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目前心血管系统疾病已成为城乡居民总死亡因素的首要位置,其中冠心病(coronary heart disease,CHD)是心血管系统中最常见且致死率最高的疾病。CHD是一种位于冠状动脉(冠脉)壁上的以炎症和斑块积累为特征的疾病,这些斑块主要由脂质、钙和炎性细胞组成,并且这些病理改变最终可导致冠脉狭窄乃至供血不足而引起心肌功能障碍甚至死亡[1-4]。尽管已经发现了许多危险因素,如不良饮食、吸烟、高血压、糖尿病、高脂血症、肥胖等,CHD的病因和发病机制仍然尚未完全了解[5-6]。
白细胞介素-33(IL-33)是近年来发现的IL-1细胞因子家族的新成员,具有生物活性,可与靶细胞膜上的生长刺激表达基因2蛋白(ST2)结合形成跨膜复合体,形成IL-33/ST2信号通路,参与Th2活化相关的多种疾病发生与进展[7-9]。IL-33表达于多种组织器官中,而主要表达IL-33的是人类不同器官和组织中的各种内皮细胞,此外,在其他类型细胞,如上皮细胞、平滑肌细胞和纤维母细胞等也有IL-33的表达[10]。目前有研究[11]表明血清IL-33与CHD的发展存在一定的联系。肿瘤坏死因子(tumor necrosis factor,TNF-α)是一个至少由19种细胞因子组成的肽介质家族的成员,具有重要的促炎特性,在先天和适应性免疫、细胞增殖和凋亡过程中发挥关键作用[12]。有研究[13]表明TNF-α在动脉粥样硬化的病理进展中发挥着极为重要的作用,它可以通过促进内皮细胞黏附分子的表达,招募和激活炎症细胞,启动动脉壁内的炎性反应。核因子κB(nuclear factor kappa B, NF-κB)家族转录因子在细胞环境中作为应激源发挥重要作用,并控制重要调控基因的表达,如免疫、炎症、死亡和细胞增殖。NF-κB蛋白位于细胞质中,可被各种细胞刺激激活[14-15]。
目前,IL-33和TNF-α在CHD中的具体致病机制尚不明确,血清中IL-33和TNF-α水平与CHD的相关性也仍待继续研究,本研究拟检测不同病变程度CHD病人血清中IL-33和TNF-α活性水平,明确CHD病人病变程度与IL-33和TNF-α活性水平之间的关系以及外周血淋巴细胞中NF-κB蛋白水平变化在其中的关系,为CHD的临床诊疗提供一些新的思路。
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收集2019年1月至2020年6月蚌埠医科大学第一附属医院心内科确诊患有CHD的病人共167例,男98例,女69例,年龄(64.05±10.218)岁。所有病人均通过常规12导联心电图或Holter、心脏彩超和冠脉造影等检查明确病情。所有病人收治入院后及时采血完成血常规、生化常规检查并记录血压和体质量指数。CHD病人按照冠脉造影中记录的冠脉各支病变情况,依据Gensini评分[16]将病人分为:轻度病变组(< 30分,LS组)84例, 中度病变组(30~ < 60分,MS组)54例, 重度病变组(≥60分,HS组)29例。其中有CHD发作症状但行冠脉造影未见明显异常者作为对照组(CON组)共27例。4组一般资料差异均无统计学意义(P>0.05)(见表 1), 具有可比性。
分组 n 年龄/岁 男 高血压 吸烟 糖尿病 CON组 27 59.67±9.841 10 15 0 3 LS组 84 64.35±10.448 44 49 1 14 MS组 54 64.87±9.905 34 38 2 18* HS组 29 65.76±9.862 15 16 2 4 χ2 — 2.10# 8.80 2.99 3.94 8.58 P — >0.05 >0.05 >0.05 >0.05 >0.05 χ2分割检验:与CON组比较*P < 0.05;#示F值 表 1 4组病人入院时基线资料比较(x±s)
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纳入标准: (1)无严重精神系统疾病者; (2)病人及其家属均知晓并自愿接受; (3)有正常认知、书写、沟通能力者。排除标准: 既往有心脏瓣膜病、心肌病、急性心肌炎病人,既往有恶性肿瘤、风湿免疫系统疾病或内分泌及代谢性疾病、免疫功能低下、患有神经系统退行性改变疾病、心房颤动及感染性疾病病人[17],不能配合治疗或中途退出者。
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记录病人的姓名、性别、年龄等基础信息,吸烟饮酒史等一般信息,有无高血压、糖尿病、肝炎等慢性病病史,以及血细胞计数[红细胞、白细胞、血小板、中性粒细胞]、总胆红素、胆固醇及三酰甘油(TG)、高密度脂蛋白(HDL)、总蛋白、C反应蛋白(CRP)、B型脑钠肽(BNP)等相关指标。
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收集入院后24 h内肘静脉血(禁食12 h后)5 mL于肝素抗凝试管中,4 ℃,3 000 r/min,5 min离心,吸取上层血清,-80 ℃冰箱中备用;将剩余试管中液体转移至15 mL离心管中,加入等体积PBS混匀后,缓慢加入淋巴细胞分离液,4 ℃,2 000 r/min,20 min离心;缓慢吸出中间白色薄膜状淋巴细胞;加5倍以上体积的PBS洗2次,每次离心1 500 r/min,10 min;细胞沉淀用冻存液保存至液氮中备用。
