Email Alerts
RSS
Volume 39 Issue 7
Jul.  2023
Turn off MathJax
Article Contents
Abstract
References
Article Navigation >  CHINESE JOURNAL OF BURNS AND WOUNDS  > 2023  >  39(7): 662-670
Yao MY,Zhang N,Zhang Q,et al.Effects of interleukin-4-modified gold nanozymes on the full-thickness skin defects in diabetic mice[J].Chin J Burns Wounds,2023,39(1):15-24.DOI: 10.3760/cma.j.cn501225-20220630-00275.
Citation: Wang XL,Li J,Bian YQ,et al.Influence of pH value on tube formation of human dermal microvascular endothelial cells and its molecular mechanism[J].Chin J Burns Wounds,2023,39(7):662-670.DOI: 10.3760/cma.j.cn501225-20220930-00429.
shu

Influence of pH value on tube formation of human dermal microvascular endothelial cells and its molecular mechanism

doi: 10.3760/cma.j.cn501225-20220930-00429

  • Department of Plastic and Burn Surgery, the Second Affiliated Hospital, Air Force Medical University, Xi'an 710038, China

Funds:

General Program of National Natural Science Foundation of China 31570986

Natural Science Basic Research Program of Shaanxi Province of China 2023-JC-QN-0916

More Information
    Abstract
  •   Objective   To explore the influence of pH value on tube formation of human dermal microvascular endothelial cells (HDMECs) and study its molecular mechanism, so as to provide theoretical basis for the study of promoting angiogenesis in the process of wound healing.   Methods   The experimental study methods were applied. HDMECs of 4 or 5 passages in the logarithmic growth phase were collected for experiments. Culture mediums with pH values of 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8 were prepared, and the cells were adaptively cultured (the same culture method below) for 24 h before further experiments being carried out. After another 36 h of culture, the relative fluorescence value of cytoplasmic pH value was measured by flow cytometry, and the correlation analysis between the relative fluorescence value of cytoplasmic pH value and the medium pH value was carried out. After another 1.5, 2.5, 3.5, 4.5, and 5.5 days of culture, the cell proliferation activity was detected with cell counting kit 8. Oris TM cell migration detection kit was used to detect the remaining area of cell migration at 0 (immediately), 24, and 48 h after removing the cell seeding stopper. Three-dimensional stromal gel cell tube formation experiment was carried out to detect the lumen diameter of tube formed by cells after another 48 h of culture. The protein expressions of phosphorylation sites 473 and 308 of protein kinase B (Akt) were detected by Western blotting after another 48 h of culture. The sample number was 3. Data were statistically analyzed with Pearson correlation analysis, one-way analysis of variance, analysis of variance for factorial design, analysis of variance for repeated measurement, and Bonferroni correction.   Results   After another 36 h of culture, the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 6.8-7.8 mediums were significantly higher than the level in pH 6.4 medium ( P<0.05); compared with those in pH 6.6-7.0 mediums, the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 7.4-7.8 mediums were significantly increased ( P<0.05), and the relative fluorescence value of cytoplasmic pH value of cells cultured in pH 6.6 medium was significantly lower than that in pH 7.0 or 7.2 mediun (with P values all <0.05); the relative fluorescence values of cytoplasmic pH value of cells cultured in pH 7.6 and 7.8 mediums were significantly higher than those in pH 7.2 and 7.4 mediums ( P<0.05). The relative fluorescence value of cytoplasmic pH value was significantly positively correlated with the medium pH value ( r=0.99, P<0.05). The proliferation activity was similar among cells cultured in 8 mediums of different pH values for another 1.5 days ( P>0.05). After another 2.5 days of culture, the proliferation activity of cells cultured in pH 6.4-6.8 mediums was significantly decreased compared with that in pH 7.6 medium ( P<0.05). After another 3.5 days of culture, the proliferation activity of cells cultured in pH 7.0-7.8 mediums was significantly higher than that in pH 6.4-6.8 mediums ( P<0.05); compared with that in pH 7.6 medium, the proliferation activity of cells cultured in pH 7.0-7.4 and 7.8 mediums was significantly decreased ( P<0.05). After another 4.5 or 5.5 days of culture, the proliferation activity of cells cultured in pH 6.8-7.8 mediums was significantly higher than that in pH 6.4 medium ( P<0.05); compared with that in pH 6.6 and 6.8 mediums, the proliferation activity of cells cultured in pH 7.0-7.8 mediums was significantly increased ( P<0.05). After another 4.5 days of culture, the proliferation activity of cells cultured in pH 7.6 medium was significantly higher than that in pH 7.0 medium ( P<0.05). After another 5.5 days of culture, the proliferation activity of cells cultured in pH 7.2-7.6 mediums was significantly increased compared with that in pH 7.0 medium ( P<0.05); the proliferation activity of cells cultured in pH 7.2 and 7.4 mediums was significantly lower than that in pH 7.6 medium (with P values all <0.05) but significantly higher than that in pH 7.6 medium (with P values all <0.05). Immediately after removing the cell seeding stopper, the remaining migration areas were similar among cells cultured in 8 mediums of different pH values ( P>0.05). At 24 h after removing the cell seeding stopper, the remaining migration areas of cells cultured in pH 6.6-7.8 mediums were significantly smaller than the area in pH 6.4 medium ( P<0.05); compared with those in pH 6.6 and 6.8 mediums, the remaining migration areas of cells cultured in pH 7.0 to 7.6 mediums were significantly reduced ( P<0.05). At 48 h after removing the cell seeding stopper, compared with those in pH 6.4 and 6.6 mediums, the remaining migration areas of cells cultured in pH 7.0-7.8 mediums were significantly reduced ( P<0.05); the remaining migration areas of cells cultured in pH 7.2 and 7.4 mediums were significantly smaller than those in pH 6.8, 7.0, and 7.8 mediums ( P<0.05) but significantly larger than the area in pH 7.6 medium ( P<0.05); the remaining migration area of cells cultured in pH 7.6 medium was significantly smaller than that in pH 6.8 or 7.8 medium (with P values all <0.05). After another 48 h of culture, the lumen diameters of tubes formed by cells cultured in pH 7.0, 7.2, 7.4, 7.6, and 7.8 mediums were (5.0±0.5), (7.6±0.9), (8.5±0.7), (11.0±0.8), and (5.3±0.8) μm, respectively, which were significantly longer than (2.8±0.8) μm in pH 6.4 medium ( P<0.05); the lumen diameters of tubes formed by cells cultured in pH 6.6 ((4.2±0.3) μm), 6.8 ((4.5±0.6) μm), 7.0, and 7.8 mediums were significantly shorter than the diameter in pH 7.6 medium ( P<0.05). After another 48 h of culture, compared with those in pH 6.4 and 6.6 mediums, the protein expressions of Akt phosphorylation sites 473 and 308 of cells cultured in pH 6.8 to 7.8 mediums were significantly increased ( P<0.05). Moreover, the protein expression of Akt phosphorylation site 308 of cells cultured in pH 6.6 medium was significantly higher than that in pH 6.4 medium ( P<0.05); compared with the expression in pH 6.8 medium, the protein expressions of Akt phosphorylation site 473 of cells cultured in pH 7.0 and 7.4-7.8 mediums were significantly increased ( P<0.05); compared with the expression in pH 7.6 medium, the protein expressions of Akt phosphorylation site 473 of cells cultured in pH 7.0-7.4 and 7.8 mediums were significantly decreased ( P<0.05); compared with the expression in pH 7.8 medium, the protein expressions of Akt phosphorylation site 308 of cells cultured in pH 7.0 to 7.6 mediums were significantly increased ( P<0.05).   Conclusions   pH value can regulate the lumen diameter of HDMEC-formed capillaries, which is closely related to the activation of Akt. 7.2-7.6 is the appropriate pH value for constructing tissue engineered capillaries.

