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急性胰腺炎患者血浆内皮细胞微粒水平的改变及其形成机制

王迪迪 刘秋圆 胡翠 王兵兵 韦亚蓉 丁浩 刘晓昌 梅俏

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Citation:

急性胰腺炎患者血浆内皮细胞微粒水平的改变及其形成机制

DOI: 10.3969/j.issn.1001-5256.2022.09.027
伦理学声明:本研究于2019年10月15日经安徽医科大学第一附属医院伦理委员会批准,批号:PJ2018-12-17,所有研究对象均签署知情同意书。
利益冲突声明:本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明:王迪迪、刘秋圆负责课题设计,资料分析,撰写论文;胡翠、王兵兵、韦亚蓉、丁浩参与收集数据,修改论文;刘晓昌、梅俏负责拟定写作思路,指导撰写文章并最后定稿。
详细信息
    通信作者:

    梅俏,meiqiao@hotmail.com

Changes and formation mechanism of plasma endothelial microparticles in patients with acute pancreatitis

More Information
  • 摘要:   目的  探讨急性胰腺炎(AP)患者血浆内皮细胞微粒(EMP)水平的改变并初步观察其形成的机制。  方法  收集安徽医科大学第一附属医院2020年8月-2021年6月60例AP患者的血液标本, 分为轻症急性胰腺炎组(MAP组, n=23)、中度重症急性胰腺炎组(MSAP组, n=23)和重症急性胰腺炎组(SAP组, n=14), 取健康体检者20例为对照组。使用差速离心法获得贫血小板血浆, 流式细胞仪检测CD31+CD41-EMP水平, ELISA检测内皮素(ET-1)、血管性血友病因子(vWF)、一氧化氮(NO)和血管内皮细胞黏附分子-1(VCAM-1)水平。使用AP患者的血浆刺激HUVEC细胞, 使用流式细胞术和qRT-PCR分别检测EMP、细胞活性氧(ROS)、线粒体膜电位的变化及内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)、细胞间黏附分子-1(ICAM-1)、VCAM-1、NADPH氧化酶及P-选择素的表达。符合正态分布的计量资料多组间比较采用单因素方差分析, 进一步两两比较采用LSD-t检验; 不符合正态分布的计量资料组间和组内两两比较采用Kruskal-WallisH秩和检验。计数资料组间比较采用χ2检验。相关分析采用Pearson相关性检验。  结果  与对照组相比, MAP组、MSAP组、SAP组EMP水平均明显升高(P值均 < 0.05)。与MAP组、MSAP组比较, SAP组EMP水平明显升高(P值均 < 0.05)。AP患者EMP水平与APACHEⅡ评分、BISAP评分、Ranson评分、CT评分、CRP均呈正相关(r值分别为0.686 2、0.777 3、0.713 8、0.771 8、0.473 9, P值均 < 0.01)。与对照组相比, MAP组、MSAP组、SAP组的ET-1、vWF、VCAM-1水平明显升高, NO水平明显降低(P值均 < 0.05)。与对照组相比, MSAP和SAP组血浆可促进HUVEC中EMP大量释放(P值均 < 0.05)。与对照组比较, 除MAP组的VCAM-1和eNOS外, 其余各组的eNOS、iNOS、ICAM-1、P-选择素、VCAM-1、NADPH氧化酶的mRNA表达水平均显著升高(P值均 < 0.05)。与对照组相比, MAP组、MSAP组、SAP组、LPS组患者HUVEC中的ROS水平升高明显, 线粒体膜电位下降显著(P值均 < 0.05)。  结论  AP患者血浆EMP水平明显升高, 与胰腺炎严重程度相关, 且AP患者血浆可刺激HUVEC形成EMP, 机制可能与细胞氧化损伤相关。

     

  • 图  1  不同严重程度AP患者的EMP水平检测

    P1,EMP所在区域;P2,藻红蛋白(PE)和别藻蓝蛋白(APC)双阳性区域;P3,阴性颗粒所在区域;P4,藻红蛋白(PE)阳性区域。

    Figure  1.  EMP level of AP patients with different severity

    图  2  AP患者血浆中ET-1、vWF、NO、VCAM-1水平改变

    Figure  2.  Plasma levels of ET-1、vWF、NO and VCAM-1 in AP patients

    图  3  AP患者血浆对HUVEC中EMP水平的影响

    注:Q2-UL, EMP所在区域;Q2-UR, 藻红蛋白(PE)和别藻蓝蛋白(APC)双阳性区域;Q2-LL, 阴性颗粒所在区域;Q2-LR, 藻红蛋白(PE)阳性区域。

    Figure  3.  Influence of plasma on EMP level in HUVEC of AP patients

    图  4  AP患者血浆对HUVEC中eNOS、iNOS、ICAM-1、VCAM -1、NADPH氧化酶及P-选择素mRNA表达的影响

    Figure  4.  The effects of plasma of AP patients on mRNA expression levels of eNOS、iNOS、ICAM-1、VCAM-1、NADPH oxidase and P-selectin in HUVEC

