M1型库普弗细胞极化在三氯乙烯致敏小鼠肝脏损伤中的作用

徐琼影, 张家祥, 杨呓, 李娜, 代玉莹, 黄丽萍, 朱启星

徐琼影, 张家祥, 杨呓, 李娜, 代玉莹, 黄丽萍, 朱启星. M1型库普弗细胞极化在三氯乙烯致敏小鼠肝脏损伤中的作用[J]. 环境与职业医学, 2019, 36(10): 927-933. DOI: 10.13213/j.cnki.jeom.2019.19387
引用本文: 徐琼影, 张家祥, 杨呓, 李娜, 代玉莹, 黄丽萍, 朱启星. M1型库普弗细胞极化在三氯乙烯致敏小鼠肝脏损伤中的作用[J]. 环境与职业医学, 2019, 36(10): 927-933. DOI: 10.13213/j.cnki.jeom.2019.19387
XU Qiong-ying, ZHANG Jia-xiang, YANG Yi, LI Na, DAI Yu-ying, HUANG Li-ping, ZHU Qi-xing. Role of M1 Kupffer cell polarization in liver injury induced by trichloroethylene in mice[J]. Journal of Environmental and Occupational Medicine, 2019, 36(10): 927-933. DOI: 10.13213/j.cnki.jeom.2019.19387
Citation: XU Qiong-ying, ZHANG Jia-xiang, YANG Yi, LI Na, DAI Yu-ying, HUANG Li-ping, ZHU Qi-xing. Role of M1 Kupffer cell polarization in liver injury induced by trichloroethylene in mice[J]. Journal of Environmental and Occupational Medicine, 2019, 36(10): 927-933. DOI: 10.13213/j.cnki.jeom.2019.19387

M1型库普弗细胞极化在三氯乙烯致敏小鼠肝脏损伤中的作用

基金项目: 国家自然科学基金(81874259,81673141)
详细信息
    作者简介:

    徐琼影(1996-), 女, 硕士生; E-mail:xqy9617@163.com

    通讯作者:

    朱启星,Email:zqxing@yeah.net

    利益冲突  无申报

Role of M1 Kupffer cell polarization in liver injury induced by trichloroethylene in mice

Funds: This study was funded
More Information
    Corresponding author:

    ZHU Qi-xing,Email:zqxing@yeah.net

  • 摘要:
    [背景] 三氯乙烯(TCE)可致职业性药疹样皮炎并伴有严重的肝脏损伤,是致死的主要原因之一,但其发生机制尚不清楚。

    [目的] 观察TCE致敏小鼠肝脏中库普弗细胞(KCs)极化的情况,探讨KCs的M1型极化在TCE免疫性肝损伤中的作用。

    [方法] 36只雌性BALB/c小鼠,6~8周龄,随机分为4组:空白对照组(n=5),溶剂对照组(n=5),TCE处理组(TCE组)(n=11),抑制剂处理组(TCE+GdCl3组)(n=15),适应性喂养一周。按照本课题组的建模方法建立TCE致敏模型。在末次激发24 h后对TCE组和TCE+GdCl3组进行皮肤评分,将TCE组和TCE+GdCl3组小鼠分为致敏阳性组和致敏阴性组;末次激发72 h后处死小鼠,取肝脏。全自动生化分析仪检测肝脏损伤指标丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)水平,HE染色法检测小鼠肝脏病理变化,免疫组化法检测KCs的表面标志物F4/80的表达情况,免疫荧光法检测小鼠肝脏M1型巨噬细胞表面标志物CD11c的表达水平,免疫组化法检测小鼠肝脏肿瘤坏死因子(TNF)-α的表达情况。

