抵抗素在川崎病患儿冠状动脉损伤中的作用及其机制研究

发布时间：2018-03-23 20:15

本文选题：川崎病　切入点：抵抗素　出处：《重庆医科大学》2013年博士论文

【摘要】：第一部分血清抵抗素水平与川崎病的相关性研究目的：川崎病是一种急性发热性疾病，主要发生在婴幼儿及年龄小的儿童，常伴有冠状动脉损伤。由于15%-25%的患儿有发生冠状动脉瘤的危险，所以近年来引起了儿科医生的极大关注。抵抗素是新近发现的一种脂肪细胞来源的肽类，属于一类富含半胱氨酸残基的分泌性蛋白家族，有研究发现抵抗素可能参与了炎症过程，且与冠脉动脉钙化，粥样硬化等疾病的发生发展密切相关。因以，本研究探讨血清抵抗素水平与川崎病患儿及其发生冠脉动脉瘤的相关性。方法:用酶联免疫吸附试验检测了165例儿童血清抵抗素水平，并将其分为4组：健康对照组（n＝85），无冠脉动脉损伤的川崎病患儿组（N=41），，伴冠脉扩张的川崎病患儿组（N=31），并发冠状动脉瘤的川崎病患儿组（N=8）。同时统计其血液学指标,包括血白细计数（WBC），红细胞计数（RBC），血红蛋白水平（HB），红细胞压积（Hct），血小板计数，C-反应蛋白（CRP）和红细胞沉降率（ESR）。结果:与正常对照组及川崎病患儿其他组相比，伴有冠肪动脉瘤的川崎病患儿血清抵抗素水平明显的升高，此外血红蛋白水平明显降低；且在患儿组，血清抵抗素水平与其CRP水平呈显著正相关关系，而与患儿红细胞水平呈负相关。结论:抵抗素和血红蛋白可能与川崎病患儿冠状动脉瘤的发生有关，血清抵抗素的表达上调可能与川崎病急性期的炎症因子增加有关。第二部分抵抗素基因启动子-420C/G多态性与儿童川崎病的相关性研究目的：川崎病（KD）是一种小儿全身性血管炎，通常伴发冠状动脉病变（CAL）。抵抗素为近年来发现的一种脂肪细胞来源的肽类，可能与炎症反应及冠脉动脉疾病的发生密切相关。-420C G多态性位点位于抵抗素基因（RETN）启动子区域，新近研究显示该区位点多态性在炎症发生及心血管疾病中起到了重要作用。然而，RETN启动子多态性与川崎病之间的关联尚不清楚。故本研究拟探讨抵抗素基因多态性（－420C/G）与儿童川崎病易感性及其临床特征的相关性。方法：共计91名川崎病患儿，115名正常对照组儿童纳入本研究。依照诊断标准将川崎病患儿分为了完全性（cKD）和不完全性川崎病（iKD）。采用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF)进行抵抗素启动子基因多态性分型。采用夹心酶联免疫法（ELISA）对所有血清样本抵抗素蛋白浓度进行测定。结果：KD组和健康对照组抵抗素基因（-420CG）的基因型和其等位基因频率进行了比较，然而差异无统计学意义（P〈0.05）。此外，该基因位点多态性与川崎病的CALS的发生也无明显差异。然而，G等位基因的频率在iKD组明显高于cKD组（P＜0.05），GG基因型也在iKD组显著增加（P＜0.05）。KD组血清抵抗素水平明显高于对照组（P＜0.05）。与基因型CC和CG和相比，GG基因型KD患儿血清抵抗素水平较高，但差异无统计学意义。结论：本研究结果表明，抵抗素可能在KD的发病机制中发挥作用，位于RETN基因启动子的-420CG基因多态性与儿童川崎病无明显相关,然而该位点等位基因G及GG基因型有助于不完全性川崎病的诊断。第三部分MAPKs信号通路在介导抵抗素致人冠状动脉内皮细胞分泌MMP-9、TIMP-1中的作用研究目的：抵抗素为脂肪细胞特异性分泌因子，可能与炎症和心血管疾病的发生相关。本课题组前期研究表明血清抵抗素水平在川崎病患儿明显升高，并与冠状动脉瘤的发生密切相关；但其致冠脉损伤的机制尚不清楚。基质金属蛋白酶-9（MMP-9）在KD患儿的炎症反应及血管损伤中起到了关键作用。因此，本课题提出假设，抵抗素可能对内皮细胞MMP-9及金属蛋白酶组织抑制剂（TIMP-1）的有调控作用，并进行机制的探讨。方法:用人重组抵抗素处理人冠状动脉内皮细胞（HCAECs）。用RT-PCR方法检测细胞MMP-9和TIMP-1的表达，用ELISA法测定MMP-9和TIMP-1蛋白表达。用Western印迹分析ERK1/2,P38MAPK,JNK的总蛋白及磷酸化蛋白表达。结果：抵抗素可刺激冠脉内皮细胞MMP-9的分泌和减少其TIMP-1的产生,并有时间和浓度依赖性。ERK1/2抑制剂（U0126），p38MAPK抑制剂(SB202190），和JNK抑制剂（SP600125）可明显减少抵抗素致HCAECsMMP-9的分泌。结论：综上所述，ERK1/2，p38和JNK信号通路可能参与了抵抗素致HCAECs MMP-9的分泌。MAPKs信号通路可能与抵抗素相关的心血管疾病有关。
[Abstract]:The first part of study on correlation between serum resistin levels and Kawasaki disease
Objective: Kawasaki disease is an acute febrile illness occurred mainly in infants and children younger, often accompanied by coronary artery injury. Due to 15%-25% with risk of coronary artery aneurysms, so in recent years has aroused great concern to pediatricians. Resistin is an adipocyte derived peptide discovered recently, belongs to a class of cysteine rich residues of secretory protein family, studies have found that resistin may be involved in the inflammatory process, and coronary artery calcification, closely related to the occurrence and development of atherosclerosis and other diseases. Due to this research, the correlation between serum resistin level and coronary artery disease in children and Kawasaki tumors.
Methods: 165 cases of children with serum resistin levels were detected by enzyme-linked immunosorbent assay, and divided into 4 groups: normal control group (n = 85), without coronary artery injury in children with Kawasaki group (N=41), with the expansion of coronary artery disease in children Kawasaki group (N=31), coronary artery aneurysm Kawasaki disease group (N=8). At the same time, the statistics of hematological indexes, including blood leukocyte count (WBC), red blood cell count (RBC), hemoglobin (HB), hematocrit (Hct), platelet count, C- reaction protein (CRP) and erythrocyte sedimentation rate (ESR).
Results: compared with normal control group and Kawasaki group with other disease patients, aneurysms Kawasaki crown fat disease in children serum resistin levels significantly increased, in addition to hemoglobin levels decreased significantly; and in group, was significant positive correlation between serum resistin levels and the CRP level, but negatively correlated with the level of red blood cells in children.
Conclusion: Resistin and hemoglobin may be related to the occurrence of coronary artery aneurysm disease Kawasaki, increased serum resistin expression may be related to the increase of inflammatory factors in acute stage of Kawasaki. The second part of the resistin gene promoter -420C/G polymorphism and Sichuan children