     

    • FullText(HTML)
  • 下载: 全尺寸图片 幻灯片

    (1)表明氧化应激参与了高压电烧伤大鼠伤后肾脏损伤的病理过程,且肾脏损伤呈进行性加重,为高压电烧伤后肾脏损伤的机制研究提供了新思路。

    (2)表明灯盏花素可以减轻高压电烧伤大鼠伤后肾脏氧化应激损伤,为高压电烧伤后肾脏损伤的临床治疗提供了依据。

    Highlights:

    (1)It showed that oxidative stress was involved in the pathological process of kidney injuries in rats after high-voltage electric burns, and the kidney injury was progressively aggravated, which provided a new idea for studying the mechanism of kidney injury after high-voltage electric burns.

    (2)It showed that breviscapine could alleviate renal oxidative stress injuries in rats with high-voltage electric burns, which provided the basis for the clinical treatment of kidney injuries after high-voltage electric burns.

    电力技术的不断进步,促使电力在工业生产、家庭生活以及其他社会各类活动中得到广泛应用,因此电烧伤的发病率呈上升趋势。尽管电烧伤患者只占所有烧伤患者的9.1%[1],但相较于热力烧伤其损害程度更重。多项研究表明,相较于低压电烧伤,高压电烧伤通常会对组织造成更深层次、更广泛的损害,并更容易导致脏器损伤[2, 3, 4, 5]。近年来的研究显示,高压电烧伤可导致渐进性微循环障碍及血管、肌肉的进行性损伤[6, 7, 8]。本课题组先前的研究证实,高压电烧伤后过度的氧化应激反应会造成心、肝、脑损伤[9, 10, 11]。肾脏作为对缺血缺氧敏感的重要脏器[12],易受高压电烧伤的影响并发生损伤,但目前相关研究较少。灯盏花素是一种从灯盏花中提取的黄酮类有效成分,具有抗氧化、抗炎以及改善循环等作用[13]。本研究旨在探讨高压电烧伤后大鼠肾脏氧化应激损伤的特点,并观测灯盏花素对高压电烧伤大鼠肾损伤的干预效果。

    1.   材料与方法

    本实验研究经河北医科大学第三医院动物实验伦理委员会审批通过,遵循河北医科大学第三医院和国家有关实验动物管理和使用的相关规定。

    1.1   动物和主要试剂及仪器来源

    160只健康清洁级8~10周龄、体重270~320 g雄性SD大鼠购自北京华阜康生物科技股份有限公司,许可证号：SCXK（冀）2019-0008。肌酐测定试剂盒、尿素氮测试盒、过氧化氢酶（catalase,CAT）测定试剂盒购自南京建成生物工程研究所,大鼠晚期氧化蛋白产物（advanced oxidation protein product,AOPP）ELISA试剂盒、大鼠Klotho ELISA试剂盒购自武汉华美生物工程有限公司,灯盏花素注射液（每5毫升注射液中含20 mg灯盏花素）购自运城市石药银湖制药有限公司。TC-30-20KVA型调压器和YDJ-10KVA型实验变压器购自武汉市得福电气有限公司,PG270A型数字钳型电流表购自杭州申华电工仪表有限公司,M200PRO型酶标仪购自瑞士TECAN集团公司,CenLee4K型低速离心机购自湖南湘立科学仪器有限公司,KZ-III-F低温型研磨仪购自武汉赛维尔生物科技有限公司,HLD-5003型电子天平购自杭州友恒称重设备有限公司,BX43型光学显微镜购自日本OLYMPUS株式会社。

    1.2   动物分组与处理

    将160只大鼠按随机数字表法分为假伤组、电烧伤组、盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组,其中假伤组、电烧伤组各60只大鼠,余4组各10只大鼠。将假伤组和电烧伤组大鼠按伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周6个时间点,分为每个时间点10只。

    按照本课题组的高压电烧伤大鼠模型制作方法[8, 9],将6组大鼠经腹腔按10 mg/kg剂量注射30 g/L戊巴比妥钠溶液麻醉后剃除左前肢及右后肢毛发,仰卧位固定于绝缘动物实验台上,连接调压器及实验变压器,大鼠左前肢连接电流入口电极板,右后肢连接电流出口电极板。除假伤组大鼠不通电致假伤外,余5组大鼠均接受输出电压3 kV、电流强度（1.92±0.24）A持续通电3 s,造成电流入口和出口处各1 cm×1 cm的高压电烧伤创面,深达肌肉、筋膜。伤后立即对6组大鼠经腹腔按5 mL/kg剂量注射生理盐水行液体复苏1次,对盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别另经腹腔按5 mL/kg剂量注射生理盐水及0.4、1.6、4.0 g/L灯盏花素,每24小时1次,至伤后72 h。模型制作成功后14只大鼠死亡,包括伤后24 h、48 h、72 h、1周电烧伤组各1、2、2、1只,伤后72 h盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组各4、1、2、1只。将各个时间点各组存活大鼠分别行下腔静脉采血2 mL后处死。

