【作者】 张驰；

【导师】 李校堃；

【作者基本信息】 吉林大学 ， 医学基因组学， 2009， 博士

【摘要】 糖尿病(diabetes mellitus,DM)是一种慢性代谢性疾病,能够引起多组织器官的损伤甚至功能衰竭。糖尿病已成为威胁人类生命健康的三大危险性疾病之一。DM的致病机理十分复杂,目前尚无明确定论,但随着研究的深入,越来越多的证据显示,氧化应激与DM及其并发症的关系密切,可能是导致DM及其并发症的主要原因之一。持续高血糖状态导致机体代谢紊乱,氧自由基产生和清除机制遭到破坏,过量产生的氧自由基由于无法及时得到清除而堆积在脏器细胞中造成氧化损伤,其损伤部位组织上皮细胞能通过多种辅助因子与外周血单核细胞发生粘附而介导炎症反应,而炎症反应也是DM并发症的主要特征。与高剂量电离辐射不同,低剂量辐射(low dose radiation,LDR)能诱导生物适应性反应,有助于提高机体的免疫力。近些年来,多项研究结论表明,LDR对DM有显著的预防和治疗作用,具有较强的抗氧化作用;另一方面,LDR还能通过多种途径有效降低炎症反应的强度。本研究在上述研究基础上,对LDR通过降低氧化应激引起的炎症反应而治疗DM及其并发症进行深入的研究,并探讨其作用机制。本研究采用了多种分子生物学研究方法,包括RT-PCR、Western-blot、酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)、免疫组化(immunohistochemistry,IHC)等,从基因、蛋白质和组织不同层面研究了LDR在不同时间点(2、4、8、12、16周)对不同脏器(心脏、肾脏、血管)、不同炎症因子(ICAM-1、IL-18、CRP、MCP-1、TNF-α)表达以及不同生理生化指标的影响。结果显示,LDR能有效降低链脲佐菌素(STZ)诱导的糖尿病小鼠血糖水平,延缓糖尿病及其并发症对组织的损伤;并在很大程度上,抑制了糖尿病小鼠不同炎症因子在不同脏器的表达。本研究的初步结论,印证了研究目的的设想,即低剂量电离辐射可以通过降低炎症反应而治疗糖尿病,今后尚需对LDR降低糖尿病炎症反应的分子机制进行深入的探索。更多还原

【Abstract】 Diabetes is one of three complicated ascites diseases in clinic at present.It epidemics all around the world and becomes international public health problem which threatens our health.Diabetes is kind of metabolic disorder disease including glucose,adipose and protein cuaused by many reasons.Long time hyperglycemia can lead to multiple system and organs damage and dysfuction.During this process, oxygen free radical plays a very important role.More and more evidences demonstrated that the hyperglycemia,hyperlipidemia and following oxidative stress are all key factors to the development of diabetic complications.The hyperglycemia can lead to the destruction of dynamic balance mechanisim on the ROS redox.The excess oxygen free radical which can not be removed deposites in cells and induces oxidative stress and the following oxidative damage.The oxidative damage always developed accompanied with inflammation.The epithelial cells of the damaged tissue caused by oxidative stress can recruit the peripheral blood mononuclear cells via the adhesion activity of different adhesive factors from both of the cells.During the inflammation process,many kinds of inflammatory factors produced by different cells formed a complicant interaction network system.On the other hand,the increased inflammatory factors stimulated the neutrophil to release more oxygen free radical to agrassive the oxidate damage.Contrast to the injuring activity of high dose ionizing radiation,the low dose radiation can induce adaptive response including improving immunity and reproductive ability,prolonging life span and preventing radiational and chemical damage.Recently there are several possible mechanism for low dose radiation induced hormesis:①Low dose radiation can improve DNA repair;②Low dose radiation can mediate the expression of many kind of protein;③Low dose radiation can mediate many kind of cell signaling pathways;④Low dose radiation can induce the expression of many kind of antioxidant enzyme;⑤Low dose radiation can mediate apoptosis.The low dose radiation induced hormesis also paly a significant role in diabetes. A large number of study show that the low dose radiation efficiently prevent and treat the development of diabetes and diabetic complications.Low dose radiation can notablely decrease the glucose level,increase insulin level,improve the survival rate ofβcells,upregulate the antioxidant enzymes and FGFs in diabetic rat.Along with deepening of the research on hormesis of low dose radiation,many evidences showed that the low dose radiation can treat many kind of benign diseases, Especially for acute and chronic inflammation with 4 possible mechamism:①Low dose radiation can supress endothelial cell adhesion efficiently.②Low dose radiation can decrease the expression of endothelial cells-derived cytokines.③Low dose radiation can stimulate the apoptosis of peripheral blood mononuclear cells surounding the damage location.④Low dose radiation can decrease macrophage and granulocyte-derived iNOS.Several kind of molecular biological techniques were used in the present study to examine the effects of low dose irradiation on the expression of inflammatory factors in diabatic mice form gene,protein and tissue level.1.Establishment of T1DM mice modelMale C57BL/6J mice,10 weeks of age(18-22 g of body weight),animals were randomly divided into two groups,streptozotocin(STZ;Sigma Chemical,St.Louis, MO) treated group and the vehicle(0.05 M sodium citrate,pH4.5).Both group of the mice fasted overnight before the injection.Mice of the STZ treated group were injected i.p.with STZ(60 mg/kg body wt) for 6 consecutive days.The STZ was dissolved in 10 mmol/L citrate buffer(pH 4.5) and injected within 5 min of dissolution.Control animals received equivalent value of the citrate buffer solution (pH 4.5).The blood glucose level was determined using a freestyle glucometer (Freestyle,USA) a week after the last injection