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急性冠状动脉综合征(ACS)是临床常见的危重疾病,是在冠状动脉粥样硬化斑块固定存在的基础上,继发斑块侵袭与破裂,形成完全或不完全闭塞性血栓导致的一组临床综合征[1]。其包括:不稳定型心绞痛(UAP)、非ST段抬高型心肌梗死和ST段抬高型心肌梗死。据报道,2015年我国农村急性心肌梗死(AMI)死亡率达到70.09/10万人,城市死亡率为56.38/10万,住院总费用高达152.40亿元[2],心血管疾病发病率及死亡率增长势头未减,已成为危害我国公民健康的主要因素,故掌握冠心病的发病机制、寻找更精准的病情评估指标具有十分重要的意义,而无创、易获取的血液学指标成为重点筛查对象。
程序性坏死是细胞受到精密的程序调控而发生的坏死性死亡,是非caspase依赖的细胞死亡通路,相关研究已证实其参与冠状动脉斑块形成、心肌缺血再灌注损伤、心肌细胞坏死及纤维化心室重塑等多个病理过程[3-5]。其中刺激受体相互作用蛋白1(RIP1)-RIP3-混合谱系激酶结构域样假激酶(MLKL)是研究较为透彻的经典的通路。KARUNAKARAN等[6]发现MLKL在动脉粥样硬化病人的斑块核内是高度活跃的,说明程序性坏死可能是导致冠状动脉易损性的始动因子之一,可作为诊断及治疗靶点。但目前仍缺乏大样本的临床试验证明MLKL及ACS之间的关系。本研究选取对照组、UAP及AMI组,对比观察RIP1、RIP3和MLKL的组间表达差异,探讨RIP1-RIP3-MLKL通路和ACS疾病进程之间的关系。现作报道。
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3组间年龄、高血压、糖尿病、白细胞(WBC)、红细胞(RBC)、血小板(PLT)、三酰甘油、高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、载脂蛋白A、丙氨酸氨基转移酶、总胆红素(TBil)、总蛋白差异均无统计学意义(P>0.05);而AMI组中血清总胆固醇(TC)、天冬氨酸氨基转移酶和血肌酐(Cr)水平明显高于UAP组和对照组(P < 0.05)(见表 1)。
分组 n 年龄/岁 高血压 糖尿病 WBC/
(×109/L)RBC/
(×1012/L)PLT/
(×109/L)TC/
(mmol/L)三酰甘油/
(mmol/L)对照组 19 59.74±7.56 18 3 6.35±2.78 4.28±0.76 235.37±116.24 3.93±1.30 1.52±1.23 UAP组 31 63.87±9.59 12 7 6.62±1.65 4.35±0.44 218.45±68.70 3.94±1.05 1.71±0.83 AMI组 20 64.75±7.78 9 3 7.62±2.97 4.18±0.69 266.55±78.50 5.04±2.12*# 1.76±0.74 F — 1.95 1.01△ 0.66△ 1.57 0.47 1.88 3.92 0.37 P — >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 < 0.05 >0.05 MS组内 — 73.699 — — 5.797 0.377 7 490.817 2.222 0.870 分组 n HDL/
(mmol/L)LDL/
(mmol/L)天冬氨酸氨基
转移酶/(IU/L)丙氨酸氨基
转移酶/(IU/L)TBil/
(μmol/L)总蛋白/
(g/L)Cr/
(mg/L)对照组 19 1.06±0.26 2.12±0.71 20.42±3.52 21.70±11.56 9.83±3.81 65.71±5.61 59.00±14.14 UAP组 31 0.93±0.17 2.21±0.69 20.97±6.90 19.16±10.60 10.32±4.39 65.87±4.97 65.52±10.72 AMI组 20 0.94±0.27 2.65±1.02 32.35±18.97*# 19.79±10.50 10.10±5.60 66.29±7.29 79.78±42.88* F — 2.14 2.57 7.59 0.33 0.07 0.05 3.59 P — >0.05 >0.05 < 0.01 >0.05 >0.05 >0.05 < 0.05 MS组内 — 0.052 0.644 126.697 117.477 21.426 34.586 626.592 △示χ2值: q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 1 病人基线资料比较(x±s)
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3组受试者血浆IL-1、IL-18含量和MLKL含量差异均有统计学意义(P < 0.01)(见表 2)。
分组 n IL-1/(pg/mL) IL-18/(pg/mL) MLKL/(ng/mL) 对照组 19 99.49±21.44 121.35±29.02 517.65±163.97 UAP组 31 126.19±20.79* 136.89±41.42 646.06±166.33* AMI组 20 146.60±17.32*# 168.45±35.13*# 745.03±180.70*# F — 27.03 52.00 8.77 P — < 0.01 < 0.01 < 0.01 MS组内 — 402.097 1 344.412 28 870.42 q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 2 3组外周血浆IL-1、IL-18和MLKL含量比较(x±s)
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PBMCs经CD14-抗体结合磁珠提纯并经流式鉴定,得到的人PBMCs纯度>90%(见图 1)。
