【作者】 朱斌；

【导师】 李平；

【作者基本信息】 北京协和医学院 ， 药理学， 2014， 博士

【摘要】 DNase I是由260个氨基酸组成的单体糖蛋白,在Ca2+存在的条件DNase I可以水解双链DNA,生成具有5’磷酸和3’羟基末端的寡核苷酸。有研究发现DNase I与细胞凋亡、个体发育及自身防御等有着密切的联系。随着研究的深入发现DNaseⅠ不仅是一种可以降解外源DNA的水解酶,同时该酶还在细胞凋亡、降解坏死细胞的染色质方面起到很大作用。在细胞凋亡过程中,死亡细胞的DNA片段会被内源性的DNase I降解形成180-200bp的DNA片段,最终完全被降解成核苷酸从而参与新的DNA的复制。除了广泛参与细胞凋亡的过程外,目前DNase I已经被发现与系统性红斑狼疮和急性心肌梗死关系密切。然而作为一种主要由胰腺所分泌的酶,DNase I与2型糖尿病之间的关系,却少有报道。在课题组前期研究过程中,我们发现DNase I在2型糖尿病患者血清中出现了明显的升高。而据报道血清中60%-65%的DNase I活性都是由胰腺所分泌的,因此我们推测血清中DNase I的升高可能与胰腺存在着密切的关系。再者由于DNase I的升高可以促进细胞的凋亡,基于此我们推测DNase I的升高可能参与了糖尿病患者胰腺的损伤。因此本研究通过体内实验与体外实验相结合,来检测DNase I在胰腺损伤过程中的重要作用。目的：观察DNase I表达升高与2型糖尿病高糖所引起的胰腺损伤之间的关系,为进一步深入了解糖尿病发病机制及其并发症的发展提供新的思路,同时为胰腺损伤的分子诊断和基因治疗提供新的科学依据。方法：1、DNase I在2型糖尿病患者血清及胰腺组织中的观察。1.1、本部分实验通过收集在我院就诊的2型糖尿病患者以及健康志愿者血清,采用辐射状酶扩散法对血清中DNase I的活性进行对比分析,来探讨DNase I在血清中的改变情况。1.2、收集本院近两年患有2型糖尿病及未患有2型糖尿病的胰腺癌患者手术切除的癌旁组织进行免疫组织化学分析,观察DNase I在胰腺组织中的定位,同时对阳性表达区域进行半定量分析。2、体内实验研究胰腺组织中DNase I的蛋白及mRNA表达变化。观察高脂饲料合并STZ诱导的2型糖尿病大鼠胰腺组织中DNase I的表达变化来探讨DNase I在高糖引起的胰腺损伤中的作用,通过免疫组织化学,Western Blot及Real-time PCR对DNase I的作用进行更加深入探讨。3、采用高糖刺激胰岛β细胞模型,验证高糖环境下DNase I表达与细胞凋亡之间的关系。通过采用高糖培养基(30mmol/L),验证糖毒性对胰岛β细胞的损伤。通过外源性给予DNase I及siRNA敲降,观察高糖环境中DNase I与胰岛β细胞损伤之间的关系。4、DNase I与糖尿病肾病损伤之间的关系探讨。本部分实验主要通过观察高脂饲料合并STZ诱导的2型糖尿病大鼠肾脏中DNase I的变化,观察DNase I与糖尿病肾病之间的关系。结果：一、临床样本结果(1)和健康对照组相比,2型糖尿病患者血清中DNase I的活性出现了明显的升高(p<0.01),且该增高的趋势在不同性别之间无显著性差异。(2)免疫组织化学结果显示,在胰腺癌不伴随糖尿病的患者胰腺组织中DNase I主要表达在胰岛细胞中,而在在胰腺癌伴随糖尿病患者的胰腺组织中,我们发现DNase I在胰岛中的表达有升高的趋势(p<0.05),同时腺泡中也出现了大量的DNaseⅠ阳性表达(p<0.001)。二、体内实验结果(1)和正常组大鼠相比,我们发现高脂合并STZ诱导的大鼠血清中DNase I的活性在第2周和第20都出现了明显的升高(p<0.05)。(2)免疫组织化学结果显示DNase I在2型糖尿病大鼠胰腺中的改变与人类胰腺组织中变化趋势较一致,和正常大鼠相比,在第2周和第20周胰岛中的DNase I均出现了明显的升高(p<0.05),同时伴随着DNase I在腺泡中出现显著性的升高(p<0.001)。(3)和正常组相比,Western Blot结果显示,糖尿病大鼠的胰腺组织中DNase I的表达在第2周和第20周中均出现了显著性的升高(p<0.05)。但是Caspase-3则仅出现了升高的趋势而无统计学意义。Bcl-2在第2周出现显著性下降(p<0.05),而在第20周两组之间则无显著性差异(p>0.05)。Realtime-PCR结果显示两组之间在第2周和第20周mRNA表达均未出现明显的改变。三、体外实验结果(1)激光共聚焦结果显示DNase I广泛存在于细胞质和细胞核中。(2)和正常对照组(11.1mmol/L)相比,高糖刺激(30mmol/L)可以明显引起INS-1细胞DNase I, Caspase-3的表达升高(p<0.05)。 DNase I敲降后则可以显著降低高糖刺激的细胞内Caspase-3的表达(p<0.05),而敲降并未对Bcl-2的蛋白表达水平造成明显改变。(3)流式细胞仪结果显示,正常培养条件下INS-1细胞的凋亡率仅为7.9%,而高糖刺激则可以明显引起细胞的凋亡,凋亡率为18.1%,敲降细胞内DNase I的表达后则可以明显减少高糖刺激后的细胞的凋亡(凋亡率为9.9%)。(4)外源性的给予胰岛β细胞5mU/ul和50mU/ul的DNase I后,采用流式细胞仪进行观察,发现正常组的凋亡率为7.2%,而5mU/ul DNaseⅠ组的凋亡率为6.9%,50mU/ul DNase I组的凋亡率为9.8%；予高糖刺激后在再进行相应浓度的DNase Ⅰ刺激,我们发现高糖组的凋亡率为17.3%,高糖+5mU/ul DNaseⅠ组的凋亡率为21.8%,高糖+50mU/ul DNaseⅠ组的凋亡率为34.6%。并且我们也通过TUNEL实验对该部分的实验结果进行了验证,并且得到了较为一致的结果。四、大鼠肾脏DNaseⅠ表达和正常组相比,20周时模型组大鼠肾脏基底膜及足突出现明显的损伤；PAS结果显示肾小球中出现了明显的糖原沉积。Western Blot结果显示在第2周和第20周时大鼠肾脏中DNaseⅠ的表达水平出现了明显的升高。Bcl-2和Caspase-3的变化趋势同胰腺组织中的变化趋势。结论：(1)2型糖尿病患者伴随着血清中DNase I活性明显升高,同时在高脂合并SZT诱导2型糖尿病大鼠血清中,也发现了血清中DNase I的活性出现显著性升高,我们推测DNase I可能参与了糖尿病的发病过程。(2)高糖刺激可以引起胰岛β细胞内DNase I的蛋白表达明显的升高,同时伴随着细胞凋亡率的显著增加；而敲降细胞内DNase I的表达水平后可以明显降低细胞的凋亡率；在体内实验中也发现高血糖可以导致胰腺和肾脏组织中DNase I的显著性升高,由此我们推测)Nase I可能参与了2型糖尿病胰岛β细胞的凋亡过程,同时DNase I的升高可能也参与糖尿病肾病的发病过程。本研究首次发现在2型糖尿病患者血清中DNase I活性出现明显的升高。同时我们也第一次发现在2型糖尿病患者和大鼠的胰腺腺泡组织中,DNase I的表达出现了极显著的升高；通过体外实验证实了高糖刺激可以导致胰腺组织内DNase I的表达明显上调,从而介导了高糖引起的胰腺细胞的凋亡的发生。通过本项研究可以为2型糖尿病胰腺损伤的预防及2型糖尿病的发病机制提供新的思路。更多还原