Study on the correlation of kazaki disease
Objective: Kawasaki disease (KD) is a systemic vasculitis, usually associated with coronary artery disease (CAL) peptide. A fat cell source for resistin found in recent years, may be related to inflammation and coronary artery disease is closely related to the occurrence of.-420C G polymorphisms in resistin gene (RETN) promoter recent research shows that this region polymorphism plays an important role in cardiovascular disease and inflammation. However, the association between RETN promoter polymorphism and sub Kawasaki disease remains unclear. Therefore, this study intends to explore the resistin gene polymorphism (420C/G) and its correlation with clinical characteristics of children with Kawasaki disease susceptibility.
Methods: a total of 91 Kawasaki disease in children, 115 normal controls were included in the study group of children. Children to complete Kawasaki disease according to the diagnostic criteria (cKD) and incomplete Kawasaki disease (iKD). By using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of resistin gene promoter polymorphism points type. Using a sandwich enzyme immunoassay (ELISA) for all serum samples of resistin protein concentrations were measured.
Results: KD group and healthy control group (-420CG) of resistin gene genotype and allele frequencies were compared, but the difference was not statistically significant (P < 0.05). In addition, there is no obvious difference between CALS gene polymorphism and the Kawasaki disease occurred. However, the frequency of G allele in the iKD group was significantly higher than that of group cKD (P < 0.05), the GG genotype was significantly increased in iKD group (P < 0.05) resistin level of serum.KD was significantly higher than the control group (P < 0.05). And the genotypes of CC and CG and compared the serum resistin level in children with KD GG genotype is higher, but no significant the difference in meaning.
Conclusion: the results of this study show that resistin may play a role in the pathogenesis of KD, located in RETN gene promoter -420CG gene polymorphism and childhood disease was not related to Kawasaki, however, the allele G and genotype GG were helpful to the diagnosis of incomplete Kawasaki disease. The third part of the MAPKs signaling pathway in resistin mediated by coronary artery
The role of endothelial cells in the secretion of MMP-9 and TIMP-1
Objective: to resistin adipocyte specific secretory factors that may be associated with inflammation and cardiovascular disease. Previous study showed that serum resistin levels were significantly increased in patients with coronary artery disease and Kawasaki, tumor is closely related to the occurrence of the damage caused by coronary artery; but the mechanism is not clear. -9 (matrix metalloproteinase MMP-9) in inflammation and vascular injury in children with KD to play a key role. Therefore, this paper put forward the hypothesis of resistin on endothelial cell MMP-9 and tissue inhibitor of metalloproteinases (TIMP-1) are used as control, and explore the mechanism.
Methods: human coronary artery endothelial cells (HCAECs) were treated with recombinant human resistin. The expression of MMP-9 and TIMP-1 was detected by RT-PCR. The expression of MMP-9 and TIMP-1 protein was detected by ELISA. The expression of total protein and phosphorylated protein in ERK1/2, P38MAPK and JNK were analyzed by Western blot.
Results: Resistin stimulated the secretion of MMP-9 and decreased the production of TIMP-1 in coronary endothelial cells, and there were time and concentration dependent.ERK1/2 inhibitors (U0126), p38MAPK inhibitors (SB202190), and JNK inhibitors (SP600125), which significantly reduced the secretion of resistin induced HCAECsMMP-9.
Conclusion: In conclusion, ERK1/2, p38 and JNK signaling pathways may be involved in resistin induced HCAECs MMP-9 secretion, and.MAPKs signaling pathway may be related to resistin related cardiovascular diseases.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R725.4

【参考文献】