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. -
证明超声清创法可显著减轻烧伤残余创面的细菌负荷，减少术后血肿形成，促进移植皮片成活，从而缩短患者病程。
烧伤，尤其是深度烧伤会遗留严重瘢痕，对患者身心造成损害［1, 2, 3］。深度烧伤创面难以愈合或一次封闭困难，均会导致残余创面形成。临床上修复残余创面常采用清创+植皮术，而传统清创手术易误伤创面周围健康组织［4］，违背现代精准医学的治疗理念。近年来，超声清创技术在糖尿病足、下肢静脉溃疡、压疮、骨外露等创面修复［5, 6, 7, 8］中取得了良好效果，但目前国内鲜见将超声清创技术应用于烧伤残余创面的相关报告。解放军联勤保障部队第980医院（以下简称本单位）近年来将超声清创技术应用于烧伤残余创面，取得了良好的临床效果。
1. 对象与方法
本回顾性队列研究符合《赫尔辛基宣言》的基本原则，根据本单位伦理委员会政策，临床资料可以在不泄露患者身份信息的前提下分析、使用。
1.1 入选标准
纳入标准：（1）原发深Ⅱ度或Ⅲ度烧伤经清创植皮后仍存在残余创面；（2）创面分布相对集中，创面总面积≥1%TBSA，且至少存在1处残余创面直径≥5 cm。（3）对残余创面行传统清创法或超声清创法+植皮治疗者。排除标准：资料不全者。
1.2 患者一般资料及烧伤创面情况
2017年8月—2021年8月，本单位收治64例符合入选标准的患者，其中男41例、女23例，年龄为9个月~67岁，平均年龄31岁，烧伤总面积25%~90%TBSA，残余创面总面积1%~17%TBSA，致伤原因：火焰烧伤者36例，热液烫伤者23例，电烧伤者5例。根据创面清创方法，将患者分为超声清创组（34例）和传统清创组（30例）。2组患者性别、年龄、致伤原因、烧伤部位等一般情况比较，差异均无统计学意义（P>0.05）；2组患者烧伤总面积、残余创面面积、伤后清创时间等创面情况均相似（P>0.05）。见表1。清创前，2组患者的所有创面中均有细菌感染：超声清创组创面中检出金黄色葡萄球菌者12例、铜绿假单胞菌者15例、鲍曼不动杆菌者5例、粪肠球菌者3例、阴沟肠杆菌者2例，其中复合感染者3例；传统清创组创面检出金黄色葡萄球菌者11例、铜绿假单胞菌者15例、鲍曼不动杆菌者3例、肺炎克雷伯菌者2例、阴沟肠杆菌者2例，其中复合感染者3例。
表1 2组烧伤残余创面患者一般资料及其烧伤创面情况比较组别 例数 性别（例） 年龄（岁， 致伤原因（例） 烧伤部位（例） 烧伤总面积（%TBSA， 残余创面面积（%TBSA， 伤后清创时间（d， 男 女 火焰烧伤 热液烫伤 电烧伤 头面颈部 前后躯干 四肢 超声清创组 34 22 12 31 19 12 3 4 20 10 51 10.9 32 传统清创组 30 19 11 32 17 11 2 3 19 8 50 10.1 33 统计量值 χ2=0.01 t=0.30 χ2=0.10 χ2=0.14 t=0.29 t=0.89 t=0.71 P值 0.909 0.972 0.949 0.932 0.707 0.907 0.169 注：TBSA为体表总面积 1.3 治疗方法
2组患者入院后均行常规补液、换药等治疗，术前纠正电解质紊乱、低蛋白血症、贫血、血糖和血压异常等情况，选择敏感抗生素防治感染，改善创面微循环。术中全身麻醉下对创面进行清创：（1）超声清创组创面，采用超声清创机根据创面大小使用500~2 000 mL生理盐水清除坏死组织或陈旧性肉芽至新鲜点状出血，清创后以1∶250 000肾上腺素盐水浸润的纱布覆盖创面以减少出血。超声机功率设置（50%~80%额定功率）及清创时长根据肉芽生长、坏死组织残存及感染情况确定。