【Abstract】 Type2diabetes mellitus is a complex metabolic disorder that afflicts hundreds of millions of people worldwide..It is estimated that there are about7million people diagnosed with diabetes each year in the world and a person dies of diabetes-related diseases every10seconds. However, the underlying mechanisms have not been elucidated. DNase I is a38-kDa glycoprotein. As one kind of Ca2+/Mg2+dependent nonrestriction nucleases, it can hydrolyze phosphodiester bonds both in single and double stranded DNA to generate single nucleotides and oligo nucleotides with5’-phospho and3’-hydroxy termini. It is physiologically a benign extracellular enzyme present ubiquitously in blood serum and body secretions. It is responsible for the degradation of the chromatin released by dead cells, which have not undergone apoptosis. In the process of cell apoptosis, the DNA of dying cells’can be degraded into180-200bp fragments and finally is completely degraded into nucleotides to participate in the new DNA replication.At present, there are two diseases-SLE (systemic lupus erythematosus) and AMI (acute myocardial infarction) known to be associated with increased levels of DNase I in serum. However, as an enzyme that is secreted by pancreas, the underline mechanisms of it with type2diabets are still in vain. Human pancreas exhibits the higher DNase I activity. It was reported that60-65%of serum DNase I was secreted by pancreas. It is still not clear whether there is some correlation between the DNase I and the diabetes. In this study, we want to explore the relationship between DNase I and T2DM using the sera and pancreas tissue both in human and rat.Objective:By using of clinical samples, animal samples and cells, we explore the relationship of DNase I increase with pancreas injuries by high glucose in type2diabetes. Through the study we hope to supply a new thought and pathogenesis reasons fot the investigation of pancreas injuries in type2diabetes. Meantime, we can provide a new molecular diagnosis marker of pancreatic injury and scientific basis for gene therapy. Methods:(1) To examine the DNase I activity in serum in patients with type2diabetes.To examine the DNase I activity in serum in patients with type2diabetes, we collected serum of patients and healthy volunteers in our hospital. The serum enzyme activity was detected by radial enzyme-diffusion method.(2) in vivo studies of DNasae I expression in pancreas.This part of the experiment mainly divided into two parts. The first part was done by collecting the the pancreas of patients with pancreatic cancer, to observe the locations of DNase I in the pancreas in human. The second part mainly through observation type2diabetes rats induced by high-fat food plus low dosage STZ injection to explore the protein expression of DNase I, Caspase-3and Bcl-2in diabetes. In this part of the experiment immunohistochemistry, Western Blot and Real-time PCR was used for the detection.(3) High glucose stimulated the beta cells proliferation and apoptosis.INS-1cells were simulated by30mmol/L glucose concentration to observe the changes of DNase I under glucose environment. Meantime, DNase I siRNA was also used to observe the Caspase-3and Bcl-2changes in the environment of DNase I knocked down. In addition, we gave the INS-1cells exogenous DNase I to explore the overexpression of DNasae I to the levels of Caspase-3and Bcl-2.