过敏性紫癜血清IgA1诱导人脐静脉内皮细胞凋亡的体外实验研究

发布时间：2018-06-08 19:09

  本文选题：儿童 + 过敏性紫癜　； 参考：《安徽医科大学》2012年硕士论文

【摘要】：目的 通过观察HSP患儿血清IgA1和正常儿童血清IgA1对体外培养的HUVECs凋亡诱导作用，及其对P53、Bax、Bcl-2和Caspase-3表达的影响，探讨IgA1诱导内皮细胞凋亡的分子机制，为明确IgA1在HSP血管损伤中的作用提供实验依据。 方法 1.血清采集及IgA1的分离提取2010年9月-2011年1月在本院儿科住院明确诊断HSP的初发患儿10例，10名与HSP患儿同时期、同地区无风湿免疫性疾病史的门诊健康体检儿童作为正常对照。所有受试患儿清晨空腹采取外周静脉非抗凝全血5ml，分离血清。亲和层析法分离提取血清IgA1。 2.实验分组用含10%胎牛血清（FBS）的RPMI-1640常规培养HUVECs，依据培养条件不同分为3组：①HSP组：用含不同浓度（0.05mg/ml、0.1mg/ml、0.25mg/ml）HSP患儿血清IgA1的RPMI-l640培养；②正常对照组：用含不同浓度（0.05mg/ml、0.1mg/ml、0.25mg/ml）正常儿童血清IgA1的RPMI-l640培养；③空白对照组：用不含IgA1的RPMI-l640培养。 3.细胞凋亡的观察与检测光学显微镜下观察IgA1诱导前后各组HUVECs形态学变化；各组于诱导因素加入后12h和24h分别收集一次细胞，行流式细胞仪（FCM）和TUNEL法检测细胞凋亡率。 4.凋亡相关基因检测0.25mg/mlIgA1诱导HUVECs24h时应用实时荧光定量聚合酶链反应（Real time PCR）和蛋白质印记技术（Western blot）检测各组HUVECs中凋亡相关基因P53、Bax、Bcl-2及Caspase-3mRNA及蛋白表达情况。 结果 1.光学显微镜下观察可见IgA1诱导24h时HUVECs圆缩，体积缩小，与周围细胞脱离，HSP组较正常对照组改变更加明显。 2.各组HUVECs诱导培养12h时，HSP组和正常对照组FCM法IgA1为0.25mg/ml，TUNEL法IgA1为0.1mg/ml时，即可诱导内皮细胞发生明显凋亡（P＜0.01），但HSP组与正常对照组比较差异无显著性（P0.05）。在诱导培养24h时，HSP组和正常对照组IgA1为0.1mg/ml时即可诱导内皮细胞发生明显凋亡（P＜0.01），HSP组凋亡率与正常对照组相比显著增加（P＜0.01），且随着IgA1浓度增加及诱导时间延长，细胞凋亡率逐渐增加（P＜0.01）。 3.0.25mg/ml IgA1诱导24h时，HSP组和正常对照组与空白对照组相比，P53、Bax、Caspase-3mRNA表达均明显增多，Bcl-2mRNA表达均明显减少，差异有统计学意义（P0.05）；HSP组P53、Bax、Caspase-3mRNA表达的增加和Bcl-2mRNA表达的减少较正常对照组变化更为显著（P0.05）。 4.0.25mg/ml IgA1诱导各组HUVECs24h时，HSP组和正常对照组与空白对照组相比，P53、Bax、Caspase-3蛋白表达明显增多，Bcl-2蛋白表达明显减少（P0.01）；HSP组与正常对照组相比也有显著性差异（P0.01）。 结论 1.IgA1可以诱导体外培养HUVECs凋亡，HUVECs凋亡率与IgA1的剂量和作用时间具有一定的量效时效关系，当作用时间及IgA1浓度相同时，，HSP患儿血清提取的IgA1对HUVECs的凋亡诱导作用更强。 2.IgA1诱导体外培养HUVECs凋亡的机制可能与上调促凋亡基因P53、Bax、Caspase-3表达，下调抑制凋亡基因Bcl-2表达有关。
[Abstract]:Objective to investigate the molecular mechanism of apoptosis induced by IgA1 in cultured HUVECs and the expression of Bcl-2 and Caspase-3 in cultured HUVECs by observing the effects of serum IgA1 and IgA1 on the expression of Bcl-2 and Caspase-3 in cultured HUVECs. To provide experimental evidence for the role of IgA1 in the vascular injury of HSP. Methods 1. Serum collection and isolation of IgA1 from September 2010 to January 2011, 10 children with HSP were diagnosed in our hospital from September 2010 to January 2011, and 10 healthy children without history of rheumatic immune diseases in the same area were used as normal control. All the children were treated with 5 ml non-anticoagulant peripheral vein blood on an empty stomach in the morning to separate the serum. Serum IgA 1.2 was isolated by affinity chromatography. HUVECs were cultured with RPMI-1640 containing 10% fetal bovine serum (FBSs). According to different culture conditions, HUVECs were divided into 3 groups: group 1: RPMI-l640 was used to culture IgA1 in serum of children with HSP0. 05 mg / ml + 0. 25 mg / ml. 2 normal control group: RPMI-l640 of serum IgA1 of normal children was cultured with RPMI-l640 containing 0. 