張曉丹 李 潞 趙紅麗

·論著·

冠心病合并2型糖尿病患者糖基化終產(chǎn)物與單核細(xì)胞TLR4的關(guān)系

張曉丹 李 潞 趙紅麗

目的 探討分析冠心病(CHD)合并糖尿病(DM)患者糖基化終產(chǎn)物(AGEs)與單核細(xì)胞toll樣受體4(TLR4)的關(guān)系。方法 CHD+DM患者58例(CHD+DM組), CHD患者60例(CHD組), 健康對(duì)照組50例。采用酶聯(lián)免疫吸附測(cè)定(ELISA)法檢測(cè)各組的AGEs, 用流式細(xì)胞儀檢測(cè)外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量及TLR4陽(yáng)性單核細(xì)胞比率。結(jié)果 與健康對(duì)照組比較, CHD+DM組及CHD組AGEs及外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量、TLR4陽(yáng)性單核細(xì)胞比率明顯增高(P＜0.05)；CHD+DM組與CHD組比較, CHD+DM組AGEs及外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量、TLR4陽(yáng)性單核細(xì)胞比率增高(P＜0.05)；相關(guān)性分析顯示升高的AGEs與升高的TLR4mRNA相對(duì)表達(dá)量及TLR4陽(yáng)性單核細(xì)胞比率呈正相關(guān)。結(jié)論 CHD+DM患者AGEs及TLR4表達(dá)高于CHD患者；CHD患者AGEs及TLR4表達(dá)高于健康對(duì)照組。AGEs與TLR4的表達(dá)呈正相關(guān)。

冠心??；糖尿??；糖基化終產(chǎn)物；toll樣受體4

流行病學(xué)調(diào)查資料顯示, 糖尿病(diabetes mellitus, DM)患者發(fā)生動(dòng)脈粥樣硬化(AS)和心血管疾病的危險(xiǎn)性較常人增加3～4倍。糖尿病患者在長(zhǎng)期高糖刺激下, 體內(nèi)蛋白質(zhì)和脂質(zhì)將發(fā)生非酶糖基化, 導(dǎo)致多種異構(gòu)體在體內(nèi)蓄積, 統(tǒng)稱為AGEs。AGEs與細(xì)胞膜上的糖基化終產(chǎn)物受體相結(jié)合, 促進(jìn)糖尿病大血管并發(fā)癥的發(fā)生發(fā)展［1］。

Toll樣受體4(toll like receptor 4, TLR4))是近年來(lái)發(fā)現(xiàn)的天然免疫識(shí)別受體, 能夠識(shí)別病原體共有的病原相關(guān)分子模式并介導(dǎo)其跨膜信號(hào)傳導(dǎo)［2］。若TLR4 被激活, 將啟動(dòng)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo), 最終激活NF-κB, 誘導(dǎo)單核/ 巨噬細(xì)胞產(chǎn)生免疫炎性細(xì)胞因子、共刺激分子以及粘附分子等, 從而參與粥樣斑塊形成和破裂。通過對(duì)人及小鼠模型的AS組織切片進(jìn)行免疫組化分析, 發(fā)現(xiàn)TLR4在粥樣斑塊組織表達(dá), 尤其在巨噬細(xì)胞浸潤(rùn)、脂質(zhì)豐富的部位明顯表達(dá)［3,4］。

1 資料與方法

1.1 一般資料 入選2013年9月～2013年12月于本科住院的冠心病(coronary heart disease, CHD)+DM(CHD+DM組)患者58例, 男37例, 女21例；入選同期住院的CHD患者(CHD組)60例, 男34例, 女26例；另健康對(duì)照組50例, 男27例,女23例。三組一般資料比較, 差異無(wú)統(tǒng)計(jì)學(xué)意義(P＞0.05),具有可比性。冠心病診斷標(biāo)準(zhǔn)根據(jù)第7版《內(nèi)科學(xué)》；糖尿病者均符合1999年WHO糖尿病診斷標(biāo)準(zhǔn)。排除標(biāo)準(zhǔn)：①心肌梗死、腦血管意外、妊娠及嚴(yán)重肝腎功能不全除外；②糖尿病急性并發(fā)癥；③甲狀腺疾病、自身免疫性疾病、感染性疾病及出凝血疾??；④惡性腫瘤及結(jié)締組織病等。

1.2 研究方法 所有入選者均于住院后首次清晨空腹抽取肘靜脈血6 ml, 無(wú)需抗凝, 3000 r/min離心20 min, 取血清置于-70℃冰箱保存, 批量測(cè)定。

1.2.1 糖基化終產(chǎn)物(AGEs)測(cè)定 采用ELISA法檢測(cè)各組的AGEs。試劑盒由上?；顦飞锟萍加邢薰咎峁? 批內(nèi)差異＜9%, 批間差異＜11%。

1.2.2 測(cè)定外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量及TLR4陽(yáng)性單核細(xì)胞比率 用流式細(xì)胞儀檢測(cè)各組外周血單核細(xì)胞TLR4 mRNA相對(duì)表達(dá)量及TLR4陽(yáng)性單核細(xì)胞比率。

1.3 統(tǒng)計(jì)學(xué)方法 使用SPSS13.0統(tǒng)計(jì)軟件進(jìn)行分析。計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差( x-±s)表示 。組間均值比較用ANOVA方差分析；計(jì)數(shù)資料采用χ2檢驗(yàn)；相關(guān)分析選用Pearson相關(guān)分析。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 各組患者AGEs及外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量、TLR4陽(yáng)性單核細(xì)胞比率 與健康對(duì)照組比較, CHD+DM組及CHD組AGEs及外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量、TLR4陽(yáng)性單核細(xì)胞比率明顯增高(P＜0.05)；CHD+DM組與CHD組比較, CHD+DM組AGEs及外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量、TLR4陽(yáng)性單核細(xì)胞比率增高(P＜0.05)。見表1。