    1.3   指标检测

    1.3.1   肾/体比

    将大鼠处死前称体重,处死后立即取下双侧肾脏组织,去除周围脂肪及包膜,纱布吸干表面液体后,迅速放于电子天平上称重,并计算肾/体比,肾/体比=肾脏质量÷体重×100%。

    1.3.2   肾脏组织病理学变化和肾小管与肾间质损伤评分

    取假伤组,伤后8 h、24 h、48 h、72 h和1周的电烧伤组,伤后72 h的盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组各4只大鼠的左肾上极组织,制备石蜡切片（厚5 μm）后行HE染色,于光学显微镜200倍放大倍数下观察肾脏组织病理学变化。

    取每只大鼠1张切片,每张切片选2个视野,采用半定量病理评分系统[14, 15]行肾小管与肾间质损伤评分,评分标准：以出现肾小管上皮细胞肿胀、空泡样变性、坏死脱落和间质炎症细胞浸润、出血为肾小管与肾间质损伤。其中0分为无肾小管与肾间质损伤,1分为肾小管与肾间质损伤病变范围<25%,2分为肾小管与肾间质损伤病变范围为25%~50%,3分为肾小管与肾间质损伤病变范围>50%。评分取均值。

    1.3.3   血清肌酐和尿素氮水平

    取大鼠下腔静脉血2 mL,于离心半径13.5 cm、3 000 r/min离心10 min,分离得到血清样本。将血清样本平均分为2份,一份按照肌酐测定试剂盒说明书,通过肌氨酸氧化酶法检测血清肌酐水平；一份按照尿素氮测试盒说明书,通过脲酶法检测血清尿素氮水平。以上实验均重复2次,结果取均值。

    1.3.4   肾组织上清液中CAT活力、AOPP水平及Klotho蛋白水平

    取大鼠右肾皮质50 mg,按照组织重量与生理盐水体积比例为1∶9,采用研磨仪制备成组织匀浆,取组织匀浆上清液,按照CAT测定试剂盒说明书,通过钼酸铵法检测大鼠肾组织上清液中CAT活力。取大鼠右肾皮质100 mg,按照组织重量与生理盐水体积比例为1∶10,采用研磨仪制备成组织匀浆,将组织匀浆上清液平均分为2份,一份按照大鼠AOPP ELISA试剂盒说明书、一份按照大鼠Klotho ELISA试剂盒说明书,通过ELISA法分别检测大鼠肾组织上清液中AOPP水平、Klotho蛋白水平。以上实验均重复2次,结果取均值。

    1.4   统计学处理

    采用SPSS 26.0统计软件进行数据分析。符合正态分布的计量资料数据用x¯±s表示,多时间点的组间比较行析因设计方差分析、独立样本t检验,电烧伤组组内比较行LSD检验；多组间总体比较行单因素方差分析,多重比较行LSD检验。不符合正态分布的计量资料数据用M（min,max）表示,2组间比较行Mann-Whitney U检验,电烧伤组组内比较行Bonferroni校正；多组间比较行Kruskal-Wallis H检验,组间两两比较行Bonferroni校正。肾小管与肾间质损伤评分为等级资料,以M（min,max）表示,组间总体比较行Kruskal-Wallis H检验,组间两两比较行Bonferroni校正。P<0.05为差异有统计学意义。

    2.   结果

    2.1   肾/体比

    伤后8 h、48 h、72 h、1周,电烧伤组大鼠肾/体比均明显高于假伤组（t值分别为-0.52、-3.75、-4.05、-2.25,P值分别为<0.001、0.002、0.001、0.038）；电烧伤组大鼠肾/体比组内各时间点比较,差异均无统计学意义（P>0.05）。见表1。

    Table  1.  2组大鼠伤后各时间点肾/体比比较（%,x¯±s）
    组别0 h8 h24 h48 h72 h1周
    假伤组0.79±0.070.76±0.030.79±0.040.78±0.040.81±0.040.82±0.10
    电烧伤组0.84±0.080.87±0.06a0.83±0.060.87±0.05a0.93±0.08a0.95±0.14a
    注：假伤组大鼠不通电致假伤,各时间点样本数均为10；电烧伤组大鼠致高压电烧伤,伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周样本数分别为10、10、9、8、8、9；处理因素主效应,F=43.43,P<0.001；时间因素主效应,F=3.88,P=0.003；两者交互作用,F=1.09,P=0.370；与假伤组比较,aP<0.05
    下载: 导出CSV 
    | 显示表格

    伤后72 h,电烧伤组、盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠肾/体比分别为（0.93±0.08）%、（0.92±0.07）%、（0.90±0.05）%、（0.88±0.05）%、（0.81±0.05）%,组间总体比较,差异有统计学意义（F=5.52,P=0.001）。与电烧伤组、盐水组、低灯盏花素组、中灯盏花素组比较,高灯盏花素组大鼠肾/体比明显降低（P值分别为<0.001、0.001、0.002、0.013）；与电烧伤组、盐水组比较,低灯盏花素组（P值分别为0.383、0.144）、中灯盏花素组（P值分别为0.617、0.287）大鼠肾/体比无明显变化；低灯盏花素组与中灯盏花素组、电烧伤组与盐水组大鼠肾/体比比较,差异均无统计学意义（P值分别为0.517、0.764）。

    2.2   肾脏组织病理学变化和肾小管与肾间质损伤评分

    与假伤组比较,电烧伤组大鼠伤后8 h~1周肾小球出现系膜基质增多、毛细血管充血和系膜细胞增生,肾小管出现上皮细胞肿胀、空泡样变性和坏死脱落,肾间质出现炎症细胞浸润和出血,且损伤程度随时间延长而加重。伤后72 h,与电烧伤组和盐水组比较,低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠上述肾组织损伤程度减轻,其中高灯盏花素组大鼠肾组织损伤减轻最为明显。见图1。

