Changes of heparin-binding protein in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells and neutrophils
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摘要:
目的 探讨严重烧伤患者休克期肝素结合蛋白（HBP）的变化及其在体外对人脐静脉血管内皮细胞（HUVEC）和中性粒细胞的影响。 方法 采用前瞻性观察性研究与实验研究方法。将2020年8—11月南京医科大学附属苏州医院烧伤整形科收治的符合入选标准的20例严重烧伤患者纳入严重烧伤组［男12例、女8例，年龄为44.5（31.0，58.0）岁］，同期招募该单位体检中心体检结果正常的20名健康志愿者纳入健康对照组［男13例、女7例，年龄为39.5（26.0，53.0）岁］。采用酶联免疫吸附测定（ELISA）法检测健康对照组志愿者血浆与严重烧伤组患者伤后48 h内血浆中HBP和组织金属蛋白酶抑制物1（TIMP-1）蛋白表达水平，分别对2组血浆中HBP和TIMP-1蛋白表达的相关性进行Pearson相关分析。取第4代对数生长期HUVEC进行实验，将细胞按随机数字表法（分组方法下同）分为常规培养的正常对照组（处理下同）与进行相应处理的重组HBP（rHBP）处理12 h组、rHBP处理24 h组、rHBP处理48 h组，采用实时荧光定量反转录PCR法检测细胞中TIMP-1的mRNA表达；将细胞分为正常对照组与进行相应处理的rHBP处理48 h组，采用蛋白质印迹法检测细胞中TIMP-1蛋白表达；将细胞分为正常对照组与加入相应试剂处理的单纯rHBP组、单纯抑蛋白酶多肽组、rHBP+抑蛋白酶多肽组（rHBP终物质的量浓度为200 nmol/L，抑蛋白酶多肽终质量浓度为20 μg/mL），培养48 h，采用ELISA法检测细胞培养上清液中TIMP-1蛋白表达水平。采用免疫磁珠分选法从前述10名健康志愿者外周静脉血中分离中性粒细胞，将细胞分为正常对照组与加入相应试剂处理的单纯重组TIMP-1（rTIMP-1）组、单纯佛波酯组、rTIMP-1+佛波酯组（rTIMP-1终质量浓度为500 ng/mL，佛波酯终物质的量浓度为10 nmol/L），培养1 h，采用免疫荧光法观测细胞中CD63蛋白表达，采用流式细胞术检测细胞中CD63蛋白阳性表达率，采用ELISA法检测细胞培养上清液中HBP和髓过氧化物酶（MPO）蛋白表达水平。正常对照组均于适宜时间点进行前述相关检测，细胞实验中各组样本数均为3。对数据行χ2检验、Mann-Whitney U检验、Kruskal-Wallis H检验、Tamhane T2检验。 结果 严重烧伤组患者血浆中HBP、TIMP-1蛋白表达水平分别为404.9（283.1，653.2）、262.1（240.6，317.4）ng/mL，均明显高于健康对照组志愿者的61.6（45.0，68.9）、81.0（66.3，90.0）ng/mL（Z值分别为-5.41、-5.21，P<0.01）。健康对照组志愿者血浆中HBP和TIMP-1蛋白表达相关性不强（P>0.05），严重烧伤组患者血浆中HBP和TIMP-1蛋白表达呈明显正相关（r=0.64，P<0.01）。与正常对照组比较，rHBP处理12 h组、rHBP处理24 h组、rHBP处理48 h组HUVEC中TIMP-1 mRNA表达水平均显著升高（t值分别为-3.58、-2.25、-1.26，P<0.05）。蛋白质印迹法检测显示，与正常对照组比较，rHBP处理48 h组HUVEC中TIMP-1蛋白表达明显增强。培养48 h，与正常对照组比较，单纯rHBP组HUVEC培养上清液中TIMP-1蛋白表达水平显著升高（t=9.43，P<0.05），单纯抑蛋白酶多肽组、rHBP+抑蛋白酶多肽组HUVEC培养上清液中TIMP-1蛋白表达水平均无明显变化（P>0.05）；与单纯rHBP组比较，rHBP+抑蛋白酶多肽组HUVEC培养上清液中TIMP-1蛋白表达水平显著下降（t=4.76，P<0.01）。培养1 h，免疫荧光法和流式细胞术检测的各组中性粒细胞CD63蛋白表达结果趋势一致。培养1 h，与正常对照组比较，单纯rTIMP-1组中性粒细胞中CD63蛋白阳性表达率及细胞培养上清液中HBP、MPO蛋白表达水平均无明显变化（P>0.05），单纯佛波酯组、rTIMP-1+佛波酯组中性粒细胞中CD63蛋白阳性表达率及细胞培养上清液中HBP、MPO蛋白表达水平均明显升高（t值分别为2.41、3.82，5.73、1.05、4.16、1.08，P<0.05或P<0.01）；与单纯佛波酯组比较，rTIMP-1+佛波酯组中性粒细胞中CD63蛋白阳性表达率及细胞培养上清液中HBP、MPO蛋白表达水平均明显下降（t值分别为5.26、2.83、1.26，P<0.05或P<0.01）。 结论 严重烧伤患者休克期血浆中HBP表达水平增加；HBP可在体外诱导HUVEC分泌TIMP-1，TIMP-1可降低人中性粒细胞CD63分子表达。 Abstract:Objective To investigate the changes of heparin-binding protein (HBP) in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells (HUVECs) and neutrophils in vitro. Methods Prospective observational and experimental research methods were used. Twenty severe burn patients who met the inclusion criteria and were admitted to the Department of Burns and Plastic Surgery of Affiliated Suzhou Hospital of Nanjing Medical University from August to November 2020 were included in severe burn group (12 males and 8 females, aged 44.5 (31.0, 58.0) years). During the same period, 20 healthy volunteers with normal physical examination results in the unit's Physical Examination Center were recruited into healthy control group (13 males and 7 females, aged 39.5 (26.0, 53.0) years). Enzyme-linked immunosorbent assay (ELISA) method was used to detect the protein expression levels of HBP and tissue inhibitor of metalloproteinase 1 (TIMP-1) in plasma of patients within 48 hours after injury in severe burn group and in plasma of volunteers in healthy control group. The correlation between protein expression of HBP and that of TIMP-1 in the plasma in the two groups was