Study For Safety Of Expression Of Recombinant Human Insulin Gene Plasmid In Diabetic Rats

ObjectiveTo study initially for safety of transfer of recombinant human insulin gene plasmid into diabetic rats induced by streptozotocin.IntroductionDiabetes mellitus is a dysbolism disease lacked relatively or absolutely insulin itself . A part of patients of diabetes need injection of exogenous insulin to sustain their lives. Because of lacking of physiological accommodation, some patients tend to develop hypoglycemia and chronic complications. That bring a-bout anguish and inconvenient to the patients.With the development of molecular biology, virology and gene technology, it becomes the hot spot in domain of endocrine that by using the method of gene transfer to reconstruct the non - beta cells to make them secrete insulin. Researchers have made a mighty advance and have met some problems after many experiments. Now the recombinant human insulin gene can express responding to physiological need in the cells in vitro or in diabetic rats;the vectors for gene transfer have been used in clinical research in spite of the need for improvement of safety of the expression of gene and vectors;whether the transfer of exogenous gene can induce insertion mutation or the intervention can generate side effect or not still need to be observed. Those are the important issue, which restrict the clinical application of gene therapy.The members of our study group have already constructed recombinant human insulin gene retroviral vector plasmid with three point mutation and physiological regulatory element. This plasmid has proven to be expressed long - term in culture in vitro and in the diabetic rats. But the safety of the plasmid is notclear. This study will detect the influence of intervention to organism, the safety of regulatory of gene expression and whether there is replication - competent retro virus or not.Materials and methodDiabetes is induced with streptozotocin (at a dose of 55 mg/ kg ) by intrap-eritoneal injection to male Wister rats. After hypodermic injection of triiodothy-ronine (T3, 2mg/kg) and intxaperitoneal injection of hepatocyte growth factor ( HGF, 0. 5mg/kg) , transfer the recombinant human insulin gene retroviral vector plasmid with three point mutation and physiological regulatory element to the diabetic rats by intraperitoneal injection. Fifteen days later, 24 - hours fasting test will be carried and ninety days after the transfer of the plasmid, the blood will be exerted from the abdominal aorta of the rats to detect alanine amin-otransferase (ALT) , albumin (ALB) , creatinine (Cr) and blood urea nitrogen (BUN) by biochemical auto - analyzer. Then the rats will be killed and some kind of tissues will be gathered for histopathology detection and for PCR to detect if there is presence of the replication - competent retrovirus.ResultBlood glucose of the group of the experiment have decreased obviously at the fifteen days after the transfer, there is no accident of hypoglycemia in the 24 - hours fasting test. The values of ALT, ALB, Cr, and BUN of the rats of experiment are not different compared to the rats of normal control ( P > 0. 05 ). No tumor or malignant cells were seen in the rats of the experiment group. There was no replication - competent retrovirus detected.Discussion1. The expression of the transferred recombinant human insulin gene retroviral vector plasmid implements safe regulation.The insulin gene segment, which can secrete mature insulin and can be regulated by blood glucose and insulin, is the center content of construction non - beta cell with pancreatic function. The regulation of expression is the important aspect of body tolerance. We transfer this recombinant to experimental animals , and 24 - hours fasting test show that there is no hypoglycemia induced by the unregulated secretion of gene product. That testifies the plasmid possesses the function of physiological regulation. The expression of recombinant human insulin gene retroviral vector plasmid is safe.2. The safety of the vector used in gene transferNow there is not a extremely suitable vector which can meet all requirements of gene transfer in all kinds of vectors in the world. Retroviral vector and its helper cell, as the gene transfer system, wont produce replication - competent retrovirus themselves. But homologous recombination may produce replication - competent retrovirus. We extract genome DNA from all sorts of tissues of experimental rats to detect whether there is replication - competent retrovirus by PCR. Our outcome testifies there is no replication - competent retrovirus in those rats.The integration between retrovirus and genome is random, which perhaps can induce cell death or destruction of anti - oncogene or activation of proto -oncogene then to make cell malignant change in theory, though there is no exact proof or final conclusion as yet. It is reported that X - linked combined immunodeficiency had been cured successfully by gene therapy with retrovirus vector in France. But two years later, there were two patients appearing leukemia alike disease. National institutes of health (NIH) discuss this problem and they think some other factors can cause that result and there is no evidence to prove its the issue of the retrovirus vector. So they still support those empirical studies involved in retrovirus vector. There is no development of tumor in our experiment and no malignant cells or obform nuclear cells are seen in the pathological section of all sorts of tissues in the experimental rats. However, we have to say that our samples are small and our experiment is the initial investigation about safe-3. The intervention factor is safe.We use hepatocyte as target cell and use triiodothyronine (T3) and hepato-cyte growth factor (HGF) to enhance the ratio of dividing phase and to promote integration. Can these drugs make harmful effects to body? In our experiment, the outcomes of ALT, ALB, Cr and BUN of diabetic rats injected with T3 and HGF are not different from those of animals in diabetic control group. T3 and HGF are the component of organism, and they themselves wont bring about severely side effects to organism.Conclusion1. At the fifteen days after the transfer, there is no accident of hypoglycemi-a in the 24 - hours fasting test. It indicates that the expression of the transferred recombinant human insulin gene retrovirus vector plasmid implements safe regulation.2. No tumor or malignant cells were seen in the rats of the experiment group. There is no evidence of the mutagenicity of the plasmid.3. There was no replication - competent retrovirus detected in the tissues of the animals which was transferred the recombinant human insulin gene retrovirus vector plasmid.4. There is no significant difference of functional values, including ALT, ALB, Cr and BUN, between the group of experiment and normal control. The intervention factors used to improve the expression efficacy may not influence the liver function and renal function.The application to rats of recombinant human insulin gene retroviral vector plasmid with three point mutation and physiological regulatory element constructed by us was safe.