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qRT-PCR结果显示UAP组MLK mRNA表达量较对照组明显升高(P < 0.05),AMI组MLKL mRNA高于对照组和UAP组(P < 0.05)(见表 3)。
分组 n MLKLmRNA 对照组 19 1.00 UAP组 31 1.17±0.17* AMI组 20 1.56±0.34*# F — 36.726 P — < 0.01 MS组内 — 3.045 q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 3 各组PBMCs中MLKL mRNA的表达(x±s)
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与对照组比较,UAP组MLKL、RIP1和RIP3的蛋白表达升高(P < 0.05);与UAP组比较AMI组MLKL、RIP1和RIP3的蛋白表达进一步升高(P < 0.05)(见表 4)。
分组 MLKL RIP1 RIP3 对照组 0.84±0.12 0.95±.08 0.82±0.10 UAP组 0.97±0.07* 1.03±.08* 0.93±0.14* AMI组 1.18±0.08*# 1.32±0.10*# 1.22±0.18*# F 72.43 91.67 41.17 P < 0.01 < 0.01 < 0.01 MS组内 0.520 0.541 0.022 q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 4 各组PBMCs中的RIP1、RIP3和MLKL蛋白表达变化(x±s)
RIP1-RIP3-MLKL信号通路在急性冠状动脉综合征病人外周血单核细胞中的表达
Expression of RIP1-RIP3-MLKL signaling pathway in peripheral blood monocytes of patients with acute coronary syndrome
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摘要:
目的研究急性冠状动脉综合征(ACS)病人外周血单核细胞(PBMCs)上刺激受体相互作用蛋白1(RIP1)-RIP3-混合谱系激酶结构域样假激酶(MLKL)信号通路的表达变化,并分析其可能机制。 方法选取初诊为ACS病人60例,其中不稳定型心绞痛(UAP)病人31例,急性心肌梗死(AMI)病人20例,对照组选取同期健康体检者19名。采集所有受试者外周静脉血,分别提取血浆和外周血单核细胞(PBMCs),酶联免疫吸附实验(ELISA)和比浊法分别检测血浆中白细胞介素(IL)-1和IL-18、MLKL的水平,运用Real Time- PCR检测MLKL的mRNA表达,免疫印迹法检测PBMCs中RIP1、RIP3和MLKL的蛋白表达。 结果3组受试者血浆IL-1、IL-18含量和MLKL含量差异均有统计学意义(P < 0.01),PBMCs中RIP1和RIP3蛋白表达增加(P < 0.05),MLKL的mRNA和蛋白表达亦增加(P < 0.05)。与UAP组比较,AMI组病人外周血浆中IL-1、IL-18和MLKL进一步升高,RIP1和RIP3的蛋白表达的进一步增加(P < 0.05),MLKL的mRNA和蛋白表达增高。 结论UAP和AMI病人体内存在不同程度的炎症活化,RIP1-RIP3-MLKL信号通路在PBMCs中的表达量随病情的严重程度逐渐增加,提示RIP1-RIP3-MLKL信号通路可能在ACS的不同类型发病中起重要作用。 -
关键词:
- 急性冠状动脉综合征 /
- RIP1-RIP3-MLKL信号通路 /
- 外周血单核细胞
Abstract:ObjectiveTo investigate the expression changes of RIP1-RIP3-MLKL signaling pathway in peripheral blood mononuclear cells(PBMCs) of patients with acute coronary syndrome(ACS) and analyze the mechanism. MethodsSixty patients with ACS were enrolled, including 31 patients with unstable angina(UAP) and 20 patients with acute myocardial infarction(AMI).The control group received 19 healthy cases.Peripheral venous blood was collected from all subjects, plasma and monocytes were extracted, and interleukin(IL)-1 and IL-18 were detected by enzyme-linked immunosorbent assay(ELISA) and turbidimetry.The level of MLKL was detected by real-time PCR.The expression of RIP1, RIP3 and MLKL in PBMCs were detected by Western blotting. ResultsThe levels of IL-1, IL-18 and MLKL in plasma of 3 groups were significantly different(P < 0.01), RIP1 and RIP3 protein expression increased