    图  1  6组大鼠伤后各时间点肾脏组织病理学变化 苏木精-伊红×200。1A.假伤组大鼠肾小管上皮细胞排列整齐、大小一致、染色均匀、界限清楚,未见水肿征象,肾间质中未见炎症细胞浸润；1B、1C、1D、1E、1F.分别为电烧伤组伤后8 h、24 h、48 h、72 h、1周情况,相较于图1A,图1B~1F出现不同程度肾小球系膜细胞增生和基质增多、肾小管上皮细胞坏死脱落、肾间质炎症细胞浸润；1G、1H、1I、1J.分别为盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组伤后72 h情况,相较于图1E、1G,图1H~1J肾小球系膜细胞增生和基质增多、肾小管上皮细胞坏死脱落、肾间质炎症细胞浸润程度减轻
    注：假伤组大鼠仅不通电致假伤,电烧伤组、盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠均致高压电烧伤,其中电烧伤组大鼠不进行药物干预,盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别经腹腔注射生理盐水或低、中、高浓度灯盏花素
    下载: 全尺寸图片 幻灯片

    电烧伤组伤后8 h、24 h、48 h、72 h和1周及假伤组大鼠肾小管与肾间质损伤评分依次为1.0（0,1.0）、1.0（0,1.0）、1.0（1.0,2.0）、1.5（1.0,2.0）、2.0（1.0,2.0）、0（0,1.0）分,组间总体比较,差异有统计学意义（H=13.03,P=0.023）。与假伤组比较,电烧伤组大鼠伤后48 h、72 h、1周肾小管与肾间质损伤评分均明显升高（P值分别为0.044、0.011、0.032）,伤后8、24 h肾小管与肾间质损伤评分无明显变化（P值均为0.320）；与电烧伤组伤后8 h比较,电烧伤组大鼠伤后1周肾小管与肾间质损伤评分明显升高（P=0.038）,伤后24、48、72 h肾小管与肾间质损伤评分无明显变化（P值分别为>0.999、0.306、0.122）；与电烧伤组伤后24 h比较,电烧伤组大鼠伤后1周肾小管与肾间质损伤评分明显升高（P=0.038）,伤后48、72 h肾小管与肾间质损伤评分无明显变化（P值分别为0.306、0.122）；与电烧伤组伤后48 h比较,电烧伤组大鼠伤后72 h、1周肾小管与肾间质损伤评分无明显变化（P值分别为0.599、0.293）；与电烧伤组伤后72 h比较,电烧伤组大鼠伤后1周肾小管与肾间质损伤评分无明显变化（P=0.599）。

    伤后72 h,电烧伤组、盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠肾小管与肾间质损伤评分依次为2.0（1.0,2.0）、1.5（1.0,2.0）、1.0（1.0,2.0）、1.0（1.0,2.0）、1.0（0,1.0）分,组间总体比较,差异无统计学意义（H=3.83,P=0.281）。

    2.3   血清肌酐和尿素氮水平

    伤后8 h、24 h、48 h、72 h、1周,电烧伤组大鼠血清肌酐和尿素氮水平均明显高于假伤组（Z值分别为-2.00、-2.37、-2.62、-2.67、-3.67和-2.34、-3.11、-3.43、-3.11、-3.51,P值分别为0.045、0.018、0.009、0.008、<0.001和0.019、0.001、0.001、0.001、<0.001）。与电烧伤组伤后0 h比较,电烧伤组大鼠伤后72 h、1周血清肌酐水平均明显升高（P<0.05）；与电烧伤组伤后8 h比较,电烧伤组大鼠伤后72 h、1周血清肌酐水平均明显升高（P<0.05）；与电烧伤组伤后24 h比较,电烧伤组大鼠伤后1周血清肌酐水平明显升高（P<0.05）。电烧伤组大鼠组内各时间点血清尿素氮水平比较,差异均无统计学意义（P>0.05）。见表2。

    Table  2.  2组大鼠伤后各时间点血清肌酐和尿素氮水平比较[M（min,max）]
    组别与指标0 h8 h24 h48 h72 h1周
    假伤组
    肌酐（μmol/L）46（35,64）45（33,63）45（31,63）51（31,66）46（30,64）47（30,59）
    尿素氮（mmol/L）9.0（6.4,11.4）8.6（5.0,10.5）7.9（5.2,11.4）8.7（7.0,11.4）8.1（6.4,12.6）8.2（4.1,12.6）
    电烧伤组
    肌酐（μmol/L）55（37,79）55（45,80）a59（54,88）a66（57,89）a78（7,95）abc74（62,102）abcd
    尿素氮（mmol/L）10.1（6.7,30.5）13.7（6.8,20.4）a12.6（9.3,44.4）a16.3（10.9,30.9）a13.9（8.9,45.6）a18.8（11.2,37.4）a
    注：假伤组大鼠不通电致假伤,各时间点样本数均为10；电烧伤组大鼠致高压电烧伤,伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周样本数分别为10、10、9、8、8、9；与假伤组比较,aP<0.05；与电烧伤组伤后0 h比较,bP<0.05；与电烧伤组伤后8 h比较,cP<0.05；与电烧伤组伤后24 h比较,dP<0.05
    下载: 导出CSV 
    | 显示表格

    伤后72 h,5组电烧伤大鼠血清肌酐水平组间总体比较,差异无统计学意义（P>0.05）；与电烧伤组比较,低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠血清尿素氮水平均明显降低（P<0.05）；与盐水组比较,中灯盏花素组、高灯盏花素组大鼠血清尿素氮水平均明显降低（P<0.05）。见表3。

    Table  3.  5组电烧伤大鼠伤后72 h血清肌酐和尿素氮水平比较[M（min,max）]
    组别样本数肌酐（μmol/L）尿素氮（mmol/L）
    电烧伤组878（7,95）13.9（8.9,45.6）
    盐水组680（18,91）12.5（8.9,30.5）
    低灯盏花素组974（6,92）10.5（7.0,11.9）a
    中灯盏花素组861（50,95）8.6（7.3,16.9）ab
    高灯盏花素组957（43,89）8.7（7.5,13.0）ab
    H值8.9715.37
    P值0.0620.004
    注：5组大鼠均致高压电烧伤,其中电烧伤组大鼠不进行药物干预,盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别经腹腔注射生理盐水或低、中、高浓度灯盏花素；H值、P值为组间各指标总体比较所得；与电烧伤组比较,aP<0.05；与盐水组比较,bP<0.05
    下载: 导出CSV 
    | 显示表格