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标本彻底融化后,采用ELISA法,使用上海羽朵生物科技有限公司的人IL-33和TNF-α ELISA试剂盒,分别检测IL-33和TNF-α的吸光度值并计算浓度。
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严格按照说明书步骤提取外周血淋巴细胞,在提取的细胞中加入适量RIPA裂解液(RIPA∶ PMSF=100∶ 1),冰上裂解30 min,4 ℃,12 000 r/min,10 min离心;取出上层蛋白悬液使用蛋白定量试剂盒(碧云天)进行定量;拟定每孔30 μg进行SDS-PAGE电泳,用PVDF膜4 ℃进行转膜,5%脱脂奶粉封闭后,4 ℃孵育一抗过夜,TBST洗膜4次,每次5 min;室温孵育二抗90 min,洗膜3次,每次10 min,应用ECL进行曝光显影。
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采用t检验、方差分析、q检验、χ2检验和χ2分割检验。
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各组间TG、总蛋白、HDL及低密度脂蛋白(LDL)、WBC、淋巴/单核细胞差异均无统计学意义(P>0.05)(见表 2)。
分组 n TC/(mmol/L) TG/(mmol/L) HDL/(mmol/L) 总蛋白/(g/L) 白细胞/(×109) 中性粒细胞/(×109) CON组 27 3.95±1.16 1.72±1.28 4.05±14.90 66.76±5.81 6.48±1.51 3.93±1.05 LS组 84 4.35±2.25 1.63±0.85 1.00±0.24 66.51±5.98 6.43±1.77 3.83±1.35 MS组 54 4.08±1.53# 1.91±2.37 0.95±0.22 67.54±8.49 6.48±1.54 3.91±1.22 HS组 29 5.90±5.35 1.78±0.72 0.96±0.15 68.94±6.92 6.60±2.08 4.58±1.64 F — 3.22 0.38 2.27 0.97 0.07 2.39 P — < 0.05 >0.05 >0.05 >0.05 >0.05 >0.05 MS组内 — 7.274 2.183 28.898 47.513 2.989 1.771 分组 n 红细胞/ (×109) 总胆红素/ μmol/L CR/ (μmol/L) CRP/ (mg/L) BNP/(pg/mL) CON组 27 4.35±0.43 12.01±8.3 62.41±17.5 5.81±7.01 271±697.65 LS组 84 4.37±0.40 11.04±5.0 68.18±21.6 5.08±10.71 315±957.28 MS组 54 4.43±0.51 9.95±3.8 67.29±16.7 4.37±7.21 338±610.86 HS组 29 4.35±0.43 10.14±4.1 71.83±17.9 4.17±4.09 380±640.12 F — 0.58 1.17 1.15 0.25 0.04 P — >0.05 >0.05 >0.05 >0.05 >0.05 MS组内 — 0.197 27.106 370.871 73.183 2 144.410 LSD-t检验:与LS组比较#P < 0.05 表 2 各组病人生化指标及血细胞计数比较(x±s)
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HS组血清IL-33水平较LS组CON组升高(P < 0.05),且随着冠脉病变严重程度加重其含量逐渐升高; 血清TNF-α水平也随着冠脉病变严重程度的加重而逐渐升高(P < 0.01),且两者之间呈正相关关系(r=0.663,P < 0.01)(见表 3)。
分组 n IL-33 TNF-α CON组 27 602.90±62.823 22.04±2.962 LS组 84 650.55±104.994* 26.65±3.76** MS组 54 687.83±126.926**# 32.69±4.462**## HS组 29 751.15±120.865**##△△ 34.30±5.096**#△ F — 10.04 65.98 P — < 0.01 < 0.01 MS组内 — 12 002.451 16.757 q检验: 与CON组比较*P < 0.05,**P < 0.01;与LS组比较#P < 0.05,##P < 0.01;与MS组比较△P < 0.05,△△P < 0.01 表 3 血清IL-33和TNF-α活性水平在CHD中的变化(x±s; pg/mL)
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Western blotting结果显示,与CON组(0.57±0.09)相比,CHD组(0.96±0.05)外周血淋巴细胞中NF-κB蛋白表达增加,差异有统计学意义(t=4.85,P < 0.01)(见图 1)。
血清IL-33和TNF-α活性及外周血淋巴细胞中NF-κB水平与冠心病不同程度病变的相关性分析