（2）传统清创组创面，采用传统锐性清创的方式，清创同时以1∶250 000肾上腺素盐水浸润的纱布湿敷，必要时行电凝止血。清创结束后，2组创面均使用过氧化氢、氯已定、生理盐水等冲洗3~5遍后植皮。功能部位或对容貌影响较大的暴露区创面首选大张皮片植皮；非功能部位创面，尤其是供皮困难者，选择移植邮票皮。植皮后四肢等部位采用无菌纱布适当加压包扎；面部等不易包扎的部位予缝线打包加压固定。术后行抗生素治疗，每2~3天换药1次。对于Ⅰ期手术后未愈合创面行二次清创+植皮术，2组创面的清创方式均分别同其Ⅰ期。
1.4 观察指标
1.4.1 创面分泌物细菌阳性率
术后3 d，于创面分泌物较多处选取标本行微生物培养及药物敏感试验检测细菌情况并计算细菌阳性率。创面细菌阳性率=细菌阳性患者例数÷患者总例数×100%。
1.4.2 皮片成活率及皮下血肿发生率
术后7 d，对于采用邮票皮治疗者，若面积较大，则统计5处约5 cm×5 cm植皮区内成活皮片数量；若面积较小，则统计所有植皮区域内成活皮片数量［4］。对于采用大张皮植皮者，行大体观察直接评估皮片成活情况。皮片成活标准为贴附良好、颜色红润、按压后充血明显、无明显滑动。皮片成活率=成活皮片数÷移植皮片总数×100%。术后7 d，大体观察创面皮下血肿发生率，皮下血肿发生率=皮下血肿形成患者例数÷患者总例数×100%。
1.4.3 创面愈合时间及二次清创率
出院时，统计患者创面愈合时长（二次清创者以Ⅱ期创面愈合时间为准）及清创次数并计算二次清创率，二次清创率=二次清创患者例数÷患者总例数×100%。
1.5 统计学处理
采用SPSS 17.0统计软件对数据进行分析。符合正态分布的计量资料数据以
2. 结果
2.1 创面分泌物细菌阳性率
术后3 d，超声清创组创面感染金黄色葡萄球菌者2例、铜绿假单胞菌者2例，传统清创组创面感染金黄色葡萄球菌者5例、铜绿假单胞菌者3例、鲍曼不动杆菌者1例、肺炎克雷伯菌者1例、阴沟肠杆菌者1例；超声清创组患者创面细菌阳性率为11.8%（4/34）明显低于传统清创组的36.7%（11/30），χ2=5.51，P=0.019。
2.2 皮片成活率及皮下血肿发生率
术后7 d，超声清创组创面的移植皮片成活率为（92
2.3 创面愈合时间及二次清创率
出院时，超声清创组创面愈合时长为（11.0
2.4 典型病例
患者男，38岁，致伤原因为电烧伤，全身多处均为Ⅲ度烧伤，总面积为26%TBSA，主要位于左下肢（14%TBSA）及头面部（3%TBSA）、左上肢（3%TBSA）、背部（4%TBSA）、左臀部（2%TBSA），伤后被送至当地医院治疗，其间行全身多处创面削痂+Meek植皮术。患者于伤后25 d转入本单位。入院时，患者部分创面已经基本封闭，已愈合区域与肉芽创面相互交错；残余创面主要位于背部，面积为2%TBSA，其表面可见肉芽组织增生及稀薄分泌物。入院后，从患者创面分泌物中检出金黄色葡萄球菌，后续采用静脉输注抗生素抗感染、纠正电解质紊乱等措施改善患者全身状况，创面每2~3天换药1次。入院后第7天，在全身麻醉下采用超声清创机对患者背部残余创面进行清创，清创机功率设置为75%额定功率，使用约500 mL生理盐水冲洗创面。然后选择右侧背部皮肤为供皮区，切取相应大小刃厚皮片行邮票皮移植术并采用无菌纱布适当加压包扎。术后行抗生素治疗，每2~3天换药1次。术后3 d，取创面中少量分泌物行微生物培养，未检出细菌。术后7 d，创面皮片成活率约为98%，皮片下无明显血肿。术后12 d创面完全愈合。见图1。
1 超声清创法+邮票皮移植治疗背部烧伤患者残余创面的疗效。1A.伤后25 d入院时，创面中较少肉芽组织增生；1B.伤后32 d背部创面清创术前，见肉芽组织增生明显，突出皮面；1C.清创术中；1D.术后12 d，邮票皮片存活良好3. 讨论
