The Effects Of Combination Therapy Of EGF And Gastrin On Pancreanic Islet PDX-1 Expression In Experimental Type Ⅰ Diabetic Rat

Objective: Exploring the associated effects of EGF/Gastrin of the PDX-1 expression on rats with type I diabetes and discuss the probably mechanism of the neogenesis synthesis of pancrease by EGF/Gastrin combination.Methods: Selecting 45 clear health male rats and breeded them for 1 week to make them get used to the environment.After this,the rats were fasted without food but water for 12h.Blood sampling the blood glucose(BG) of the rats through their tail vein and weighing them.The rats were devided into 2 groups(15 for control group and 30 for experimental group).The experimental group were treated with STZ-natrium citricum buffer(55mg.kg-1) once by peritoneal injection and the control group were treated with citricum buffer(PH4.0) by peritoneal injection.On the 7th day after the STZ injection,the BG of the experimental group are measured(BG>16.65mmol.L-1 is the diagnosis of a diabetes rat model).Counting the modeling rate and death rate to evaluate the result of diabetes model establishment.The vestigial rats of the experimental group were then be divided randomly into 2 group(14 for Diabetes Group and 14 for EGF/Gastrin Association Group).The Control group and the Diabetes Group were subcutaneous injected with PH8.0Tris-HCl buffer respectively while the EGF/Gastrin Association Group were subcutaneous injected with EGF(1μg.kg-1)/Gastrin(3μg.kg-1)once a day for 14 days.Observe the foraging,drinking and emiction activity of the rats and measure their weights and urine glucose (UGlu) during these 14 days.After the last injection,the rats of the EGF/Gastrin group were fasted without food but water for 12h and their BG were tested.Later on the rats were executed and blood serum were abstracted from the abdominal aorta and preserved in -20℃.The amount of insulin and C-Peptide were tested by radio-immunity to appraise the function of pancrease.The pancrease tissue of the rats in each group were separated immediately after the execution and washed in cold normal saline.Connective tissue of the pancrease were flush-trimmed and the rest tissue were preserved in liquid nitrogen and kept in -80℃ultra cold freezer.The pancrease were homogenated at the proceeding of testing the content of Insulin mRNA and PDX-1 mRNA by Real-time Fluorescent Quantitative PCR and the rest parts were formalin fixed and paraffin imbeded for histology examination.The morphology changes were observed with HE stain while the expression of Insulin and PDX-1 were staimed with immunal histochemistry methods,and the contents of Insulin mRNA and PDX-1 mRNA by Real-time Fluorescent QuantitativePCR.The amplification curve and melting curve of Real Time One Step qRT-PCR were drawed with the data above.Results:1)The result of establishment of type I diabetes rat model:26 of 30 rats reached the stardards of type I diabetes(BG>16.65 mmol.L-1) after the injection of STZ on the 7th day(achievement ratio was 87%) with no rats died during these days.2)The status of the rats:the weights of the rats in the Control Group increased significantly while the weights of the rats in the Experimental Group showed a decrease.The status of the EGF/Gastrin Group is similar to the Control Group except one or two rats showed an maransis.The UGlu of each group were all negative at the beginning time,but UGlu of the Diabetes group increased to +++ 48h after the injection of STZ and remained stable(+++~++++) till the end.The UGlu of the EGF/Gastrin group turned to negative at the end of the experiment.3)Blood-fasting sugar change:After the experiment,the average BG of the Control Group is 4.78±0.49mmol.L-1,the average BG of the Diabetes Group is 23.57±3.69mmol.L-1 and the average BG of the EGF/Gastrin Group is 13.43±6.38mmol.L-1.The data showed the BG in the Diabetes group was much higher than the Control Group(P<0.01)while the BG of the EGF/Gastrin group was much lower than the Diabetes Group(P<0.05)but still higer than the Control Group.4)Changes of the contents of the insulin and C-Peptide in the serum:the contents of the insulin and C-Peptide of the Control group were 12.46±1.12(uIU.ml-1)and 1.07±0.15(ng.ml-1),the contents of the insulin and C-Peptide of the Diabetes group were 3.21±0.62(uIU.ml-1),0.26±0.18(ng.ml-1)while the contents of the insulin and C-Peptide of the EGF/Gastrin group were 7.65±1.81(uIU.ml-1)and 0.77±0.21(ng.ml-1)The data showed the BG in the Diabetes group much lower than the Control Group(P<0.01)while the the contents of the insulin and C-Peptide of the EGF/Gastrin group was much higher than the Diabetes Group(P<0.05)but still lower than the Control Group.5)HE stain:most of the cells in the Control Group were round or oval shape and distributed evenly in the pancreatic acinus.The structures of the pancrese islets in the Diabetes Group were clear.The pancreatic cells were abundant in plasma and the nuclears were round,the amount ofβcell in this group was obviously less than the Control Group with the apperance of