冠心病合并2型糖尿病患者糖基化終產物與單核細胞TLR4的關系

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物與單核細胞TLR4的關系

張曉丹 李 潞 趙紅麗

目的 探討分析冠心病(CHD)合并糖尿病(DM)患者糖基化終產物(AGEs)與單核細胞toll樣受體4(TLR4)的關系。方法 CHD+DM患者58例(CHD+DM組), CHD患者60例(CHD組), 健康對照組50例。采用酶聯免疫吸附測定(ELISA)法檢測各組的AGEs, 用流式細胞儀檢測外周血單核細胞TLR4mRNA相對表達量及TLR4陽性單核細胞比率。結果 與健康對照組比較, CHD+DM組及CHD組AGEs及外周血單核細胞TLR4mRNA相對表達量、TLR4陽性單核細胞比率明顯增高(P＜0.05)；CHD+DM組與CHD組比較, CHD+DM組AGEs及外周血單核細胞TLR4mRNA相對表達量、TLR4陽性單核細胞比率增高(P＜0.05)；相關性分析顯示升高的AGEs與升高的TLR4mRNA相對表達量及TLR4陽性單核細胞比率呈正相關。結論 CHD+DM患者AGEs及TLR4表達高于CHD患者；CHD患者AGEs及TLR4表達高于健康對照組。AGEs與TLR4的表達呈正相關。

冠心病；糖尿病；糖基化終產物；toll樣受體4

流行病學調查資料顯示, 糖尿病(diabetes mellitus, DM)患者發生動脈粥樣硬化(AS)和心血管疾病的危險性較常人增加3～4倍。糖尿病患者在長期高糖刺激下, 體內蛋白質和脂質將發生非酶糖基化, 導致多種異構體在體內蓄積, 統稱為AGEs。AGEs與細胞膜上的糖基化終產物受體相結合, 促進糖尿病大血管并發癥的發生發展［1］。

Toll樣受體4(toll like receptor 4, TLR4))是近年來發現的天然免疫識別受體, 能夠識別病原體共有的病原相關分子模式并介導其跨膜信號傳導［2］。若TLR4 被激活, 將啟動胞內信號轉導, 最終激活NF-κB, 誘導單核/ 巨噬細胞產生免疫炎性細胞因子、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。通過對人及小鼠模型的AS組織切片進行免疫組化分析, 發現TLR4在粥樣斑塊組織表達, 尤其在巨噬細胞浸潤、脂質豐富的部位明顯表達［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；另健康對照組50例, 男27例,女23例。三組一般資料比較, 差異無統計學意義(P＞0.05),具有可比性。冠心病診斷標準根據第7版《內科學》；糖尿病者均符合1999年WHO糖尿病診斷標準。排除標準：①心肌梗死、腦血管意外、妊娠及嚴重肝腎功能不全除外；②糖尿病急性并發癥；③甲狀腺疾病、自身免疫性疾病、感染性疾病及出凝血疾病；④惡性腫瘤及結締組織病等。

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, 無需抗凝, 3000 r/min離心20 min, 取血清置于-70℃冰箱保存, 批量測定。

1.2.1 糖基化終產物(AGEs)測定 采用ELISA法檢測各組的AGEs。試劑盒由上海活樂生物科技有限公司提供, 批內差異＜9%, 批間差異＜11%。

1.2.2 測定外周血單核細胞TLR4mRNA相對表達量及TLR4陽性單核細胞比率 用流式細胞儀檢測各組外周血單核細胞TLR4 mRNA相對表達量及TLR4陽性單核細胞比率。

1.3 統計學方法 使用SPSS13.0統計軟件進行分析。計量資料以均數±標準差( x-±s)表示 。組間均值比較用ANOVA方差分析；計數資料采用χ2檢驗；相關分析選用Pearson相關分析。P＜0.05為差異有統計學意義。

2 結果

2.1 各組患者AGEs及外周血單核細胞TLR4mRNA相對表達量、TLR4陽性單核細胞比率 與健康對照組比較, CHD+DM組及CHD組AGEs及外周血單核細胞TLR4mRNA相對表達量、TLR4陽性單核細胞比率明顯增高(P＜0.05)；CHD+DM組與CHD組比較, CHD+DM組AGEs及外周血單核細胞TLR4mRNA相對表達量、TLR4陽性單核細胞比率增高(P＜0.05)。見表1。

表1 三組AGEs及外周血單核細胞TLR4mRNA相對表達量、TLR4陽性單核細胞比率比較( x-±s)

2.2 CHD+DM、CHD患者AGEs與單核細胞TLR4相關性分析 CHD患者單核細胞TLR4表達增高, 合并DM時增高更明顯, 且與AGEs呈正相關(r=0.643, P＜0.05)。單核細胞TLR4與CHD合并DM的炎癥反應加重有關。

3 討論

大量研究表明TLR4信號通路的激活介導了眾多炎癥因子的表達。TLR4在動脈粥樣斑塊的內皮細胞、巨噬細胞、血管平滑肌細胞和血管外膜成纖維細胞表達均增高, 在冠心病患者外周血單核細胞表達增高。Devaraj等［5］研究發現, DM患者微血管病變者炎癥介質表達水平高, 從而造成血管內皮細胞的嚴重損傷, 隨后脂質沉積、血小板粘附, 血管舒縮受限等參與了AS的病理發展過程。

目前與糖尿病及AS關系最密切的一個物質就是AGEs。大量的研究證明：AGEs可促進AS的發生發展。AGEs與蛋白質的周轉, 組織結構, 細胞氧化應激以及炎癥等疾病有關聯［6］。本研究結果所顯示出的單純冠心病患者血清AGEs水平升高也證實了AGEs與AS有關。目前認為AGEs可能通過以下途徑促進AS發揮作用：①損傷血管內皮［7］。②促進白細胞粘附、聚集及活化［8,9］。③刺激血管平滑肌細胞增殖。④加快血小板聚集和血栓形成［10］。本研究結果顯示各組間的AGEs水平差異均有統計學意義(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、氧化應激及炎性反應等因素刺激可促進AGEs的形成［11］。AGEs作為致病因素, 可以提示人們在治療相關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分子的表達呈正相關, 可揭示AGEs與AS的內在聯系、作用方式,為防治糖尿病心血管并發癥提供新的理論依據, 同時為新的診療技術及藥物開發提供目標靶點。

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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］

沈陽市科技計劃項目(項目編號：F11-262-9-18)

110002 沈陽醫學院附屬第二醫院