of STZ.We considered mice to be diabetic when blood glucose was equal to or more than 12 mmol/L for 2 weeks.Both mice of DM group and age-matched control group were randomly divided into 2 groups respectively again.One group was exposed to multiple LDR and the other group was sham for exposure.The mice of DM/LDR group and LDR group were given LDR from a Philip deep therapy apparatus(Model X.S.S.205 FZ) with 200 kvp and 10 mA using 0.5mm Cu and 1.0 Al as filter.We selected 25 mGy for one time dose of irradiation.The distance between X-ray source and animals was 212 cm with a dose rate of 12.5 mGy/min.The LDR was given every other day at the same time. There were 5 checkpoints(2 weeks,4 weeks,8 weeks,12 weeks and 16weeks) for the effect of the LDR.2.Effects of LDR on the body weightThe pre-LDR body weight of each group are approximately equal.After 2 weeks low dose radiation,the body weight of DM and DM/LDR groups were significantly higher than LDR and Control gorups.Aong with the time passing,the body weight was increasing in each group.Compare with the non-diabetic group,the diabetic group body weight increaed more significant.There is no significant differernce between DM And DM/LDR group(P＞0.05).3.Effects of LDR on the blood glucose levelAfter diabetic model established,the blood glucose of DM and DM/LDR group were both beyond 12 mol/L,and there is no significant differernt between the two groups.The blood glucose level of LDR and Control group stayed in the normal range. The blood glucose level of DM increased quickly at 2 weeks.However,the blood glucose level of DM/LDR was significantly supressed,which showed the blood glucose in diabetic mice was sensitive to low dose radiation stimulation.Moreover the significant difference was kept to the 16weeks which showed the long effectiveness of low dose radiation to blood glucose in diabetic mice.4.Effects of low dose radiation to the renal function parameters The part of study was focusing on two parameter of renal function,and the result showed that there were significant differernce of Cre and MALB between diabetic and non-diabetic group(P＜0.05) which implied the renal dysfunction in diabetic group.After treated by low dose radiation,the content of Cre and Malb in urea were significantly changed compared with the low dose radiation-free group.And the difference was increased continuously demonstrated that low dose radiation played a key role in renal function protection and delayed the renal dysfunction.5.Effects of LDR on the concentration of inflammatroy factors in serumThe result showed the content of TNF-αand MCP-1 in serum of diabtic group was significantly higher than non-diabatic group(P＜0.05),but there is no significantly differernce between DM and DM/LDR group as well as the Control group and LDR group(P＜0.05).After 2 weeks low dose radiation,the content of the two factors increased significantly compared with Control group.Contrast to DM group,the content of TNF-αand MCP-1 were significantly supressed in DM/LDR group(P＜0.05) which showed that low dose radiation could siginificantly decreased the content of TNF-αand MCP-1 with the characteristics of rapid available.Moreover, the supressed function were still significant at 16 weeks,which showed the inhibitory effect was long effectiveness.In additon,There is no significantly difference of IL-18 and ICAM-1 in serum between diabetic and non-diabetic group(P＞0.05) before treated by low dose radiation the same as treated for 2 weeks by low dose radiation.After treated by low dose radiaton for 4 weeks,the IL-18 content of each group increased in different degree except Control group.The IL-18 content of DM group was significantly higher than DM/LDR group which showed low dose radiation can decrease the IL-18 content of diabetic mice in serum.But different phnomenon appeared in non-diabetic group,the content of TNF-α,MCP-1 and IL-18 in serum of LDR group is higher than Control group.After treated by low dose radiation for 4 weeks,there was significantly difference of the ICAM-1 content in serum between diabetic and non-diabetic group(P＜0.05).Along with the time passing,the differernt increased continuously.Contrast to other inflammatory factors,there is no significant difference of ICAM-1 content between DM and DM/LDR,LDR and Control(P＞0.05).the result showed low dose radiation had no effect on the ICAM-1 level in diabetic mice.6.Eeffect of LDR on the morphology of diabetic organKidney:HE staining,PAS staining and Masson’s trichrome were applied in the part of study.The result showed that the structure of glomerular and Renal Tubular were normal.In the kidney of diabetic mice,however,there were increased glomerular size, mesangial cell proliferation,mesangial matrix expansion,capillary wall thickness and capillary collapse,and the renal tubule dilation,epithelial cells degeneration and