analyzed by Pearson correlation analysis. The fourth passage of HUVECs in logarithmic growth phase were used for the experiment. The HUVECs were divided into normal control group with routine culture (the same treatment below) and recombinant HBP (rHBP)-treated 12 h group, rHBP-treated 24 h group, and rHBP-treated 48 h group with corresponding treatment according to the random number table (the same grouping method below), and the mRNA expression of TIMP-1 in cells was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. The HUVECs were divided into normal control group and rHBP-treated 48 h group with corresponding treatment, and the protein expression of TIMP-1 in the cells was detected by Western blotting. The HUVECs were divided into normal control group, rHBP alone group, aprotinin alone group, and rHBP+aprotinin group treated with the corresponding reagents (with the final molarity of rHBP being 200 nmol/L and the final concentration of aprotinin being 20 μg/mL, respectively), cultured for 48 h, and ELISA was used to detect the protein expression of TIMP-1 in the culture supernatant of cells. The neutrophils were isolated from the peripheral venous blood of the aforementioned 10 healthy volunteers by immunomagnetic bead sorting, and the cells were divided into normal control group, recombinant TIMP-1 (rTIMP-1) alone group, phorbol acetate (PMA) alone group, and rTIMP-1+PMA group treated with corresponding reagents (with the final concentration of rTIMP-1 being 500 ng/mL and the final molarity of PMA being 10 nmol/L, respectively). After being cultured for 1 h, the expression of CD63 protein in cells was detected by immunofluorescence method, the positive expression rate of CD63 protein in cells was detected by flow cytometry, and the protein expression levels of HBP and myeloperoxidase (MPO) in the culture supernatant of cells were detected by ELISA. The normal control group underwent the above-mentioned related tests at appropriate time points. The number of samples was 3 in each group of cell experiment. Data were statistically analyzed with chi-square test, Mann-Whitney U test, Kruskal-Wallis H test, and Tamhane's T2 test. Results The protein expression levels of HBP and TIMP-1 in the plasma of patients in severe burn group were 404.9 (283.1, 653.2) and 262.1 (240.6, 317.4) ng/mL, respectively, which were both significantly higher than 61.6 (45.0, 68.9) and 81.0 (66.3, 90.0) ng/mL of volunteers in healthy control group (with Z values of -5.41 and -5.21, respectively, P<0.01). The correlation between the protein expression of HBP and that of TIMP-1 in the plasma of volunteers in healthy control group was not strong (P>0.05). The protein expression of HBP was significantly positively correlated with that of TIMP-1 in the plasma of patients in severe burn group (r=0.64, P<0.01). Compared with that in normal control group, the mRNA expression of TIMP-1 in HUVECs was significantly increased in rHBP-treated 12 h group, rHBP-treated 24 h group, and rHBP-treated 48 h group (with t values of -3.58, -2.25, and -1.26, respectively, P<0.05). Western blotting detection showed that compared with that in normal control group, the protein expression of TIMP-1 in HUVECs in rHBP-treated 48 h group was significantly enhanced. After 48 h of culture, compared with that in normal control group, the