Correlation analysis of serum IL-33, TNF-α activity and NF-kB level in peripheral blood lymphocytes with different degrees of coronary heart disease
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摘要:
目的分析冠心病(CHD)冠状动脉病变严重程度与血清白细胞介素33(IL-33)和肿瘤坏死因子α(TNF-α)活性水平及外周血淋巴细胞中核因子κB(NF-κB)蛋白表达之间的相关性,探讨其在CHD发展中的作用及价值。 方法选择就诊于心内科并行冠状动脉造影检查并确诊为CHD的病人167例(CHD组),同时期行冠脉造影正常对照27例(CON组)。根据Gensini评分将167例CHD病人分成轻微病变组(< 30分,LS组)84例、中度病变组(30~ < 60分,MS组)54例和重度病变组(≥60分,HS组)29例。实验室行ELISA检测血清中IL-33和TNF-α活性水平,分析在不同冠状动脉病变程度下IL-33和TNF-α的活性水平差异,探讨IL-33和TNF-α活性水平与CHD的关联性。同时取CHD组及CON组的外周血淋巴细胞,提取蛋白,使用Western blotting检测NF-κB蛋白表达情况。 结果HS组血清总胆固醇(TC)与CON组和LS组、MS组相比升高(P < 0.05),HS组血清IL-33与TNF-α水平较MS组、LS组和对照组升高,并且随着冠状动脉病变程度的加重逐渐升高(P < 0.01),两者之间呈正相关关系(r=0.663,P < 0.01);CHD组中NF-κB蛋白表达低于CON组(P < 0.01)。 结论随着冠状动脉病变程度的加重,血清IL-3和TNF-α水平也逐渐升高,且两者间存在一定程度的线性相关,同时与CON组相比,CHD病人NF-κB蛋白表达显著升高。 Abstract:ObjectiveTo analyze the correlation of serum interleukin 33 (IL-33), tumor necrosis factor α (TNF-α) levels and nuclear factor κB (NF-κB) protein expression in peripheral blood lymphocytes with the severity of coronary heart disease (CHD), and explore its role and value in the development of CHD. MethodsA total of 167 patients (CHD group) admitted to Department of Cardiology with coronary angiography and confirmed as CHD were selected, and during the same period, 27 cases with normal coronary angiography were confirmed as control group(CON group).According to Gensini score, 167 CHD patients were divided into mild lesion group(< 30 points, LS group, 84 cases), moderate lesion group(≥30 points to < 60 points, MS group, 54 cases), and severe lesion group(≥60 points, HS group, 29 cases).Serum IL-33 and TNF-α levels were detected by ELISA, the differences of IL-33 and TNF-α levels in different degrees of coronary artery disease were analyzed, and the correlation between IL-33, TNF-α levels and CHD was analyzed.Meanwhile, the peripheral blood lymphocytes in CHD group and CON group were collected for protein extraction, and NF-κB protein expression was detected by Western blotting. ResultsSerum total cholesterol(TC) level in HS group was increased compared with that in CON group, LS group and MS group(P < 0.05), serum IL-33 and TNF-α levels in HS group were increased compared with those in MS group, LS group and CON group, and these parameters were