Experimental Study Of The Effects Of Combination Therapy Of EGF And Gastrin On Islet β Cell Regeneration In Diabetic Rats

Diabetes is the common and frequently-occurring diseases which make serious harm to human health. The existing treatment measures can not fundamentally solve the physical demand of insulin of diabetic patients and prevent the occurrence and development of acute and chronic complications. Therefore, the reconstruction of endogenous insulin secretion system is the research focus over the years. Application research of the stem cell has opened up a new avenues for the diabetes treatment in addition to pancreas transplantation and islet cell transplantation. The problems of immune rejection and lack of pancreas and islet sources can be resolved by stem cell therapy. However, there is no well established and defined technique of induction and differentiation. The number of the differentiated insulin-secreting cell is too small to meet the requirement of clinical treatment. Therefore, it is meaningful to find differentiation promoting factors to stimulate the regeneration of isletβ-cell. EGF and Gastrin play important role in the development of fetal pancreas and injury repair of pancreas. This hints their significance. In this study, diabetic rats model was established by multiple low-dose injection of STZ. These diabetic rats were treated with combination of EGF and Gastrin. Then effects on isletβcell regeneration were observed and its preliminary mechanism was investigated.The results were as follow:1. Establishment of diabetic rats modelDiabetic rats model was established by multiple low-dose injection of STZ in this study. The modeling result was evaluated through detecting general status of the rats, blood sugar, urine sugar, the survival rate and pathological observation. The general status showed that the rats of STZ-induced group presented polydipsia ,polyuria, polyphagia, tousle and tarnish of hair at four weeks after STZ induction. The body weight of these rats of STZ-induced group increased slower than the control group. There was no death of rats during modeling time. Blood glucose test showed that there were 33 rats presented higher glucose than 11.1 mmol·L^-1 in 35 rats. It took 94% of the total. The average blood glucose value of these 33 rats which defined to be diabetic rats was 15.6±3.01 mmol·L^-1. It was significantly higher than the control group(P <0.05). HE staining showed that vacuolar degeneration appeared in isletβ-cell. The isletβ-cell number decreased obviously. Islet became empty. Inflammatory cells such as lymphocytes and monocytes infiltration could be observed in the islet. Immunohistochemical staining of insulin showed that the number of the insulin-positive cell in the islet decreased significantly. The insulin staining intensity of the residual isletβcells also decreased compared with control group. It suggested that the islet showed obvious damage after STZ injection. Most isletβcells were destroyed and decreased in number. The insulin producing ability of the residual isletβcells also decreased. All of this demonstrated that multiple low-dose STZ injection induced rats diabetic model could simulate pathogenesis of human diabetes. It showed high modeling rate, low mortality. It would be an ideal animal model for diabetes research.2. Effects of combination therapy of EGF and Gastrin on isletβcell regenerationThe diabetic rats were randomly divided into DM group, Insulin therapy group(Insulin group) and EGF/Gastrin combined therapy group. EGF/Gastrin group was treated with EGF (1ug·kg·d^-1)and gastrin (3 ug·kg^-1·d^-1) for 14 days subcutaneously. Insulin group was treated with long-acing insulin(2U·d^-1) for 14 days. DM group and normal control group was treated with PH8.0 Tris-cl buffer. After treatment, serum insulin and C-peptide content were tested. Pancreases were fixed, embedded and sliced. Glut-2/Insulin, Ki67/Insulin and PDX-1/insulin expression were detected in pancreas by double immunofluorescence staining technique. Blood glucose value, serum insulin and C-peptide content test showed that the average blood glucose value of EGF/Gastrin group was significantly lower than that of DM group(P<0.05). The serum insulin and C-peptide average content of EGF/Gastrin group were significantly higher than that of DM group. These results suggested that EGF/Gastrin could make contribution to the restoration of islet function and production of endogenous insulin. Immunofluorescence staining showed that expression of Glut-2 and insulin in EGF/Gastrin group increased obviously compared with DM group. It suggested that EGF/Gastrin could increased theβcell mass. Ki67/Insulin staining showed that there were increased number of Ki67 positive cells in EGF/Gastrin group. They mainly located in the periphery of the islet. Most of the cells did not presented the expression Ki67 and insulin at the same time. There were only a few of Ki67 positive and insulin positive cells in the pancreas. Ki67 and insulin did not expressed in the same cell at the same time. These results hinted that EGF/Gastrin could promote the proliferation of non-βcells in islet periphery but not the residualβcells. PDX-1/Insulin staining showed that there were a lot of PDX-1 positive cells located in the periphery of islet and pancreatic duct while some cell co-expressed PDX-1 and insulin in EGF/Gastrin group. There were a very small number of PDX-1 positive cells in the DM group. These results demonstrated that EGF/Gastrin could promote the appearance of PDX-1 positive cells and promoted their differentiation to insulin secreting cells. The average content of serum insulin and C-peptide of Insulin group were significantly higher than DM group. Immunofluorescence staining results showed that Glut-2/Insulin expression increased obviously in Insulin group compared with DM group. Some PDX-1 positive cells appeared around the islet and showed potential to differentiate to insulin secreting cells. Exogenous insulin could promote the isletβcell regeneration in addition to insulin supplement and blood glucose regulation. However, the mechanism of the function of exogenous insulin treatment onβcell regeneration required further identification. Exogenous insulin might act directly on islet progenitor cells to differentiate or realize this function indirectly through blood glucose regulation.In conclusion, combination therapy of EGF and gastrin on STZ induced diabetic rats could induce the appearance of PDX-1 positive progenitor cells in pancreas and promote differentiation of these cells to insulin secreting cells. EGF/Gastrin combination therapy could promote the proliferation of non-βcells abroud islet but not the residualβcells. The number of the insulin secreting cells increased viously after therapy. At the meantime, the biochemical indicator test showed that serum insulin and C-peptide content increased significantly after therapy. This demonstrated that EGF/Gastrin combination therapy could promote the maturity of newβcells in addition to its differentiation promoting ability. Therefore, combination therapy of EGF and gastrin could promote the regeneration of diabetic rats isletβcells. This would supply its clinical application theoretically.