    2.4   肾组织上清液中CAT活力、AOPP水平及Klotho蛋白水平

    伤后48 h、72 h、1周,电烧伤组大鼠肾组织上清液中CAT活力均明显低于假伤组（Z值分别为-2.22、-2.13、-3.51,P值分别为0.026、0.034、<0.001）；伤后8 h、24 h、48 h、72 h、1周,电烧伤组大鼠肾组织上清液中AOPP水平均明显高于假伤组（Z值分别为-2.00、-3.15、-2.71、-2.04、-2.33,P值分别为0.045、0.002、0.007、0.041、0.020）；伤后0 h~1周,假伤组与电烧伤组大鼠肾组织上清液中Klotho蛋白水平比较,差异均无统计学意义（P>0.05）。与电烧伤组伤后0 h比较,电烧伤组大鼠伤后72 h和1周肾组织上清液中CAT活力及伤后48 h、72 h、1周肾组织上清液中Klotho蛋白水平均明显降低（P<0.05）；与电烧伤组伤后8 h比较,电烧伤组大鼠伤后72 h和1周肾组织上清液中CAT活力及伤后48 h、72 h、1周肾组织上清液中Klotho蛋白水平均明显降低（P<0.05）；与电烧伤组伤后24 h比较,电烧伤组大鼠伤后72 h和1周肾组织上清液中CAT活力均明显降低（P<0.05）；与电烧伤组伤后48 h比较,电烧伤组大鼠伤后1周肾组织上清液中CAT活力明显降低（P<0.05）。电烧伤组大鼠组内各时间点肾组织上清液中AOPP水平比较,差异均无统计学意义（P>0.05）。见表4。

    Table  4.  2组大鼠伤后各时间点肾组织上清液中CAT活力、AOPP水平及Klotho蛋白水平比较[M（min,max）]
    组别与指标0 h8 h24 h48 h72 h1周
    假伤组
    CAT活力（U/mgprot）36.2（17.9,109.1）40.0（13.7,94.6）37.5（3.6,124.4）44.6（15.1,61.5）38.5（3.6,140.2）43.0（14.7,120.9）
    AOPP水平（μmol/L）43.8（7.2,117.4）44.4（16.3,92.4）50.5（25.7,60.1）47.2（38.3,74.4）48.6（36.5,96.0）46.3（8.9,92.4）
    Klotho蛋白水平（ng/mL）1.8（0.3,3.5）2.2（0.3,3.6）1.4（0.1,5.5）1.8（0.2,4.1）1.8（0.3,5.4）1.6（0,7.8）
    电烧伤组
    CAT活力（U/mgprot）45.9（9.7,84.9）47.2（17.3,89.6）31.8（18.0,43.6）27.3（11.7,35.0）a14.6（12.6,23.6）abcd9.0（3.9,21.9）abcde
    AOPP水平（μmol/L）53.1（42.4,88.7）72.7（38.5,127.3）a64.9（52.7,116.3）a71.3（46.8,97.6）a76.0（32.6,88.7）a75.2（55.9,92.5）a
    Klotho蛋白水平（ng/mL）1.8（1.1,3.5）2.3（1.5,2.8）1.8（1.2,2.2）1.2（0.6,1.8）bc0.7（0.5,2.0）bc0.7（0.2,2.7）bc
    注：假伤组大鼠不通电致假伤,各时间点样本数均为10；电烧伤组大鼠致高压电烧伤,伤后0 h（即刻）、8 h、24 h、48 h、72 h和1周样本数分别为10、10、9、8、8、9；CAT为过氧化氢酶,AOPP为晚期氧化蛋白产物；与假伤组比较,aP<0.05；与电烧伤组伤后0 h比较,bP<0.05；与电烧伤组伤后8 h比较,cP<0.05；与电烧伤组伤后24 h比较,dP<0.05；与电烧伤组伤后48 h比较,eP<0.05
    下载: 导出CSV 
    | 显示表格

    伤后72 h,5组电烧伤大鼠肾组织上清液中Klotho蛋白水平组间总体比较,差异无统计学意义（P>0.05）；与电烧伤组比较,低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠肾组织上清液中CAT活力均明显升高（P<0.05）,中灯盏花素组、高灯盏花素组大鼠肾组织上清液中AOPP水平均明显降低（P<0.05）；与盐水组比较,中灯盏花素组、高灯盏花素组大鼠肾组织上清液中CAT活力均明显升高（P<0.05）,AOPP水平均明显降低（P<0.05）；与低灯盏花素组比较,高灯盏花素组大鼠肾组织上清液中CAT活力明显升高（P<0.05）。见表5。

    Table  5.  5组电烧伤大鼠伤后72 h肾组织上清液中CAT活力、AOPP水平及Klotho蛋白水平比较[M（min,max）]
    组别样本数CAT活力（U/mgprot）AOPP水平（μmol/L）Klotho蛋白水平（ng/mL）
    电烧伤组814.6（12.6,23.6）76.0（32.6,88.7）0.7（0.5,2.0）
    盐水组615.7（13.7,25.6）75.7（47.0,82.9）0.8（0.7,1.2）
    低灯盏花素组920.5（18.0,39.8）a59.0（46.9,82.9）0.7（0.5,1.2）
    中灯盏花素组824.9（14.7,28.9）ab54.1（48.8,76.6）ab0.7（0.2,0.9）
    高灯盏花素组928.0（21.9,39.1）abc52.7（46.8,71.6）ab0.7（0.2,0.9）
    H值23.9013.004.31
    P值<0.0010.0110.366
    注：5组大鼠均致高压电烧伤,其中电烧伤组大鼠不进行药物干预,盐水组、低灯盏花素组、中灯盏花素组、高灯盏花素组大鼠分别经腹腔注射生理盐水或低、中、高浓度灯盏花素；CAT为过氧化氢酶,AOPP为晚期氧化蛋白产物；H值、P值为组间各指标总体比较所得；与电烧伤组比较,aP<0.05；与盐水组比较,bP<0.05；与低灯盏花素组比较,cP<0.05
    下载: 导出CSV 
    | 显示表格

    3.   讨论

    氧化应激是指机体受到有害刺激时,体内活性氧生成过度,导致机体内氧化系统和抗氧化防御系统之间的平衡失调,使平衡更偏向于氧化状态,从而引起机体组织或细胞功能的紊乱[16]。烧伤可通过凋亡[17]、炎症[18]、微循环障碍[19]、横纹肌溶解[20]、线粒体损伤[21]引起肾损伤,其中氧自由基与上述过程均有重要联系,因此推测氧化应激反应在高压电烧伤引起的肾损伤中发挥重要作用。