widened interstitium were found,all which were more evident for the group of diabetes at 8 to 16 weeks.Compared with diabetic mice,in the kidneys of diabetic mice with DM/LDR,although the pathological changes were also noticeable,these changes were significantly relieved.Heart:HE staining was applied in the part of study.The result showed that themyocardial fibers were tininess in Control and LDR groups.In DM group,the myocardial fiber were obviously not tininess,the intercelluar matrix was expanded. The edema appeared in the area of intercelluar matrix.In DM/LDR group,most of the myocardial fibers were tidiness,the intercelluar matrix area expandance is not obvious.7.Effects of LDR on the expression of inflammatory factors in heart,kidney and vascularKidney:The result showed the tendency of the change for expression of mRNA level and protein were coincident of each inflammatory factor in kidney.After treated by low dose radiation for 2 weeks,the expression of these inflammatory factors at mRNA level and protein level from one group was different from others group.The expression level of DM group was higher than others gorup(P＜0.05).For TNF-αthe expression level of DM/LDR is also significantly higher than non-diabetic groups,but significantly lower than in DM group.In another hand,there is no significant difference between DM/LDR group and non-diabetic groups for IL-18 and ICAM-1, but lower than LDR groups for MCP-1 and PAI-1.Along with the time passing,these kind of inflammatory factors increased continuously both in DM and DM/LDR group, but the significant difference between the 2 groups appeared at differernt check point. The expression of LDR group stayed at a approximate stable level but significantly higher than Control.Heart:The result showed the tendency of the change for expression of mRNA level and protein were coincident of each inflammatory factor in heart.After treated by low dose radiation for 2 weeks,there is no significant difference on the expression level of IL-18,ICAM-1 of 4 groups.But the expression of TNF-α,MCP-1 and PAI-1 at mRNA level and protein level from one group was different from others group.The expression level of DM group is significantly higher than other groups(P＜0.05). Along with the time passing,these kind of inflammatory factors(without TNF-α) increased continuously both in DM and DM/LDR group,but there was no noticeable change in LDR and Control group.So the significant difference increased between diabetic and non-diabetic groups.The TNF-αtime-history curve with zigzag shape may due to the mediation of low dose radiation on the expression of TNF-α.but the significant difference between the DM/LDR and non-diabetic groups appeared at differernt check point.Compared with DM group,the expression of IL-18.MCP-1 and PAI-1 of DM/LDR were supressed significantly.However,there is no significant diference between DM and DM/LDR at each time point,but the oppsite phenomenon were detected in LDR and Control group.vascellum:The result showed the tendency of the change for expression of mRNA level and protein were coincident of each inflammatory factor in vascellum.The expression of inflammatory factors at mRNA level and protein level from one group was different from others group.The expression of IL-18,TNF-αand MCP-1 in DM group is higher than other groups(P＜0.05).And the expression of IL-18 and TNF-αin DM/LDR were also higher than Control group(P＜0.05),different form MCP-1 and PAI-1 which expression level of DM/LDR group approximated to Control group (P＞0.05).But at the time point,there are no significant difference on the expression of ICAM-1 among each group.Along with the time passing.These kind of inflammatory factors(without TNF-α) increased continuously both in DM and DM/LDR group.The significant difference was detected between DM/LDR and non-diabetic gorup at different time point(P＜0.05).Comparing the expression of these inflammatory factors between DM and DM/LDR group at different check point, the result showed the low dose radiation can supress the expression of these inflammatory factors at different time.However,the expression of these inflammatory factors in LDR group was significantly higher than in Control group.The phenomenon showed the low dose radiation can paly different roles in diabetic and non-diabetic mice.For ICAM-1,there is no significant differernce between DM and DM/LDR at any check point(P＞0.05).The total results of the study demonstrated that low dose radiation can supress the blood glucose level of diabetic mice efficiently,mediate the content of the renal function parameters,significantly delay the damage of diabetes to the heart,kidney and vascular of diabetic mice.In additon,low dose radiation can supress the expression of several kind of inflammatory factors in serum and main organs,which can play different role in non-diabetic mice.更多还原