    [结果] TCE组致敏率为45.45%(5/11),TCE+GdCl3组致敏率为33.33%(5/15),差异无统计学意义(P>0.05)。血清ALT和AST水平在空白对照组、溶剂对照组之间差异无统计学意义(P>0.05);与溶剂对照组和TCE阴性组相比,TCE阳性组血清ALT和AST升高有统计学意义(P < 0.05),TCE阳性组ALT及AST水平分别为(115.05±13.74)U/L和(224.68±30.98)U/L;TCE+GdCl3阳性组ALT及AST水平分别为(97.59±9.16)U/L和(124.69±11.26)U/L,低于TCE阳性组(P < 0.05)。HE染色结果显示:对照组及阴性组小鼠肝脏细胞形态正常,染色均匀;TCE阳性组小鼠肝脏细胞形态异常,细胞空泡样变性;TCE+GdCl3阳性组小鼠肝脏部分细胞出现水肿。肝脏F4/80的免疫组化结果显示:空白对照组、溶剂对照组表达较低,TCE阳性组F4/80大量沉积,TCE+GdCl3阳性组表达较TCE阳性组减少(P < 0.05)。肝脏CD11c免疫荧光结果显示:空白对照组、溶剂对照组CD11c表达较少,TCE阳性组CD11c表达水平较高,而TCE+GdCl3阳性组该指标表达水平下降。免疫组化结果显示:空白对照组和溶剂对照组TNF-α表达水平极低,TCE阳性组肝脏TNF-α大量沉积,TCE+GdCl3阳性组肝脏中TNF-α表达水平较TCE阳性组下降(P < 0.05)。

    [结论] KCs在TCE致敏小鼠免疫性肝损伤中发挥重要作用,其M1型极化会加重小鼠肝脏损伤。

     

    Abstract:
    [Background] Trichloroethylene (TCE) can cause occupational drug-like dermatitis with severe liver damage, which is one of the leading causes of death, but its mechanism is still unclear.

    [Objective] This study observes the polarization of Kupffer cells (KCs) in the liver of TCE sensitized mice, and to explore the role of M1 type polarization of KCs in immune liver injury induced by TCE.

    [Methods] Thirty-six female BALB/c mice, 6-8 weeks old, were randomly divided into four groups:blank control group (n=5), vehicle control group (n=5), TCE-treated group (TCE group, n=11), and inhibitor-treated group (TCE+GdCl3 group, n=15), with adaptive feeding for one week. A TCE sensitization model was established according to an protocol developed by our team. Twenty-four hours after the last challenge, the skin of the TCE group and the TCE+GdCl3 group was scored and the mice in the TCE group and the TCE+GdCl3 group were divided into sensitization positive group and sensitization negative group. Seventy-two hours after the last challenge, the mice were sacrificed and the livers were taken. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) as selected liver injury indicators were determined using automatic biochemical analyzer; the pathological changes of liver were observed after HE staining; the expression level of surface marker F4/80 of KCs was detected by immunohistochemistry; the expression level of surface marker CD11c in M1 type macrophages was detected by immunofluorescence; and the expression level of tumor necrosis factor (TNF)-α in mouse liver was detected by immunohistochemistry.

    [Results] The sensitization rate was 45.45% (5/11) in the TCE group and 33.33% in the TCE+GdCl3 group (5/15), with no significant difference (P>0.05). The serum ALT and AST levels were not significantly different between the blank control group and the vehicle control group (P>0.05). Compared with the vehicle control group and the TCE negative group, the serum ALT and AST levels were significantly increased in the TCE positive group (P < 0.05). The ALT level and AST level in the TCE positive group were (115.05±13.74) U/L and (224.68±30.98) U/L, respectively; the levels in the TCE+GdCl3 positive group were (97.59±9.16) U/L and (124.69±11.26) U/L, respectively, lower than those in the TCE positive group (P < 0.05). The results of HE staining showed that the liver cells of the control groups and the negative groups were normal in morphology and color; in the TCE positive group, the cells had abnormal cell morphology and vacuolar degeneration; some cells in the liver of the TCE+GdCl3 positive group showed edema. The immunohistochemical results of liver F4/80 showed that the expression levels in the blank control group and the vehicle control group were low, the TCE positive group had more deposition of F4/80 than the vehicle control group, while the TCE+GdCl3 positive group had less than the TCE positive group (P < 0.05). The results of liver CD11c immunofluorescence showed that the expression levels were low in the blank control group and the vehicle control group, the expression level of CD11c was higher in the TCE positive group than in the vehicle control group, while the expression level of this index was lower in the TCE+GdCl3 positive group than in the TCE positive group. The results of immunohistochemistry showed that the expression levels of TNF-α were very low in the blank control group and the vehicle control group, and higher in the TCE positive group than in the vehicle control group, and lower in the TCE+GdCl3 positive group than in the TCE positive group (P < 0.05).

    [Conclusion] KCs play an important role in immune liver injury induced by TCE in mice, and M1 type polarization may aggravate the injury.