    表  1  qRT-PCR引物序列

    Table  1.   Primers of qRT-PCR

    引物名称 引物序列(5′-3′)
    GAPDH F:AACAGCGACACCCACTCCTC
    R:CATACCAGGAAATGAGCTTGACAA
    eNOS F:GTGGCTGTCTGCATGGACCT
    R:CCACGATGGTGACTTTGGCT
    iNOS F:ATGGAACATCCCAAATACGA
    R:GTCGTAGAGGACCACTTTGT
    ICAM-1 F:CCTTCCTCACCGTGTACTGC
    R:AGCGTAGGGTAAGGTTCTTGC
    VCAM-1 F:ACTGGTGGCCTCCTGAATGG
    R:CTGTGTCTCCTGTCTCCGCT
    NADPH氧化酶 F:CTGTGGTGTTACTATCTGTATTTTCTC
    R:CTTGCTGCATTCAGTTCAACA
    P-选择素 F:CTGTTACCCTGGATTCTATGGGC
    R:GCTGCACTGCGAGTTAAAAGAG
    注:GAPDH,内参。
    下载: 导出CSV

    表  2  AP患者临床资料

    Table  2.   Clinical data of acute pancreatitis patients

    指标 对照组(n=20) MAP组(n=23) MSAP组(n=23) SAP组(n=14) 统计值 P
    性别(例) χ2=6.344 0.096
       男 13 17 9 9
      女 7 6 14 5
    年龄(岁) 46.10±17.15 45.22±15.61 45.00±16.13 50.93±21.45 F=0.510 0.747
    WBC(×109/L) 5.95±1.66 12.55±4.541) 15.15±6.241) 12.91±3.471) F=16.220 <0.01
    中性粒细胞(×109/L) 3.35±0.97 10.66±4.301) 12.99±5.341) 11.39±3.501) F=23.350 <0.01
    RBC(×1012/L) 4.51±0.36 4.87±0.631) 4.64±0.79 4.66±0.88 F=1.056 0.370
    HB(g/L) 135.00(124.50~145.25) 149.00(131.00~164.00)1) 140.00(120.00~158.00) 138.50(125.75~172.25) H=5.929 0.115
    HCT(%) 40.12±4.34 43.33±5.161) 40.62±6.26 42.01±8.54 F=1.261 0.294
    CRP(mg/L) 0.53(0.43~0.65) 21.88(6.01~121.27)1) 60.31(5.82~196.19)1) 141.28(44.19~252.83)1) H=46.070 <0.01
    Alb(g/L) 42.48±2.87 42.89±5.45 40.68±5.50 34.10±6.831) F=9.520 <0.01
    TBil(μmol/L) 13.55(9.66~15.58) 15.85(10.89~22.85) 17.50(11.94~36.17)1) 25.08(12.83~33.32)1) H=7.348 0.062
    ALT(U/L) 15.50(10.25~25.25) 43.00(33.00~70.00)1) 37.00(19.00~264.00)1) 30.50(19.75~45.50)1) H=19.529 <0.01
    AST(U/L) 17.00(15.25~20.00) 26.00(22.00~53.00)1) 41.00(23.00~116.00)1) 38.50(25.25~78.25)1) H=26.067 <0.01
    ALP(U/L) 81.00(68.00~97.75) 102.00(87.00~137.00)1) 101.00(84.00~141.00)1) 90.50(76.25~97.75) H=12.667 <0.01
    LDH(U/L) 161.00(148.00~174.00) 207.00(177.00~244.00)1) 247.00(183.40~323.00)1) 521.50(220.00~733.25)1) H=48.475 <0.01
    BUN(μmol/L) 5.18±1.28 5.55±1.36 4.56±2.02 7.05±2.991) F=5.054 0.003
    CRE(μmol/L) 66.04±9.94 59.80±18.90 54.64±27.07 86.08±58.23 F=3.372 0.023
    钾(mmol/L) 3.93±0.33 4.28±0.421) 3.80±0.31 4.08±0.90 F=3.635 0.017
    钠(mmol/L) 140.95(139.40~142.18) 136.20(134.10~138.40)1) 136.60(131.80~138.50)1) 136.50(131.18~140.55)1) H=24.179 <0.01
    钙(mmol/L) 2.30(2.23~2.40) 2.32(2.19~2.45) 2.14(2.03~2.23)1) 1.78(1.14~2.10)1) H=30.525 <0.01
    血糖(mmol/L) 4.83(4.53~5.18) 7.42(6.33~13.16)1) 8.18(7.07~9.76)1) 8.99(6.63~11.56)1) H=31.061 <0.01
    APACHE-Ⅱ评分≥3[例(%)] 16(69.6) 21(91.3) 14(100.0) χ2=7.485 <0.05
    BISAP评分≥3[例(%)] 0 1(4.3) 10(71.4) χ2=34.528 <0.05
    Ranson评分≥3[例(%)] 0 12(52.2) 12(85.7) χ2=28.944 <0.05
    CT评分>3[例(%)] 5(21.7) 13(56.5) 14(100.0) χ2=21.568 <0.05
    注:与对照组比较,1)P<0.05。
    下载: 导出CSV
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  • 收稿日期:  2022-01-17
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