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中华妇幼临床医学杂志(电子版) ›› 2022, Vol. 18 ›› Issue (01) : 30 -39. doi: 10.3877/cma.j.issn.1673-5250.2022.01.004

论著

高迁移率族蛋白1在子痫前期母胎界面表达及其对巨噬细胞的作用
邓茜茜, 徐婷婷, 詹泳池, 王晓东†()   
  • 收稿日期:2021-11-18 修回日期:2022-01-10 出版日期:2022-02-01
  • 通信作者: 王晓东

Expression of high mobility group box 1 at maternal-fetal interface and its effect on macrophages in preeclampsia

Xixi Deng, Tingting Xu, Yongchi Zhan, Xiaodong Wang†()   

  • Received:2021-11-18 Revised:2022-01-10 Published:2022-02-01
  • Corresponding author: Xiaodong Wang
  • Supported by:
    Science and Technology Plan Project by Science and Technology Department of Sichuan Province(2022YFS0042); Chengdu Science and Technology Project(2019-YF05-00711-SN)
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引用本文：

邓茜茜, 徐婷婷, 詹泳池, 王晓东. 高迁移率族蛋白1在子痫前期母胎界面表达及其对巨噬细胞的作用[J]. 中华妇幼临床医学杂志(电子版), 2022, 18(01): 30-39.

Xixi Deng, Tingting Xu, Yongchi Zhan, Xiaodong Wang. Expression of high mobility group box 1 at maternal-fetal interface and its effect on macrophages in preeclampsia[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2022, 18(01): 30-39.

目的

探究高迁移率族蛋白(HMGB)1在子痫前期(PE)母胎界面的表达及其对巨噬细胞的作用。

方法

选择2020年6月至2021年12月，在四川大学华西第二医院确诊为PE的24例单胎孕妇为研究对象。按照孕妇PE严重程度，将其分别纳入PE组(n＝13)和重度PE(sPE)组(n＝11)。按照随机数字表法，选择同期在本院剖宫产术分娩的正常妊娠孕妇纳入对照组(n＝13)。采用免疫组织化学(IHC)法、蛋白质免疫印迹法(Western blotting)、实时荧光定量PCR(qPCR)检测3组患者胎盘蜕膜组织HMGB1相对表达水平。采用免疫磁珠富集法分离对照组原代蜕膜巨噬细胞，以不同剂量人重组HMGB1(rHMGB1)对其进行处理48 h，采用流式细胞术检测巨噬细胞表型变化。研究组与对照组PE孕妇年龄、产次等一般临床资料比较，差异均无统计学意义(P>0.05)。本研究遵循的程序符合四川大学华西第二医院伦理委员会规定，通过该伦理委员会审查，并获得批准[审批文号：2021(181)]。所有受试者均签署临床研究知情同意书。

结果

①HMGB1定位于细胞滋养层细胞、合体滋养层细胞和蜕膜基质细胞的细胞质及细胞核中。②经Western blotting检测sPE组、PE组和对照组HMGB1蛋白相对表达水平分别为3.10(1.50~5.14)、0.63(0.45~2.41)、0.72(0.07~1.49)，3组总体比较，差异有统计学意义(χ2＝9.222，P＝0.010)。③IHC检测结果发现，sPE组、PE组和对照组HMGB1强阳性比例分别为54.6%(6/11)、30.8%(4/13)、0，3组总体比较，差异有统计学意义(χ2＝15.024，P＝0.001)。④sPE组、PE组和对照组HMGB1 mRNA相对表达水平分别为4.49±1.38、3.01±2.08、1.67±1.48，3组总体比较，差异有统计学意义(F＝8.291，P＝0.001)，其中sPE组相对表达水平分别高于PE组及对照组，并且差异亦有统计学意义(P＝0.039、P<0.001)。⑤原代胎盘蜕膜巨噬细胞纯度为97.39%，经rHMGB1处理后，M1型巨噬细胞百分比增加， M1型巨噬细胞标记CD86平均荧光强度(MFI)与rHMGB1含量与呈正相关关系(r＝0.808，P<0.001)。

结论

PE母胎界面HMGB1表达增加，促进蜕膜巨噬细胞M1型极化，可能导致母胎界面炎性微环境。

关键词: 高迁移率族蛋白质类, 先兆子痫, 母胎界面, 巨噬细胞, 极化
Objective

To investigate the expression of high mobility group box (HMGB)1 at the maternal-fetal interface of pre-eclampsia (PE), and the possible regulatory effect on macrophage polarization.