    本研究首先对高压电烧伤大鼠肾脏结构及功能的损伤程度进行评估,观察到电烧伤组大鼠伤后8 h、48 h、72 h、1周肾/体比均明显高于假伤组；伤后8 h~1周肾小球、肾小管及肾间质较假伤组出现不同程度损伤,且损伤程度随时间延长而加重；伤后48 h、72 h、1周肾小管与肾间质损伤评分较假伤组明显升高。血清肌酐和尿素氮水平通常反映肾小球滤过功能[22]。本研究显示,与假伤组相比,电烧伤组大鼠伤后血清肌酐和尿素氮水平均呈现上升趋势；电烧伤组大鼠伤后1周血清肌酐水平较组内伤后0、8、24 h均明显升高,电烧伤组大鼠伤后72 h血清肌酐水平较组内伤后0、8 h升高,提示高压电烧伤后大鼠发生肾小球滤过功能障碍。

    以上假伤组与电烧伤组各时间点的肾/体比、组织病理学变化、肾小管与肾间质损伤评分、血清肌酐和尿素氮水平结果共同说明,高压电烧伤后肾脏结构损伤与功能障碍程度随伤后时间的延长,呈进行性加重变化。

    CAT是重要的抗氧化酶,可以保护细胞膜结构及功能[23],当发生氧化应激反应时,CAT活性可呈现先升高后降低或只降低的趋势[24, 25]。在本研究中,大鼠发生高压电烧伤后肾组织上清液中CAT活性开始下降,且随时间延长呈进行性下降,本课题组认为这是由于高压电烧伤后持续生成的自由基过多导致CAT无法清除,CAT活性逐渐降低。这一结果说明高压电烧伤后产生的氧化物质抑制抗氧化物质活性,导致机体处于氧化状态,从而引发过度氧化应激反应。

    AOPP是血浆蛋白被体内活性氧攻击后形成的氧化修饰产物,被认为是烧伤引起的氧化应激反应中的新型氧化物质[26],其含量增多会诱导更多的活性氧产生,加重氧化应激,由此形成一个正反馈机制相互促进,故使机体呈现持续氧化应激状态[27]。研究显示,大鼠在接受电离辐射后会产生氧化应激反应,AOPP水平升高,同时造成脏器损伤[28]。上述结果与本研究结果一致,高压电烧伤后大鼠肾组织上清液中AOPP水平升高,证明大鼠高压电烧伤后其肾脏发生氧化应激反应,减少AOPP可以通过减轻氧化应激反应的途径保护肾脏。

    Klotho主要来源于肾脏,在正常肾小管上皮细胞中高表达可发挥抗氧化作用[29]。当发生急性肾损伤时,Klotho蛋白水平降低,1周后可通过自身调节作用恢复至正常水平[30]。在本研究中,当大鼠发生高压电烧伤后肾组织上清液中Klotho蛋白水平随伤后时间的延长呈现逐渐降低趋势,考虑是由于高压电烧伤后肾小管上皮细胞随时间出现变性、坏死脱落等现象使Klotho蛋白无法正常表达；且Klotho蛋白作为抗氧化物质,也会因高压电烧伤后大鼠肾脏发生的氧化应激反应而表达降低。

    研究表明,灯盏花素能够降低心脏和肾脏等重要脏器中的氧化物质水平,并提高抗氧化酶活性,从而在氧化应激反应中发挥抗氧化作用,保护脏器[31, 32]。目前采用灯盏花素治疗肾脏疾病的研究多集中在糖尿病肾病[33]和肾脏纤维化[34]方面,在烧伤方面主要是对角膜的治疗,关于肾脏的研究较少。一项关于灯盏花素毒性剂量的研究显示,对大鼠经尾静脉注射最大剂量（120 mg/kg）的灯盏花素,未导致其肾脏结构损伤；而对比格犬经尾静脉注射100 mg/kg剂量的灯盏花素,比格犬出现了肾脏结构损伤[35]。根据赵丽雅等[32]的研究,采用灌胃给药方案治疗庆大霉素所致的大鼠急性肾损伤时,灯盏花素的剂量包括6、12、24 mg/kg。考虑到腹腔注射吸收速度较灌胃更快,因此,本实验选择的经腹腔注射灯盏花素的剂量分别为2、8、20 mg/kg,这些剂量均为安全有效的治疗剂量。在本实验中,大鼠高压电烧伤后应用灯盏花素,其肾功能及肾组织病理损伤均得到有效改善,CAT活力略有恢复,AOPP水平降低,且高灯盏花素组大鼠此改变更为明显。这表明灯盏花素的抗氧化作用,可以对抗高压电烧伤引起的氧化应激反应。因此,早期应用灯盏花素有望减轻高压电烧伤引起的肾功能受损和肾组织病理损伤,同时改善预后并减少不良反应,具有积极的临床意义。

    综上所述,结合高压电烧伤后大鼠肾功能损伤、肾组织病理结构改变、CAT活性降低及AOPP水平升高的情况,证明了高压电烧伤会通过氧化应激引起肾脏损伤。肾脏损伤程度、血清肌酐水平、肾组织CAT活性和Klotho蛋白水平与伤后时间密切相关,伤后48 h、72 h及1周时损伤往往较烧伤早期更为严重,这表明高压电烧伤导致的大鼠肾脏损伤随着时间的延长呈进行性加重。在此背景下,灯盏花素的应用有效改善了肾功能和肾组织病理损伤,因此,灯盏花素可以作为治疗高压电烧伤所致肾脏损伤的有效药物。但本研究中未针对肌红蛋白等其他因素对肾脏损伤造成的影响进行检测,需在今后的研究中进一步完善。

