synergistic effect on the prevention or treatment of T1DM.