Methods

Twenty-four singleton pregnant women admitted to the obstetrics clinic in West China Second University Hospital, Sichuan University from June 2020 to December 2021 were selected as research subjects. They were divided into PE group (n=37) and severe PE (sPE) group (n=11) based on diagnostic criteria for PE. In addition, 13 healthy pregnant women were selected as controls by the random number table method. Immunohistochemistry (IHC), Western blotting and quantitative real-time polymerase chain reaction (qPCR) were used to detect placental HMGB1 protein and mRNA levels. Decidual macrophages were isolated using immunomagnetic positive selection. Further polarization characteristics of macrophages were analyzed by flow cytometry (FCM) after being treated with various doses of recombinant human HMGB1 (rHMGB1). The procedure followed in this study was in accordance with the regulations of the Ethics Committee of West China Second University Hospital, Sichuan University, which was reviewed and approved [Approval No. 2021(181)]. Informed consent was obtained from each participant.

Results

①HMGB1 were observed both in the cytoplasm and nucleus of cytotrophoblast and decidual stromal cells. ②Western blotting showed that the relative expression levels of HMGB1 protein in sPE group, PE group and control group were 3.10 (1.50-5.14), 0.63 (0.45-2.41) and 0.72 (0.07-1.49), respectively, and the difference was statistically significant (χ2=9.222, P=0.010). ③The proportion of sPE, PE and control group strongly positive for HMGB1 was 54.6% (6/11), 30.08% (4/13) and 0, respectively, and the difference of HMGB1 was significant (χ2=15.024, P=0.001). ④The relative expression levels of HMGB1 mRNA in sPE group, PE group and control group were 4.49±1.38, 3.01±2.08 and 1.67±1.48, respectively. Compared with the three groups, the difference was statistically significant (F=8.291, P=0.001). The relative expression level of sPE group was higher than that of PE group and control group respectively, and both the differences were statistically significant (P=0.039, P<0.001). ⑤ The isolation purity of decidual macrophages was 97.39%. The percentage of M1-related marker (CD86+ ) increased after rHMGB intervention. The mean fluorescence intensity (MFI) of CD86 was positively correlated with rHMGB dose(r=0.808, P<0.001).

Conclusions

HMGB1 is significantly up-regulated at the maternal-fetal interface in women with preeclampsia. HMGB1 induces polarization of M1 macrophages and regulates inflammatory cytokines profile at the maternal-fetal interface.