05 mg / ml of 0. 05 mg 路ml / ml of different concentrations of 0. 05 mg / ml). 3 the control group was cultured with RPMI-l640 without IgA1. The morphological changes of HUVECs before and after IgA1 induction were observed under optical microscope, and the cell apoptosis rate was detected by flow cytometry (FCM) and Tunel assay at 12 h and 24 h after induction. The expression of apoptosis-related genes (P53, Baxan-2, Caspase-3 mRNA and protein) in HUVECs was detected by real-time fluorescence quantitative polymerase chain reaction (Real time PCR) and protein imprinting technique during 24h after induced by 0.25 mg / ml IgA1.Results 1. Under the optical microscope, HUVECs were reduced in volume and circle at 24 h induced by IgA1. The changes in HSP group were more obvious than those in the control group. 2. After 12 h culture of HUVECs, apoptosis of endothelial cells was induced in HSPgroup and normal control group when FCM IgA1 was 0.25 mg / ml / ml Tunel IgA1 as 0.1mg/ml (P < 0.01), but there was no significant difference between HSPgroup and normal control group (P0.05). The apoptotic rate of HSP1 group was significantly higher than that of normal control group (P < 0.01), and the apoptosis rate of HSP1 group was significantly higher than that of normal control group (P < 0.01), and with the increase of IgA1 concentration and induction time, the apoptotic rate of HSP1 group was significantly higher than that of normal control group (P < 0.01). The rate of apoptosis increased gradually (P < 0.01). The expression of Bcl-2 mRNA in HSP2 group and normal control group was significantly increased compared with the control group, and the expression of Bcl-2 mRNA was significantly decreased in 3.0.25mg/ml IgA1 induced HSP2 group and normal control group. There was a significant difference in the expression of P53 BaxCaspase-3 mRNA and the decrease of Bcl-2 mRNA expression in HSPgroup compared with the control group. The expression of Bcl-2 protein in HSPgroup and normal control group was significantly higher than that in control group at 24 h after induction by 4.0.25mg/ml IgA1. Conclusion IgA1 can induce apoptosis of HUVECs in vitro and the apoptosis rate of HUVECs has a dose-effect time-dependent relationship with the dose and time of action of IgA1. When the time of action and the concentration of IgA1 were the same, IgA1 extracted from serum of HSPs had stronger effect on the apoptosis of HUVECs. 2. The mechanism of IgA1 induced apoptosis of HUVECs in vitro may be related to the up-regulation of the expression of P53nBax-Caspase-3 and the down-regulation of Bcl-2 expression of apoptosis-promoting gene.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.5

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