protein expression level of TIMP-1 in the culture supernatant of HUVECs in rHBP alone group was significantly increased (t=9.43, P<0.05), while the protein expression level of TIMP-1 in the culture supernatant of HUVECs didn't change significantly in aprotinin alone group or rHBP+aprotinin group (P>0.05); compared with that in rHBP alone group, the protein expression level of TIMP-1 in the culture supernatant of HUVECs in rHBP+aprotinin group was significantly decreased (t=4.76, P<0.01). After 1 h of culture, the trend of CD63 protein expression in neutrophils detected by immunofluorescence method and that by flow cytometry were consistent in each group. After 1 h of culture, compared with that in normal control group, the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells in rTIMP-1 alone group all had no significant changes (P>0.05), while the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells were all significantly increased in PMA alone group and rTIMP-1+PMA group (with t values of 2.41, 3.82, 5.73, 1.05, 4.16, and 1.08, respectively, P<0.05 or P<0.01); compared with that in PMA alone group, the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells in rTIMP-1+PMA group were all significantly decreased (with t values of 5.26, 2.83, and 1.26, respectively, P<0.05 or P<0.01). Conclusions The expression level of HBP in the plasma of severe burn patients is increased during shock stage. HBP can induce HUVECs to secrete TIMP-1 in vitro, and TIMP-1 can reduce the expression of CD63 molecule in human neutrophils. -
参考文献
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1 健康对照组20名志愿者血浆与严重烧伤组20例患者伤后48 h血浆中HBP和TIMP-1蛋白表达的相关性。1A.健康对照组，二者表达相关性不强，r=-0.19，P=0.434；1B.严重烧伤组，二者表达呈明显正相关，r=0.64，P=0.002
注：TIMP-1为组织金属蛋白酶抑制物1，HBP为肝素结合蛋白
2 3种分组处理后人脐静脉血管内皮细胞的TIMP-1的mRNA和蛋白表达。2A.4组细胞中TIMP-1 mRNA表达箱式图[样本数为3，M（Q1，Q3）]；2B.蛋白质印迹法检测的2组细胞中TIMP-1蛋白表达条带图；2C.培养48 h后4组细胞培养上清液中TIMP-1蛋白表达箱式图[样本数为3，M（Q1，Q3）]
注：TIMP-1为组织金属蛋白酶抑制物1；图2A中横坐标1、2、3、4分别为正常对照组（于适宜时间点检测，后同）、重组肝素结合蛋白（rHBP）处理12 h组、rHBP处理24 h组、rHBP处理48 h组；与正常对照组比较，aP<0.01；图2B中1、2分别为正常对照组、rHBP处理48 h组；图2C中横坐标1、2、3、4分别为正常对照组、单纯rHBP组、单纯抑蛋白酶多肽组、rHBP+抑蛋白酶多肽组，与正常对照组比较，aP<0.05；与单纯rHBP组比较，bP<0.01
3 4组从健康志愿者外周静脉血分离的中性粒细胞培养1 h后CD63蛋白表达 异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚×630，图中标尺为10 μm。3A、3B、3C.分别为正常对照组（于适宜时间点检测）CD63染色、细胞核染色、CD63与细胞核染色重叠图片，细胞核完整，CD63蛋白表达较多；3D、3E、3F.分别为单纯重组组织金属蛋白酶抑制物1（rTIMP-1）组CD63染色、细胞核染色、CD63与细胞核染色重叠图片，细胞核完整，图3D中CD63蛋白表达与图3A相近；3G、3H、3I.分别为单纯佛波酯组CD63染色、细胞核染色、CD63与细胞核染色重叠图片，细胞核完整，图3G中CD63蛋白表达明显多于图3A；3J、3K、3L.分别为rTIMP-1+佛波酯组CD63染色、细胞核染色、CD63与细胞核染色重叠图片，细胞核完整，图3J中CD63蛋白表达明显多于图3A但明显少于图3G
注：CD63阳性染色为黄绿色，细胞核阳性染色为蓝色
4 4组从健康志愿者外周静脉血分离的中性粒细胞培养1 h后细胞的CD63、HBP和MPO蛋白表达。4A.细胞中CD63蛋白阳性表达率流式直方图；4B.细胞培养上清液中HBP蛋白表达箱式图[样本数为3，M（Q1，Q3）]；4C.细胞培养上清液中MPO蛋白表达箱式图[样本数为3，M（Q1，Q3）]
注：rTIMP-1为重组组织金属蛋白酶抑制物1，HBP为肝素结合蛋白，MPO为髓过氧化物酶；图4B、4C横坐标中1、2、3、4分别为正常对照组（于适宜时间点检测）、单纯rTIMP-1组、单纯佛波酯组、rTIMP-1+佛波酯组；图4B中，与正常对照组比较，aP<0.01；与单纯佛波酯组比较，bP<0.05；图4C中，与正常对照组比较，aP<0.01，bP<0.05；与单纯佛波酯组比较，cP<0.05
表1 严重烧伤组患者与健康对照组志愿者一般资料比较
组别 例数/人数 性别（例） 年龄［岁，M（Q1，Q3）］ 平均动脉压［mmHg，M（Q1，Q3）］ 中性粒细胞计数 ［×109/L，M（Q1，Q3）］ 男 女 严重烧伤组 20 12 8 44.5（31.0，58.0） 92.8（85.1，100.5） 17.70（12.16，23.24） 健康对照组 20 13 7 39.5（26.0，53.0） 87.1（74.8，99.4） 7.35（6.48，8.21） 统计量值 χ2=0.11 Z=-0.21 Z=-0.66 Z=-3.17 P值 0.744 0.827 0.513 0.034 注：1 mmHg=0.133 kPa 
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