in PBMCs(P < 0.05), mRNA and protein expression of MLKL were also increased(P < 0.05).Compared with the UAP group, IL-1, IL-18 and MLKL were increased in peripheral blood of patients with AMI, and the protein expression of RIP1 and RIP3 were increased(P < 0.05).The expressions of MLKL mRNA and protein expression were also increased. ConclusionsThere are different degrees of inflammatory activation in UAP and AMI patients.The expression of RIP1-RIP3-MLKL signaling pathway in PBMCs gradually increases with the severity of the disease, suggesting that RIP1-RIP3-MLKL signaling pathway may play an important role in ACS. -
表 1 病人基线资料比较(x±s)
分组 n 年龄/岁 高血压 糖尿病 WBC/
(×109/L)RBC/
(×1012/L)PLT/
(×109/L)TC/
(mmol/L)三酰甘油/
(mmol/L)对照组 19 59.74±7.56 18 3 6.35±2.78 4.28±0.76 235.37±116.24 3.93±1.30 1.52±1.23 UAP组 31 63.87±9.59 12 7 6.62±1.65 4.35±0.44 218.45±68.70 3.94±1.05 1.71±0.83 AMI组 20 64.75±7.78 9 3 7.62±2.97 4.18±0.69 266.55±78.50 5.04±2.12*# 1.76±0.74 F — 1.95 1.01△ 0.66△ 1.57 0.47 1.88 3.92 0.37 P — >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 < 0.05 >0.05 MS组内 — 73.699 — — 5.797 0.377 7 490.817 2.222 0.870 分组 n HDL/
(mmol/L)LDL/
(mmol/L)天冬氨酸氨基
转移酶/(IU/L)丙氨酸氨基
转移酶/(IU/L)TBil/
(μmol/L)总蛋白/
(g/L)Cr/
(mg/L)对照组 19 1.06±0.26 2.12±0.71 20.42±3.52 21.70±11.56 9.83±3.81 65.71±5.61 59.00±14.14 UAP组 31 0.93±0.17 2.21±0.69 20.97±6.90 19.16±10.60 10.32±4.39 65.87±4.97 65.52±10.72 AMI组 20 0.94±0.27 2.65±1.02 32.35±18.97*# 19.79±10.50 10.10±5.60 66.29±7.29 79.78±42.88* F — 2.14 2.57 7.59 0.33 0.07 0.05 3.59 P — >0.05 >0.05 < 0.01 >0.05 >0.05 >0.05 < 0.05 MS组内 — 0.052 0.644 126.697 117.477 21.426 34.586 626.592 △示χ2值: q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 2 3组外周血浆IL-1、IL-18和MLKL含量比较(x±s)
分组 n IL-1/(pg/mL) IL-18/(pg/mL) MLKL/(ng/mL) 对照组 19 99.49±21.44 121.35±29.02 517.65±163.97 UAP组 31 126.19±20.79* 136.89±41.42 646.06±166.33* AMI组 20 146.60±17.32*# 168.45±35.13*# 745.03±180.70*# F — 27.03 52.00 8.77 P — < 0.01 < 0.01 < 0.01 MS组内 — 402.097 1 344.412 28 870.42 q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 3 各组PBMCs中MLKL mRNA的表达(x±s)
分组 n MLKLmRNA 对照组 19 1.00 UAP组 31 1.17±0.17* AMI组 20 1.56±0.34*# F — 36.726 P — < 0.01 MS组内 — 3.045 q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 表 4 各组PBMCs中的RIP1、RIP3和MLKL蛋白表达变化(x±s)
分组 MLKL RIP1 RIP3 对照组 0.84±0.12 0.95±.08 0.82±0.10 UAP组 0.97±0.07* 1.03±.08* 0.93±0.14* AMI组 1.18±0.08*# 1.32±0.10*# 1.22±0.18*# F 72.43 91.67 41.17 P < 0.01 < 0.01 < 0.01 MS组内 0.520 0.541 0.022 q检验:与对照组比较*P < 0.05;与UAP组比较#P < 0.05 -
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