gradually increased with the aggravation of coronary artery disease(P < 0.01).There was a positive correlation between serum IL-33, TNF-α levels and coronary artery disease(r=0.663, P < 0.01).The NF-κB protein expression in CHD group was significantly higher than that in CON group(P < 0.01). ConclusionsWith the increasing degree of coronary artery disease, serum IL-3 and TNF-α levels were gradually increased, and there was a certain degree of linear correlation.NF-κB protein expression in CHD group was significantly increased compared with that in CON group. -
Key words:
- coronary heart disease /
- interleukin-33 /
- tumor necrosis factor α /
- nuclear factor-κB
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表 1 4组病人入院时基线资料比较(x±s)
分组 n 年龄/岁 男 高血压 吸烟 糖尿病 CON组 27 59.67±9.841 10 15 0 3 LS组 84 64.35±10.448 44 49 1 14 MS组 54 64.87±9.905 34 38 2 18* HS组 29 65.76±9.862 15 16 2 4 χ2 — 2.10# 8.80 2.99 3.94 8.58 P — >0.05 >0.05 >0.05 >0.05 >0.05 χ2分割检验:与CON组比较*P < 0.05;#示F值 表 2 各组病人生化指标及血细胞计数比较(x±s)
分组 n TC/(mmol/L) TG/(mmol/L) HDL/(mmol/L) 总蛋白/(g/L) 白细胞/(×109) 中性粒细胞/(×109) CON组 27 3.95±1.16 1.72±1.28 4.05±14.90 66.76±5.81 6.48±1.51 3.93±1.05 LS组 84 4.35±2.25 1.63±0.85 1.00±0.24 66.51±5.98 6.43±1.77 3.83±1.35 MS组 54 4.08±1.53# 1.91±2.37 0.95±0.22 67.54±8.49 6.48±1.54 3.91±1.22 HS组 29 5.90±5.35 1.78±0.72 0.96±0.15 68.94±6.92 6.60±2.08 4.58±1.64 F — 3.22 0.38 2.27 0.97 0.07 2.39 P — < 0.05 >0.05 >0.05 >0.05 >0.05 >0.05 MS组内 — 7.274 2.183 28.898 47.513 2.989 1.771 分组 n 红细胞/ (×109) 总胆红素/ μmol/L CR/ (μmol/L) CRP/ (mg/L) BNP/(pg/mL) CON组 27 4.35±0.43 12.01±8.3 62.41±17.5 5.81±7.01 271±697.65 LS组 84 4.37±0.40 11.04±5.0 68.18±21.6 5.08±10.71 315±957.28 MS组 54 4.43±0.51 9.95±3.8 67.29±16.7 4.37±7.21 338±610.86 HS组 29 4.35±0.43 10.14±4.1 71.83±17.9 4.17±4.09 380±640.12 F — 0.58 1.17 1.15 0.25 0.04 P — >0.05 >0.05 >0.05 >0.05 >0.05 MS组内 — 0.197 27.106 370.871 73.183 2 144.410 LSD-t检验:与LS组比较#P < 0.05 表 3 血清IL-33和TNF-α活性水平在CHD中的变化(x±s; pg/mL)
分组 n IL-33 TNF-α CON组 27 602.90±62.823 22.04±2.962 LS组 84 650.55±104.994* 26.65±3.76** MS组 54 687.83±126.926**# 32.69±4.462**## HS组 29 751.15±120.865**##△△ 34.30±5.096**#△ F — 10.04 65.98 P — < 0.01 < 0.01 MS组内 — 12 002.451 16.757 q检验: 与CON组比较*P < 0.05,**P < 0.01;与LS组比较#P < 0.05,##P < 0.01;与MS组比较△P < 0.05,△△P < 0.01 -
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