本文選題：兒童 + 過(guò)敏性紫癜��； 參考：《安徽醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的 通過(guò)觀察HSP患兒血清IgA1和正常兒童血清IgA1對(duì)體外培養(yǎng)的HUVECs凋亡誘導(dǎo)作用，及其對(duì)P53、Bax、Bcl-2和Caspase-3表達(dá)的影響，探討IgA1誘導(dǎo)內(nèi)皮細(xì)胞凋亡的分子機(jī)制，為明確IgA1在HSP血管損傷中的作用提供實(shí)驗(yàn)依據(jù)。 方法 1.血清采集及IgA1的分離提取2010年9月-2011年1月在本院兒科住院明確診斷HSP的初發(fā)患兒10例，10名與HSP患兒同時(shí)期、同地區(qū)無(wú)風(fēng)濕免疫性疾病史的門診健康體檢兒童作為正常對(duì)照。所有受試患兒清晨空腹采取外周靜脈非抗凝全血5ml，分離血清。親和層析法分離提取血清IgA1。 2.實(shí)驗(yàn)分組用含10%胎牛血清（FBS）的RPMI-1640常規(guī)培養(yǎng)HUVECs，依據(jù)培養(yǎng)條件不同分為3組：①HSP組：用含不同濃度（0.05mg/ml、0.1mg/ml、0.25mg/ml）HSP患兒血清IgA1的RPMI-l640培養(yǎng)；②正常對(duì)照組：用含不同濃度（0.05mg/ml、0.1mg/ml、0.25mg/ml）正常兒童血清IgA1的RPMI-l640培養(yǎng)；③空白對(duì)照組：用不含IgA1的RPMI-l640培養(yǎng)。 3.細(xì)胞凋亡的觀察與檢測(cè)光學(xué)顯微鏡下觀察IgA1誘導(dǎo)前后各組HUVECs形態(tài)學(xué)變化；各組于誘導(dǎo)因素加入后12h和24h分別收集一次細(xì)胞，行流式細(xì)胞儀（FCM）和TUNEL法檢測(cè)細(xì)胞凋亡率。 4.凋亡相關(guān)基因檢測(cè)0.25mg/mlIgA1誘導(dǎo)HUVECs24h時(shí)應(yīng)用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)（Real time PCR）和蛋白質(zhì)印記技術(shù)（Western blot）檢測(cè)各組HUVECs中凋亡相關(guān)基因P53、Bax、Bcl-2及Caspase-3mRNA及蛋白表達(dá)情況。 結(jié)果 1.光學(xué)顯微鏡下觀察可見IgA1誘導(dǎo)24h時(shí)HUVECs圓縮，體積縮小，與周圍細(xì)胞脫離，HSP組較正常對(duì)照組改變更加明顯。 2.各組HUVECs誘導(dǎo)培養(yǎng)12h時(shí)，HSP組和正常對(duì)照組FCM法IgA1為0.25mg/ml，TUNEL法IgA1為0.1mg/ml時(shí)，即可誘導(dǎo)內(nèi)皮細(xì)胞發(fā)生明顯凋亡（P＜0.01），但HSP組與正常對(duì)照組比較差異無(wú)顯著性（P0.05）。在誘導(dǎo)培養(yǎng)24h時(shí)，HSP組和正常對(duì)照組IgA1為0.1mg/ml時(shí)即可誘導(dǎo)內(nèi)皮細(xì)胞發(fā)生明顯凋亡（P＜0.01），HSP組凋亡率與正常對(duì)照組相比顯著增加（P＜0.01），且隨著IgA1濃度增加及誘導(dǎo)時(shí)間延長(zhǎng)，細(xì)胞凋亡率逐漸增加（P＜0.01）。 3.0.25mg/ml IgA1誘導(dǎo)24h時(shí)，HSP組和正常對(duì)照組與空白對(duì)照組相比，P53、Bax、Caspase-3mRNA表達(dá)均明顯增多，Bcl-2mRNA表達(dá)均明顯減少，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；HSP組P53、Bax、Caspase-3mRNA表達(dá)的增加和Bcl-2mRNA表達(dá)的減少較正常對(duì)照組變化更為顯著（P0.05）。 4.0.25mg/ml IgA1誘導(dǎo)各組HUVECs24h時(shí)，HSP組和正常對(duì)照組與空白對(duì)照組相比，P53、Bax、Caspase-3蛋白表達(dá)明顯增多，Bcl-2蛋白表達(dá)明顯減少（P0.01）；HSP組與正常對(duì)照組相比也有顯著性差異（P0.01）。 結(jié)論 1.IgA1可以誘導(dǎo)體外培養(yǎng)HUVECs凋亡，HUVECs凋亡率與IgA1的劑量和作用時(shí)間具有一定的量效時(shí)效關(guān)系，當(dāng)作用時(shí)間及IgA1濃度相同時(shí)，，HSP患兒血清提取的IgA1對(duì)HUVECs的凋亡誘導(dǎo)作用更強(qiáng)。 2.IgA1誘導(dǎo)體外培養(yǎng)HUVECs凋亡的機(jī)制可能與上調(diào)促凋亡基因P53、Bax、Caspase-3表達(dá)，下調(diào)抑制凋亡基因Bcl-2表達(dá)有關(guān)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.5

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本文編號(hào)：1996883