浅Ⅱ度烧伤经适当处理后可愈合，但深Ⅱ度及Ⅲ度等深度烧伤创面愈合困难，勉强愈合后也会遗留瘢痕或反复破溃，严重影响机体外观和功能［9, 10, 11］。因此，对深度烧伤常在早期行外科手术修复，常形成残余创面需在Ⅱ期行清创植皮术。理想的清创方式应具备高效且尽量保留健康组织、减少出血、降低住院费用、减轻创面中细菌负荷及提高愈合率等特点［12］。烧伤残余创面存在已愈合区域与坏死组织或肉芽组织相互交叉、镶嵌分布、边缘不规则等特点，清创难度大，传统锐性清创法的应变效能差，会造成周围正常组织不必要的损伤，出血较多。这导致经验较少的年轻医师在治疗残余创面的过程中存在较多突出问题［4］。
超声清创法是近年来兴起的清创方式，为残余创面清创提供了新的选择［13］。高频超声以热效应为主，低频超声以机械效应为主，即分别具有空化效应与声流作用［14, 15, 16］。超声清创机采用低频、高能超声波加载喷射流技术，在冲洗创面过程中会产生空化效应，即通过超声波形成的空化泡崩塌产生的微射流及高压可清除坏死组织［17, 18, 19］。超声清创机操作灵活，可用于平面或腔隙等不同类型创面；还可根据坏死组织或肉芽组织情况选择不同的功率，达到最佳清创效果。同时，超声清创机可将液体以雾化的形式喷出，使液体全面接触创面以更精确地清除非活性组织，又避免了对周围正常组织的附带损伤［5，17，20］。
创面细菌负荷的减轻程度是反映清创效果的一个重要指标。创面细菌负荷与细菌生物膜形成密切相关，生物膜可保护细菌免受抗生素、防腐剂和宿主免疫的影响，还可逐步释放浮游细菌导致创面持续感染，严重影响创面皮片成活率［21, 22, 23, 24, 25, 26］。有研究显示超声不仅对革兰阳性、革兰阴性、需氧和厌氧细菌具有明显抗菌作用［27, 28］，还可以有效清除细菌生物膜［21］，显著减少细菌负荷，改善创面条件［29, 30, 31］。本研究结果显示，超声清创组术后3 d创面细菌阳性率显著低于传统清创组，同时前者的二次清创率也显著低于后者，提示超声清创法在清除创面坏死组织，减轻细菌负荷等方面具有显著作用，其结果与前述既往国内外研究结果一致。
此外，超声清创法可有效减轻术后炎症反应和疼痛，还可促进伤口组织中碱性FGF和EGF的高表达，改善创面微循环，促进血管生成和胶原沉积，提升组织再生和修复能力，这可能与超声产生的空化效应和声流作用等机械能刺激信号转导，继发广泛的细胞效应有关［18, 19，32, 33, 34］。本研究中超声清创组的皮片成活率显著高于传统清创组，且创面愈合时间显著短于传统清创组。移植皮片的成活率与皮下积血、积液等密切相关。相关研究指出，超声清创机可通过热效应达到减少出血的效果［5，35, 36］，但由于术中出血与组织液、膨胀液或冲洗液等混合在一起，难以精确估计出血量。但是本研究显示，超声清创组皮下血肿发生率显著低于传统清创组，提示减少术中出血和皮下血肿形成可能是超声清创法提高皮片成活率，促进创面愈合的机制之一。
综上，与传统清创法相比，超声清创法可在烧伤残余创面清创中彻底清除坏死组织和陈旧性肉芽组织、减轻细菌负荷、降低皮下血肿发生率，促进移植皮片成活，从而缩短创面愈合时间，为烧伤残余创面的修复提供了新的方法和选择。超声清创法对于提高烧伤残余创面的修复效果具有重要意义，值得临床推广。
戚欣欣、刘璐：实验操作、论文撰写；杨云稀、黄佳敏：数据整理与分析；孙炳伟：研究指导、论文修改、经费支持所有作者均声明不存在利益冲突 -
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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