【作者】 吴英；

【导师】 王力宁；

【作者基本信息】 中国医科大学 ， 肾内科， 2007， 硕士

【摘要】 近年来，世界范围内糖尿病（DM）的发病率明显上升。糖尿病是一组以高血糖为特征的代谢性疾病，长期高血糖状态可导致肾脏组织损伤，功能障碍和衰竭。糖尿病肾病（DN）是DM患者致残和死亡主要原因之一。据统计我国DN年发生率高达30％，而据美国肾脏资料统计，约50％的终末期肾衰（ESRD）由糖尿病所致，并呈逐年上升趋势。但目前研究尚未完全探明DN的发生发展机制，亦缺少有效的防治措施。因此，进一步探讨DN发病机制并且寻找阻止DN进展的方法已成为国内外学者研究的热点问题。目前认为糖尿病肾病的发病过程中有多种酶和转录因子被激活，涉及多条细胞内信号转导通路（DAG／PKC、MAPK、JAK／STAT）杀伤靶器官，即高糖可以通过激活多种细胞内信号因子而导致靶器官损伤。其中Janns激酶／信号转导与转录激活子（Jams kinase／signal transducers and activators of transcription，JAK／STAT）信号传导通路在近年来越来越引起人们的重视，大量研究表明，其与系膜细胞的增殖，肥大及细胞外基质分泌有关。但此信号通路与肾脏疾病的关系的报道尚不十分清楚目前的研究证实，氧化应激在糖尿病的发病过程中为一原发且独立的参与因素。活性氧簇（ROS）在糖尿病肾病（DN）的发生和发展过程中起着重要的作用，高糖可上调ROS，转化生长因子-β（TGF-β）在肾小球系膜细胞中的表达。ROS作为氧化应激的中心环节，参与DN的病理生理过程。还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶通过其产生的ROS，可改变肾脏血流动力学，激活细胞内信号转导等，导致糖尿病肾病（DN）的发生、发展。然而，在DN时ROS是否能够通过调控JAK₂／STAT₃通路的活性而影响DN的病理生理过程，还有待于进一步研究。为此我们设计了以下实验：目的1．观察高糖培养大鼠肾小球系膜细胞（GMC）P-STAT₃的表达变化，并利用JAK₂阻断剂Ag-490阻断JAK₂／STAT₃通路，观察其对P-STAT₃表达变化的影响，探讨糖尿病状态下GMC JAK₂／STAT₃途径的活性变化。2．观察高糖培养大鼠肾小球系膜细胞在不同时间点下活性氧簇（ROS）产生的量的变化。3．通过NADPH氧化酶抑制剂（Apocynin）阻断ROS的产生，观察高糖培养下系膜细胞P-STAT₃的表达变化，探讨JAK₂／STAT₃通路与ROS之间的相互作用的影响。方法1．细胞培养：购买的大鼠肾小球系膜细胞株，选用DMEM培养基，在0.25％胰蛋白酶+0.02％EDTA消化液消化并吹打均匀后，按1：3比例进行传代，3-5天传代一次2．分组：（1）GMC同步化后分成：正常糖浓度组（含糖5.5mmol／L）；高糖浓度（25mmol／L）；甘露醇组（5.5mmol／L糖+19.5mmol甘露醇）；正常糖+AG-490（浓度10umol／L）组；高糖+AG-490（浓度10umol／L）组。继续观察培养24、48小时后用Western Blot及细胞免疫化学方法检测系膜细胞STAT₃、P-STAT₃表达的变化。（2）GMC同步化后分成：正常糖浓度组（N，含糖5.5mmol／L）；高糖浓度组（H，含糖25mmol／L）；甘露醇组（M，含5.5mmol／L葡萄糖+19.5mmol／L甘露醇）；正常糖+Apocynin（N+A，Apocynin浓度为100umol／L）；高糖+Apocynin（H+A，Apocynin浓度为100umol／L），分别于0、12h及36h收集上清液，用比色法检测系膜细胞产生ROS的量。（3）NADPH氧化酶抑制剂Apocynin预处理，GMC同步化后分为正常糖浓度组（N，含糖5.5mmol╱L）；高糖浓度组（H，含糖25mmol／L）；甘露醇组（M，含糖5.5mmol／L，甘露醇19.5 mmol／L）：正常糖+Apocynin（N+A，Apocynin浓度为100umol／L）组；高糖+Apocynin（H+A，Apocynin浓度100umol／L）组；Apocynin提前1小时加入，与正常糖或高糖共同培养GMC48小时，培养结束后，用Western Blot方法检测系膜细胞P-STAT₃表达。3．检测方法：（1）用细胞免疫化学方法定性检测高糖环境下大鼠肾小球系膜细胞STAT₃、P-STAT₃表达的变化。（2）用免疫印记法（Western Blot）半定量检测高糖状态下大鼠肾小球系膜细胞STAT₃、P-STAT₃表达的变化。（3）用比色法检测高糖作用后培养系膜细胞上清液中ROS产生的水平。结果1．高糖培养大鼠肾小球系膜细胞在24小时和48小时P-STAT₃的表达较正常糖浓度组明显升高。甘露醇组与正常糖浓度组相比差异无统计学意义。而各组之间STAT₃表达差异无统计学意义。JAK₂阻断剂Ag-490可抑制高糖下P-STAT₃的表达，但对STAT₃表达无明显影响。2．高糖条件下，ROS产生明显升高，NADPH氧化酶抑制剂Apocynin可明显降低ROS的产生水平。3．高糖条件下，Apocynin经预处理后，在正常糖浓度和高糖浓度共同培养48小时，正常糖浓度组和正常糖+Apocynin组对比P-STAT₃的表达差异无明显差别；高糖+Apocynin组较正常糖浓度组P-STAT₃明显增加，但与高糖组相比P-STAT₃显著降低。结论1．高糖通过磷酸化方式激活大鼠肾小球系膜细胞（GMC）JAK₂／STAT₃信号转导通路，Ag-490可有效抑制GMC JAK₂／STAT₃信号转导通路活性的变化。2．高糖作用下，肾小球系膜细胞ROS产生增加，并具有时间依赖性，NADPH氧化酶抑制剂Apocynin可有效抑制ROS的产生。3．高糖激活JAK₂／STAT₃通路后，阻断ROS增加可部分抑制大鼠肾小球系膜细胞P-STAT₃的表达。更多还原

【Abstract】 In recent years, the incidence of diabetes which is a chronic metabolic diseases characterized by hyperglycemia, in the world is significantly increased. Tissue Injury, dysfunction and failure can be found when the kidney is emerged in the long-term hyperglycemia. Today, diabetic nephropathy （DN） becomes one of most important factors causing patients disabled and death. And the incidence of DN-induced renal failure is rising annually. According to statistics, the incidence rate of DN has caught up to 47.66%, while in the United States more than 30% of end-stage renal failure （ESRD） is caused by diabetes, and the ratio is stepping upward annually.However, the present study shows neither definitely clear targets that could be blamed for the development of DN nor effective prevention and control