    • References(22)
  • [1]
    NovoselEC,KleinhansC,KlugerPJ.Vascularization is the key challenge in tissue engineering[J].Adv Drug Deliv Rev,2011,63(4/5):300-311.DOI: 10.1016/j.addr.2011.03.004.
    [2]
    魏亚婷,吴军.创面修复中的皮肤组织再生研究进展[J].中华烧伤杂志,2021,37(7):670-674.DOI: 10.3760/cma.j.cn501120-20200604-00296.
    [3]
    FangS,EllmanDG,AndersenDC.Review: tissue engineering of small-diameter vascular grafts and their in vivo evaluation in large animals and humans[J].Cells,2021,10(3):713.DOI: 10.3390/cells10030713.
    [4]
    WeekesA,BartnikowskiN,PintoN,et al.Biofabrication of small diameter tissue-engineered vascular grafts[J].Acta Biomater,2022,138:92-111.DOI: 10.1016/j.actbio.2021.11.012.
    [5]
    TuckerWD,AroraY,K.AnatomyMahajan, vesselsblood[M]. Treasure Island(FL):StatPearls Publishing,2022.
    [6]
    MunissoMC,YamaokaT.Circulating endothelial progenitor cells in small-diameter artificial blood vessel[J].J Artif Organs,2020,23(1):6-13.DOI: 10.1007/s10047-019-01114-6.
    [7]
    HuangWL,TungSY,TsengCS,et al.The combination of blood flow velocity and vascular diameter: the arterial flow index is a better parameter in the evaluation of arteriogenic erectile dysfunction[J]. European Urology,2021,79:S653-S654. DOI: 10.1016/S0302-2838(21)00858-7.
    [8]
    ShahinH,ElmasryM,SteinvallI,et al.Vascularization is the next challenge for skin tissue engineering as a solution for burn management[J/OL].Burns Trauma,2020,8:tkaa022[2022-09-30].https://pubmed.ncbi.nlm.nih.gov/32766342/.DOI: 10.1093/burnst/tkaa022.
    [9]
    BoykinJVJr,HokeGD,DriscollCR,et al.High-dose folic acid and its effect on early stage diabetic foot ulcer wound healing[J].Wound Repair Regen,2020,28(4):517-525.DOI: 10.1111/wrr.12804.
    [10]
    GerweckLE,SeetharamanK.Cellular pH gradient in tumor versus normal tissue: potential exploitation for the treatment of cancer[J].Cancer Res,1996,56(6):1194-1198.
    [11]
    BaiY,ZhaoJ,WangS,et al.Carbon dots with absorption red-shifting for two-photon fluorescence imaging of tumor tissue pH and synergistic phototherapy[J].ACS Appl Mater Interfaces,2021,13(30):35365-35375.DOI: 10.1021/acsami.1c08076.
    [12]
    肖仕初,郑勇军.组织工程皮肤现状与挑战[J].中华烧伤杂志,2020,36(3):166-170.DOI: 10.3760/cma.j.cn501120-20191202-00449.
    [13]
    RecktenwaldSM,GraesselK,MaurerFM,et al.Red blood cell shape transitions and dynamics in time-dependent capillary flows[J].Biophys J,2022,121(1):23-36.DOI: 10.1016/j.bpj.2021.12.009.
    [14]
    SchulzE,MünzelT.Lumen size matters: role of protein disulfide isomerase A1 in vascular remodeling[J].Hypertension,2016,67(3):488-489.DOI: 10.1161/HYPERTENSIONAHA.115.06296.
    [15]
    MaX,HeZ,LiL,et al.Development and in vivo validation of tissue-engineered, small-diameter vascular grafts from decellularized aortae of fetal pigs and canine vascular endothelial cells[J].J Cardiothorac Surg,2017,12(1):101.DOI: 10.1186/s13019-017-0661-x.
    [16]
    WertheimerS,SharabiM,ShelahO,et al.Bio-composites reinforced with unique coral collagen fibers: towards biomimetic-based small diameter vascular grafts[J].J Mech Behav Biomed Mater,2021,119:104526.DOI: 10.1016/j.jmbbm.2021.104526.
    [17]
    FlauraudV,MastrangeliM,BernasconiGD,et al.Nanoscale topographical control of capillary assembly of nanoparticles[J].Nat Nanotechnol,2017,12(1):73-80.DOI: 10.1038/nnano.2016.179.
    [18]
    KharaziAZ,AtariM,VatankhahE,et al. A nanofibrous bilayered scaffold for tissue engineering of small‐diameter blood vessels[J]. Polymers for Advanced Technologies,2018,29(12): 2849-3185.DOI: 10.1002/pat.4437.
    [19]
    GeelhoedWJ,van der BogtKEA,RothuizenTC,et al.A novel method for engineering autologous non-thrombogenic in situ tissue-engineered blood vessels for arteriovenous grafting[J].Biomaterials,2020,229:119577.DOI: 10.1016/j.biomaterials.2019.119577.
    [20]
    SmithGA,HowellGJ,PhillipsC,et al.Extracellular and luminal pH regulation by vacuolar H+-ATPase isoform expression and targeting to the plasma membrane and endosomes[J].J Biol Chem,2016,291(16):8500-8515.DOI: 10.1074/jbc.M116.723395.
    [21]
    YERC.The relationship of pH of the granulation tissue and the take of the skin graft[J].Plast Reconstr Surg (1946),1957,19(3):213-217.DOI: 10.1097/00006534-195703000-00004.
    [22]
    QuadeBN,ParkerMD,OcchipintiR.The therapeutic importance of acid-base balance[J].Biochem Pharmacol,2021,183:114278.DOI: 10.1016/j.bcp.2020.114278.
    • Relative Articles