vacuolar degeneration and lymph cytes infusion.The amount ofβcell in the EGF/Gastrin group was much more than the Diabetes Group and the pancrese islets in the this group were also clear and with no capsule.6)Immunohistochemistry stain of insulin:Plasma of positive insulinβcell showed brown.Positive cells in the Control Group were distributed evenly in the pancrease while the positive cells in the Diabetes Group were much less and stain intensity were much weaker.Positive cells in the EGF/Gastrin group were much more than the Diabetes Group but still less than the Control Group.The percentage(β/T area,%) of the insulin stained area in the Control Group was (78.39±3.79)%;The percentage(β/T area,%) of the insulin stained area in the Diabetes Group was (15.51±6.09)% while the percentage(β/T area,%) of the insulin stained area in the EGF/Gastrin Group was (51.65±12.79) %.The percentage of the insulin stained area in the Diabetes Group was much lower than the Control Group(P<0.001) ;while the percentage of the insulin stained area in the EGF/Gastrin Group was higher than the Diabetes Group,but it was still lower than the Control Group(P<0.001).7)Immunohistochemistry stain of PDX-1:PDX-1 were expressed both in the plasma and nuclear of the pancrease cell.Positive cells in the Control Group were distributed evenly in the pancrease while the content of the positive cell was much less in the Diabetes Group.Positive cells in the EGF/Gastrin group were much more than the Diabetes Group but still less than the Control Group. The percentage(β/T area,%) of the PDX-1 stained area in the Control Group was (76.44±2.42)%;The percentage(β/T area,%) of the PDX-1 stained area in the Diabetes Group was (24.62±13.75)% while the percentage (β/T area , %)of the PDX-1 stained area in the EGF/Gastrin Group was (64.30±6.85)%.The percentage of the PDX-1 stained area in the Diabetes Group was much lower than the Control Group(P<0.001); while the percentage of the PDX-1 stained area in the EGF/Gastrin Group was higher than the Diabetes Group(P<0.001),but it was still lower than the Control Group.8)Immunohistochemistry stain of insulin: Positive cells in the Control Group were distributed evenly in the pancrease.There were almostly no insulin positive cells in the Diabetes Group while the survival insulin positive cells expressed in a low intensity.In contrast with the Control Group,the insulin positive cells in the EGF/Gastrin group were much more and expressed in a high intensity but still less than the Control Group.9)Immunofluorescent stain:PDX-1 were expressed both in the plasma and nuclear of the pancrease cell and mainly in the cytoplasm but occasionally expressed in the nuclear.Positive cells in the Control Group were distributed evenly in the pancrease while there were only a few PDX-1 expression in the Diabetes Group.PDX-1 positive cells showed in the duct epithelial cells and around the pancrease in the EGF/Gastrin group.10)Real time One Step qRT-PCR:①abstraction,quantitation and Integrity test of total RNA:OD260/OD280>1.8,the RNA extract is 1% agarose gel for electrophoresis.18s and 28s stripes showed under the uviol lamp proved the mRNA in the total DNA of pancrease tissue was complete and with no lysis.②Test of the content of Insulin mRNA and PDX-1 mRNA by Real-time Fluorescent Quantitative PCR in the pancrease tissue:Insulin,PDX-1 andβ-actin mRNA genes expressed in all of the 3 groups.β-actin acted as the reference in the test and the melting curve of the gene amplification showed specificities of the gene amplifications of Insulin,PDX-1 andβ-actin mRNA. The Diabetes Group was the least.Transcriptional level of Insulin mRNA in the Diabetes Group was the lowest among the three group and it was 2.4 times lower than the Control Group which was the highest one(P<0.05).Transcriptional level of Insulin mRNA in the Control Group was the most(2.1 times higher than the Control Group,P<0.05) while the Diabetes Group was the least.Transcriptional level of PDX-1 mRNA in the Diabetes Group was the lowest among the three group and it was 2.8 times lower than the Control Group which was the highest one(P<0.05).Transcriptional level of PDX-1 mRNA in the EGF/Gastrin group increased obviously and was 2.2 times higher than the Diabetes Group(P<0.05) but was still lower than the Control Group.Conclusion:①PDX-1 expression in the Diabetes Group decreased a lot while the expression of Insulin mRNA和PDX-1 mRNA also decreased.②The association of EGF/Gastrin could lower the BG of rats with diabetes and increse the contents of C-peptide andβcells to recover the functions.③The association of EGF/Gastrin could promote the expression of PDX-1 to recover the functions ofβcells and decrease the level of BG.And the increased expression of PDX-1 could promote the neogenesis ofβcells in pancrease.④Investigating the PDX-1 expression alteration in the progress of diabetes and find new drugs for regulating the expression and functions of PDX-1 is significant for illustrating the mechanism of diabetes and finding more efficient ways to cure diabetes.