measures. Therefore, to further exploring the pathogenesis of diabetic nephropathy and find ways to prevent DN progress is inevitable.The activation of a variety of enzymes and transcription factors was involved in the process of diabetic nephropathy, which has been proved by many studies. That is to say, many cellular signal transduction pathways, such as DAG / PKC, MAPK, and JAK / STAT, were also involved in, which gave us a further explanation that target organs were damaged through activation of multiple intracellular signaling factors. Among them, Janns Kinase/signal transducers and activators of transcription （JAK / STAT） signaling pathway has attracted people’s attentions and numerous studies indicated that the proliferation of mesangial cells and the secretion of extracellular matrix were closely related to the pathway. But so far, few studies have focused on the relation between JAK / STAT pathway and the renal diseases, which awaits our further studies.Oxidative stress is a primary and independent factor in the pathogenesis of diabetes.The deadly complication of diabetes - DN, has become a tremendous threat to human health. More and more evidences have proved that the reactive oxygen species （ROS） play an most important role in the start-up and development process of DN. Under high-glucose conditions, ROS can be raised, and then transforming growth factors-β（TGF-β） and extracellular matrix （ECM） express in the mesangial cells. As the central part, ROS is involved in the DN pathophysiological process. Reduced nicotinamide adenine dinucleotide phosphate （NADPH） oxidase can change the renal hemodynamics, impacting the remodeling of ECM and intracellular signal transductions, through its product - ROS. Whether can ROS regulate the signal pathway and leads to end-stage DN or not and if so what molecular mechanism it depends on is not described and proved clearly. And also, that whether ROS can affect the DN pathophysiological process by activating the JAK2/STAT3 pathway or not still stays unknown and awaits our further studies. To this end we have devised the following experiments:1. To study the changes of JAK2/STAT3 pathway in the diabetic state by observing the changes of p-STAT₃ in the rat glomerular mesangiai cells （GMC） cultured in high glucose state.2. To observe the levels of ROS in the GMC treated with high glucose.3. Through the use of NADPH oxidase inhibitor （Apocynin） blocking the combination of ROS with its receptors, observe the P-STAT3 expression changes in the GMC cultured in high glucose state and study the effects of JAK₂/STAT₃ pathway on the generation of ROS and the secretion of ECM. 1. High glucose can induce the activation of JAK2/STAT3 pathway in the GMC:Objective: To study the changes of JAK2/STAT3 signal pathway by observation the expression changes of p-STAT3 in rat GMC cultured in high-glucose state.Methods: Synchronized GMCs are divided into following groups: the normal group （5.5mmol/L Glu）, High glucose group （25mmol/L Glu）, Mannitol group （5.5mmol/LGlu and 19.5mmol mannitol）, the normal + AG-490 （10μmol / L）, the high glucose + AG-490 （10μmol / L）. After 24-48 hours’ incubation, the GMCs were analyzed by Westem Blot and Immunocytochemistry to observe the changes of STAT3 and P-STAT3 expressions in mesangial cells.Results: At 24h and 48h, the