(4) DNase I expression in kidneyTo observe the relationship of DNase I with diabetic nephropathy, we had observed the DNase I, Bcl-2and Caspase-3expression in kidney of type2diabetic rats.Results:(1) Compared with healthy people, the activity of serum DNase I in patients with type2diabetes showed a significantly increased (p<0.01). And this increase seems do not affected by gender. At the same time in rats serum, we also found that the activity in2weeks and20weeks had both significantly increased in diabetic group (p<0.05)(2) Immunohistochemistry results of both human and rats showed that DNase I was mainly expressed in islets. However, in type2diabetes the DNase I expressions in islet were increased. And high glucose can induced the DNase I expression in acinus increased significantly (p<0.001). Western Blot results showed that the protein level of DNase I was significantly increased in rats pancreas both in2weeks and20weeks (p<0.05). However, compared with normal group, the Bcl-2in diabetic rats in the second week dropped significantly (p<0.05), while in20weeks, no significant difference was observed. In addition, there was no significant difference of caspase in both2weeks and20weeks.(3) Cell experiments have shown that DNase Ⅰ was widely expressed in cytoplasm and nucleus. High glucose can induced DNase Ⅰ and Caspase3expression significantly increased in INS-1cells (p<0.05). Meantime, we had used DNase Ⅰ siRNA to knock down the DNase Ⅰ expression, and we had found that once DNase Ⅰ was knocked down, the cell apoptosis rate and Caspase-3level can be greatly decreased even in high glucose simulated group (p<0.05). Flow cytometry results showed that the cell apoptosis rate in Normal group was only7.9%, the high glucose group was18.1%, whereas the apoptosis rate in the knocked down group was only9.9%.(4) INS-1cells in normal group was given5mU/ul and50mU/ul of DNase Ⅰ, then we had observed the apoptosis rate was only7.2%in normal group, and6.9%in5mU/ul DNase Ⅰ group and9.8%in50mU/ul DNase Ⅰ group. In addition, we had foun that the apoptosis rate in high glucose group was17.3%,21.8%with5mU/ul DNase Ⅰ group and34.6%in50mU/ul DNase Ⅰ group.(5) Electron microscopy results showed that at20weeks, rat kidney basement membrane and podocyte can be observed with serious damage; PAS results showed that there were obviously glycogen depositions in the glomerular in diabetic rats. Further analysis found that there was a significantly increase of DNase Ⅰ expression in both2weeks and20weeks of rat kidney (p<0.05).Conclusion:(1) Patients with type2diabets showed a significantly increase of DNase Ⅰ activity in serum, while this increase was also observed in type2diabetic rats, we speculate that the DNase Ⅰ may participate in the process of diabetes and it may involved in the injuries of islets and acinus induced by high glucose.(2) High glucose stimulation can lead to DNase Ⅰ expression increased, at the same time, cell apoptosis rate were also increased significantly. However, knowed down of DNase Ⅰ can significantly alleviate the cell apoptosis rate. Base on the results, we speculated that DNase Ⅰ may participate in the process of pancreas and kidney damage caused by high glucose.This is the first study that had discoveryed the DNase Ⅰ activity was increased in serum of type2diabetic patients. Meantime, for the first time we also found that high glucose can induced the significant increase of DNase Ⅰ in pancreatic acinus; through the in vitro study, we also proofed that DNase Ⅰ increase can promote β cells apoptosis under high glucose condition. By doing this researches we can supply a new thought for the mechanism study of type2diabetes.更多还原