  • Relative Articles

    [1]Zhu Dongzhen, Yao Bin, Yan Ziqiang, Huang Sha, Fu Xiaobing. Research advances on the construction of an ideal scar model in vitro based on innovative tissue engineering technology[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2022, 38(10): 983-988. doi: 10.3760/cma.j.cn501120-20210723-00257
    [2]Zeng Yingnan, Kang Yangbo, Xu Yong'an. Research advances on skin sweat gland regeneration induced by stem cells and tissue engineering[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2021, 37(9): 900-904. doi: 10.3760/cma.j.cn501120-20200624-00328
    [3]Zhang Junhui, Zhang Qiong, Jia Jiezhi, Li Hongmei, Zhang Can, Hu Jiongyu, Zhang Dongxia, Huang Yuesheng. Effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 on the migration and motility of human dermal microvascular endothelial cells under hypoxia and the mechanism[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2021, 37(1): 9-16. doi: 10.3760/cma.j.cn501120-20200927-00425
    [4]Xiao Shichu, Zheng Yongjun. Status and challenges of tissue-engineered skin[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2020, 36(3): 166-170. doi: 10.3760/cma.j.cn501120-20191202-00449
    [6]Huang Hong, Qiu Wei, Zhu Ming, Zhang Ya, Cui Wenhui, Xing Wei, Li Xiangyun, An Tianchen, Chen Minjia, Guo Wei, Xu Xiang. Expressions of the key proteins of the protein kinase B/mammalian target of rapamycin signaling pathway in skin tissue and wound tissue of diabetic rats[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2016, 32(10): 588-593. doi: 10.3760/cma.j.issn.1009-2587.2016.10.004
    [7]Li Ran, Wang Hong, Leng Chongyan, Wang Kuan, Xie Ying. Advances in the research of natural polymeric materials and their derivatives in the manufacture of scaffolds for dermal tissue engineering[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2016, 32(5): 316-318. doi: 10.3760/cma.j.issn.1009-2587.2016.05.014
    [9]Ruan Qiongfang, Zhao Chaoli, Ye Ziqing, Zhang Wei-dong, Xie Qionghui, Xie Weiguo. Role of phosphoinositide 3-kinase/protein kinase B signal pathway in monocyte-endothelial adhesion induced by serum of rats with electrical burn[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2014, 30(3): 237-242. doi: 10.3760/cma.j.issn.1009-2587.2014.03.013
    [10]HAN Chun-mao, WANG Xin-gang. Tissue engineered skin and regenerative wound repair[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2013, 29(2): 122-125. doi: 10.3760/cma.j.issn.1009-2587.2013.02.008
    [12]QIU Xue-wen, WANG Jia-han, WANG Ying, LIU Liang, WU Qi, MA Jun. Clinical observation on repairing of wounds of skin graft donor site with acellular tissue engineering dermal matrix[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2013, 29(1): 46-49. doi: 10.3760/cma.j.issn.1009-2587.2013.01.016
    [13]WANG Xin-gang, FENG Zhan-zeng, GUO Ming-feng, HAN Chun-mao. Advances in research of the strategies for promoting angio- genesis in tissue engineering[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2012, 28(5): 374-377. doi: 10.3760/cma.j.issn.1009-2587.2012.05.016
    [16]MA Zhong-feng, CHAI Jia-ke, YANG Hong-ming, LIANG Li-ming, XU Ming-huo. Construction of tissue-engineering skin with carrier of active composite dermal matrix[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2008, 24(4): 272-274.
    [17]LV Gen-fa, CHEN Bi, ZHANG Wan-fu, WANG Yun-ehuan, ZHU Xiong-xiang, HU Da-hai. Study on crosstalk between phosphatidylinositol 3-kinase/Akt pathway and p38 mitogen-activated protein kinase pathway in cardiomyocyte with challenge of burn serum[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2008, 24(4): 263-267.
    [19]XIE Wei-guo, TAN Hong, ZHAO Chao-li, WANG Hui. The histological changes and the revascularization process in the grafted dermal substitutes[J]. CHINESE JOURNAL OF BURNS AND WOUNDS, 2005, 21(1): 37-39.
    • Supplements(0)
    • Cited By
  • Created with Highcharts 5.0.7Amount of accessChart context menuAbstract Views, HTML Views, PDF Downloads StatisticsAbstract ViewsHTML ViewsPDF Downloads2024-052024-062024-072024-082024-092024-102024-112024-122025-012025-022025-032025-04010203040
    Created with Highcharts 5.0.7Chart context menuAccess Class DistributionFULLTEXT: 32.9 %FULLTEXT: 32.9 %META: 62.3 %META: 62.3 %PDF: 4.9 %PDF: 4.9 %FULLTEXTMETAPDF
    Created with Highcharts 5.0.7Chart context menuAccess Area Distribution其他: 6.7 %其他: 6.7 %其他: 1.2 %其他: 1.2 %Ataturk: 0.7 %Ataturk: 0.7 %上海: 2.3 %上海: 2.3 %东莞: 0.5 %东莞: 0.5 %伊利诺伊州: 0.2 %伊利诺伊州: 0.2 %佛山: 0.5 %佛山: 0.5 %北京: 1.9 %北京: 1.9 %南京: 0.7 %南京: 0.7 %南宁: 0.5 %南宁: 0.5 %台北: 0.2 %台北: 0.2 %哈尔滨: 0.2 %哈尔滨: 0.2 %哥伦布: 0.2 %哥伦布: 0.2 %天津: 0.5 %天津: 0.5 %太原: 1.2 %太原: 1.2 %安康: 0.5 %安康: 0.5 %山景城: 0.5 %山景城: 0.5 %广元: 0.5 %广元: 0.5 %广州: 0.2 %广州: 0.2 %张家口: 2.8 %张家口: 2.8 %成都: 0.7 %成都: 0.7 %扬州: 0.2 %扬州: 0.2 %新竹: 0.9 %新竹: 0.9 %昆明: 0.5 %昆明: 0.5 %本溪: 0.2 %本溪: 0.2 %梧州: 0.5 %梧州: 0.5 %武汉: 1.6 %武汉: 1.6 %沃思堡: 0.2 %沃思堡: 0.2 %沈阳: 0.2 %沈阳: 0.2 %济南: 0.5 %济南: 0.5 %海得拉巴: 0.7 %海得拉巴: 0.7 %温州: 1.4 %温州: 1.4 %漯河: 1.4 %漯河: 1.4 %石家庄: 0.9 %石家庄: 0.9 %绥化: 0.5 %绥化: 0.5 %芒廷维尤: 52.3 %芒廷维尤: 52.3 %芝加哥: 0.5 %芝加哥: 0.5 %衡水: 0.2 %衡水: 0.2 %西宁: 3.7 %西宁: 3.7 %西安: 0.7 %西安: 0.7 %郑州: 0.5 %郑州: 0.5 %重庆: 6.5 %重庆: 6.5 %金华: 0.2 %金华: 0.2 %长沙: 3.2 %长沙: 3.2 %其他其他Ataturk上海东莞伊利诺伊州佛山北京南京南宁台北哈尔滨哥伦布天津太原安康山景城广元广州张家口成都扬州新竹昆明本溪梧州武汉沃思堡沈阳济南海得拉巴温州漯河石家庄绥化芒廷维尤芝加哥衡水西宁西安郑州重庆金华长沙

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Figures(6)  / Tables(3)

    Get Citation
    PDF
    XML

    Article Metrics

    Article views (268) PDF downloads(21) Cited by()
    Proportional views
    Related

    Copyright © Chinese Journal of Burns京ICP备07035254号-14

    E-mail：shaoshangzazhi@163.com

    Website：https://zhsszz.xml-journal.net/

    Supported by: Beijing Renhe Information Technology Co. Ltd    

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return