p-STAT3 expression in the GMC that cultured with high glucose is significantly higher than that in the normal group. And there is no statistical difference between the mannitol and the normal group; and also there are no significant differences among the groups on the STAT3 expressions. AG-490 inhibited P-STAT3 expression in the cells cultured with high glucose, but had no obvious effects on STAT3 expressions.2.The levels of ROS in GMC cultured with high glucose:Objective: To study the effects of high glucose on the generation of ROS in the rat GMC cultured under high-glucose state.Methods: Synchronized GMCs are divided into following groups: the normal group （5.5mmol/L Glu）, High glucose group （25mmol/L Glu）, Mannitol group （5.5mmol/LGlu and 19.5mmol mannitol）, the normal + Apocynin （100μmol/L）, the high glucose + Apocynin （100μmol/L）. Collect the cells at Oh, 12h and 36h respectively, and then determine the O₂^- levels with colorimetric method. Results: At On, O₂^- level in each group is almost same and at 12h the O₂^-1evel in the High glucose group is obviously increased and higher than that in the normal （p<0.05）. there is no significant difference between the Mannitol and the normal group on the O₂^- level and the O₂^- level in the high glucose + Apocynin is lower than that in the high glucose group （p<0.05）. At 36h, the O₂^- levels in both the high glucose and the high glucose + Apocynin group are higher than that in the normal （p<0.01）, but the O₂^- level of the high glucose + Apocynin group is significantly lower than that in the high glucose （p<0.01） and there are no statistical differences among the groups including the Mannitol, the normal, and the the normal + Apocynin.3.Effects of high glucose on the ROS expression in GMC after activation of JAK2/STAT3 pathwayObserve: the changes of p-STAT3 expression in GMC under the high glucose condition when using the ROS blocker Apocynin to study the relation between ROS and JAK2/STAT3 pathway.Methods: Synchronized GMCs are divided into following groups: the normal group （N, 5.5mmol/L Glu）, High glucose group （H, 25mmol/L Glu）, the normal + Apocynin （NA, 100μmol/L）, the high glucose + Apocynin （HA, 100μmol/L）. After the treatment with Apocynin for 1h, incubate the GMC of each group for 48 hours. The change of p-STAT3 expression in GMC was detected by Western Blot.Results: The p-STAT3 expression in GMC of the H group is obviously higher than that in the N group at 48h; there is no difference between the NA and the N groups; the p-STAT3 expression in the HA group is significantly decreased but still higher than that in the N group.Conclusion 1. Under high glucose conditions, JAK2/STAT3 signal transduction pathway can be activated in the phosphorylation manner but AG-490 can inhibit the activation. The JAK₂/STAT₃ pathway may be involved in the regulation of the secretion of ECM.2. ROS in GMC can be increased in a time-dependent manner under high glucose condition and NADPH oxidase is the stimulator for the generation of ROS so the Apocynin ofNADPH oxidase inhibitors can inhibit ROS level effectively.3. Under high glucose condition with the activated JAK2/STAT3 pathway, blockage of ROS can only decrease the p-STAT3 expression in the GMC partly, which means the pathway may be involved in the regulation of the secretion of ECM.更多还原