表1 三組AGEs及外周血單核細(xì)胞TLR4mRNA相對(duì)表達(dá)量、TLR4陽(yáng)性單核細(xì)胞比率比較( x-±s)

2.2 CHD+DM、CHD患者AGEs與單核細(xì)胞TLR4相關(guān)性分析 CHD患者單核細(xì)胞TLR4表達(dá)增高, 合并DM時(shí)增高更明顯, 且與AGEs呈正相關(guān)(r=0.643, P＜0.05)。單核細(xì)胞TLR4與CHD合并DM的炎癥反應(yīng)加重有關(guān)。

3 討論

大量研究表明TLR4信號(hào)通路的激活介導(dǎo)了眾多炎癥因子的表達(dá)。TLR4在動(dòng)脈粥樣斑塊的內(nèi)皮細(xì)胞、巨噬細(xì)胞、血管平滑肌細(xì)胞和血管外膜成纖維細(xì)胞表達(dá)均增高, 在冠心病患者外周血單核細(xì)胞表達(dá)增高。Devaraj等［5］研究發(fā)現(xiàn), DM患者微血管病變者炎癥介質(zhì)表達(dá)水平高, 從而造成血管內(nèi)皮細(xì)胞的嚴(yán)重?fù)p傷, 隨后脂質(zhì)沉積、血小板粘附, 血管舒縮受限等參與了AS的病理發(fā)展過程。

目前與糖尿病及AS關(guān)系最密切的一個(gè)物質(zhì)就是AGEs。大量的研究證明：AGEs可促進(jìn)AS的發(fā)生發(fā)展。AGEs與蛋白質(zhì)的周轉(zhuǎn), 組織結(jié)構(gòu), 細(xì)胞氧化應(yīng)激以及炎癥等疾病有關(guān)聯(lián)［6］。本研究結(jié)果所顯示出的單純冠心病患者血清AGEs水平升高也證實(shí)了AGEs與AS有關(guān)。目前認(rèn)為AGEs可能通過以下途徑促進(jìn)AS發(fā)揮作用：①損傷血管內(nèi)皮［7］。②促進(jìn)白細(xì)胞粘附、聚集及活化［8,9］。③刺激血管平滑肌細(xì)胞增殖。④加快血小板聚集和血栓形成［10］。本研究結(jié)果顯示各組間的AGEs水平差異均有統(tǒng)計(jì)學(xué)意義(P＜0.05), 以糖尿病合并冠心病組為最高, 支持了上述理論, 說明AGEs在血管病變中起了一定的作用。衰老、高血糖、腎功能衰竭、氧化應(yīng)激及炎性反應(yīng)等因素刺激可促進(jìn)AGEs的形成［11］。AGEs作為致病因素, 可以提示人們?cè)谥委熛嚓P(guān)疾病中采取新的策略, 在新藥篩選中具有重要意義［12］。

綜上所述, 冠心病合并2型糖尿病AGEs與TLR4分子的表達(dá)呈正相關(guān), 可揭示AGEs與AS的內(nèi)在聯(lián)系、作用方式,為防治糖尿病心血管并發(fā)癥提供新的理論依據(jù), 同時(shí)為新的診療技術(shù)及藥物開發(fā)提供目標(biāo)靶點(diǎn)。

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Relationship between advanced glycation end products and monocyte TLR4 in patients of coronary heart disease complicated with type 2 diabetes mellitus

ZHANG Xiao-dan, LI Lu, ZHAO Hong-li.
The Second Affiliated Hospital of Shenyang Medical College, Shenyang 110002, China

Objective To investigate the relationship between advanced glycation end products (AGEs) and monocyte Toll-like receptor 4 (TLR4) in patients with coronary heart disease (CHD) combined with diabetes mellitus (DM).Methods There were 58 patients with CHD+DM (CHD+DM group), 60 patients with CHD only (CHD group), and 50 patients in healthy control group.Enzyme-linked immuno sorbent assay (ELISA) was applied to detect AGEs in all the groups, and flow cytometry was used to detect relative expression of peripheral blood monocytes TLR4mRNA and ratio of TLR4 positive monocytes.Results Compared with the healthy control group, CHD+DM group and CHD group all had remarkably increased AGEs, relative expression of peripheral blood monocytes TLR4mRNA, and ratio of TLR4 positive monocytes (P＜0.05).Between the CHD+DM group and CHD group, the former had higher increased levels of AGEs, relative expression of peripheral blood monocytes TLR4mRNA, and ratio of TLR4 positive monocytes (P＜0.05).The results of correlated analysis showed that there was positive correlation between increased AGEs, increased relative expression of TLR4mRNA, and ratio of TLR4 positive monocytes.Conclusion The expressions of AGEs and TLR4 of CHD+DM patients are higher than CHD patients, and those of CHD patients are also higher than healthy control group.AGEs and expression of TLR4 are positively correlated.

Coronary heart disease; Diabetes mellitus; Advanced glycation end products; Toll-like receptor 4

10.14163/j.cnki.11-5547/r.2015.04.001

2014-10-17］

沈陽(yáng)市科技計(jì)劃項(xiàng)目(項(xiàng)目編號(hào)：F11-262-9-18)

110002 沈陽(yáng)醫(yī)學(xué)院附屬第二醫(yī)院