Key words: High mobility group proteins, Pre-eclampsia, Maternal-fetal interface, Macrophages, Polarization
表1 实时荧光定量PCR引物序列
引物名称 上游引物(5′→3′) 下游引物(3′→5′) 产物长度(bp)
HMGB1 TATGGCAAAAGCGGACAAGG CTTCGCAACATCACCAATGGA 196
ACTB GAGAAAATCTGGCACCACACC GGATAGCACAGCCTGGATAGCAA 177
表2 3组孕妇一般及相关临床资料比较
组别 例数 年龄(岁，±s) 产次[例数(%)] 孕龄(周，±s) 最高收缩压(mmHg，±s) 最高舒张压(mmHg，±s)
0次 1次
sPE组 11 32.3±2.6 9(81.8) 2(18.2) 33.4±2.1 167.4±6.1 106.4±7.2
PE组 13 31.5±2.2 10(76.9) 3(23.1) 37.5±0.8 155.3±3.7 101.7±6.9
对照组 13 30.5±1.7 10(76.9) 3(23.1) 38.4±0.8 129.5±4.4 78.5±5.2
检验值   F＝1.888 χ2＝0.106 χ2＝25.528 F＝200.847 F＝66.498
P值   0.167 0.948 <0.001 <0.001 <0.001
组别 例数 (g/24 h，±s) 最高随机尿蛋白[例数(%)] 新生儿出生体重(g，±s)
- + ++ +++
sPE组 11 4.1±2.5 0(0) 0(0) 7(63.6) 4(36.4) 1 812.6±514.1
PE组 13 1.1±0.6 0(0) 8(61.5) 5(38.5) 0(0) 2 852.3±146.9
对照组 13 0±0 13(100.0) 0(0) 0(0) 0(0) 3 197.7±116.3
检验值   χ2＝31.595 χ2＝31.595 χ2＝30.777
P值   <0.001 <0.001 <0.001
表3 3组胎盘蜕膜组织HMGB1蛋白及HMGB1 mRNA相对表达水平比较
组别 例数 Western blotting检测HMGB1结果[M(P25~P75)] IHC检测HMGB1结果[例数(%)] HMGB1 mRNA(±s)
— + ++ +++
sPE组 11 3.10(1.50~5.14) 0(0) 2(18.2) 3(27.3) 6(54.6) 4.49±1.38
PE组 13 0.63(0.45~2.41) 0(0) 3(23.1) 6(46.2) 4(30.8) 3.01±2.08
对照组 13 0.72(0.07~1.49) 5(38.5) 5(38.5) 3(23.1) 0( 0) 1.67±1.48
总体比较              
  检验值   χ2＝9.222 χ2＝15.024 F＝8.291
  P值   0.010 0.001 0.001
两两比较(P值)              
  PE组vs对照组   0.140 0.001 0.052
  sPE组vs PE组   0.057 0.353 0.039
  sPE组vs对照组   0.002 <0.001 <0.001
图1 3组胎盘组织HMGB1蛋白相对表达水平(图1A：HMGB1电泳条带图；图1B：HMGB1相对表达水平柱状图)注：HMGB1为高迁移率族蛋白1，sPE为重度子痫前期，PE为子痫前期
图2 3组胎盘组织HMGB1 mRNA相对表达水平柱状图注：HMGB1为高迁移率族蛋白1，sPE为重度子痫前期，PE为子痫前期
图3 HMGB1表达及细胞定位(IHC)[图3A~3D: sPE组、PE组、对照组、阴性对照组胎盘蜕膜组织HMGB1的表达(×100)；图3E：连续组织切片(蜕膜基质细胞标记波形蛋白表达定位，×200)；图3F：连续组织切片(滋养层细胞标记角蛋白7表达定位，×200)；图3G：连续组织切片(HMGB1表达定位，×200)]注：HMGB1为高迁移率族蛋白1，IHC为免疫组织化学染色
图4 原代胎盘蜕膜巨噬细胞鉴定流式细胞术散点图(图4A：根据FSC和SSC，筛选了表达CD14的均质细胞群；图4B：巨噬细胞同时表达CD68及CD14占97.39%；图4C：阴性对照，CD14单染、CD68单染对照及CD14、CD68双染对照)注： FSC表示正向散射，SSC表示侧向散射
图5 rHMGB1对蜕膜巨噬细胞M1型标记(CD86)诱导作用的流式细胞术示意图(图5A：空白处理；图5B：25 ng/mL rHMGB1处理；图5C：50 ng/mL rHMGB1处理；图5D：100 ng/mL rHMGB1处理；图5E：200 ng/mL rHMGB1处理；图5F：rHMGB1处理前)(红色矩形区域所示为M1型巨噬细胞阳性细胞比例)注： rHMGB1为重组高迁移率族蛋白1，FSC表示正向散射，SSC表示侧向散射
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