     

  • 三氯乙烯(trichloroethylene,TCE)是一种卤代烃类有机溶剂,相对分子质量为131.5,由于其脂溶性、挥发性、不易燃的特点,在五金、电子、电镀、塑胶等多种行业用作干洗剂和除脂剂[1-2]。包括我国在内的很多发展中国家在日常生产活动中广泛使用TCE,少数职业工人接触TCE后会出现类似于严重药物过敏反应所引起的全身性皮肤病,称之为职业性三氯乙烯药疹样皮炎(occupational medicamentosalikedermatitis due to TCE,OMLDT)[3]。OMLDT患者除严重的皮肤损伤外,还伴有肝脏损伤,是其致死的主要原因之一。因此,开展TCE介导的免疫性肝脏损伤的机制研究,以及OMLDT免疫性脏器损伤的救治已经成为职业卫生领域的重要任务和难题之一[3-5]。有研究发现TCE致敏动物的免疫性肝脏损伤过程中伴有库普弗细胞(Kupffer cells,KCs)活化和CD68表达增高[6]

    KCs是机体重要的先天性免疫细胞,是肝脏最主要的巨噬细胞,外源性和/或内源性危险信号都会诱发KCs的极化并触发炎症反应,导致促炎细胞因子的级联释放并造成肝脏损伤[7]。KCs被激活后,其表面标志物F4/80的表达上升[8]。不同的微环境和疾病状态下,KCs会出现M1和M2两种不同的极化类型,分别发挥不同的功能。CD11c是M1型巨噬细胞的主要标志物,在受到外界刺激时,KCs可以极化成为M1型巨噬细胞,分泌肿瘤坏死因子(tumor necrosis factor,TNF)-α、白介素(interleukin,IL)-1β、IL -6等细胞因子和趋化因子,发挥促进炎症,清除病原体的功能;而KCs极化成的M2型巨噬细胞,分泌IL-10、转化生长因子-β等细胞因子,发挥抑制炎症,修复受损组织以及免疫调节的功能[9-10]。氯化钆(GdCl3)在动物实验中常用作KCs的抑制剂,可以特异性消除KCs和(或)阻断KCs的功能[11]。本研究采取整体动物实验的方法,在建立TCE经皮致敏小鼠模型的基础上腹腔注射KCs的抑制剂GdCl3,检测小鼠肝脏内F4/80、CD11c等相关指标的表达情况,探索KCs的M1型极化在TCE经皮致敏小鼠免疫性肝损伤中的作用。

    参照前期研究建立小鼠模型[12],选择36只雌性BALB/c小鼠,6~8周龄,购自安徽医科大学实验动物中心,体重(20.28±1.36)g。将小鼠圈养在经过认证的微隔离笼中,在温度20~25℃和湿度(50±5)%条件下饲养,每笼5只动物,并在12 h光照下自由接受标准小鼠食物和水。每天按12 h光照12 h黑暗的周期循环,适应性喂养1周。将小鼠随机分为4组:空白对照组(n=5),溶剂(橄榄油和丙酮)对照组(n=5),TCE处理组(TCE组,n=11),抑制剂处理组(TCE+GdCl3组,n=15)。

    TCE与弗氏完全佐剂(FCA)(Sigma,美国),丙酮与橄榄油(上海化学试剂公司,中国),GdCl3(西亚试剂,中国),链霉卵白素-生物素免疫组化检测试剂盒、二氨基联苯胺(DAB)显色液(北京中杉金桥生物技术有限公司,中国),天冬氨酸氨基转移酶(aspartatetransaminase,AST)、丙氨酸氨基转移酶(alaninetransaminase,ALT)检测试剂盒(南京建成生物工程研究所,中国),F4/80抗体(Abcam,英国),CD11c抗体(Santa cruz,美国),TNF-α抗体(北京博奥森生物技术有限公司,中国),AlexaFluor®594标记山羊抗小鼠IgG(H & L)、DAPI(Abcam,英国),放射免疫沉淀法(RIPA)裂解缓冲液与苯甲基磺酰氟(PMSF)蛋白酶抑制剂(碧云天生物技术,中国),0.45 μm聚偏氟乙烯(PVDF)膜(GE Healthcare,德国),WB显影试剂盒(Advansta,美国),苏木素与伊红染色液(无锡市江源实业技贸总公司,中国),BX53光学显微镜(奥林巴斯,日本),全自动生化分析仪(Bio-Tek,美国)。

    建立TCE小鼠致敏模型。第1天,TCE组小鼠皮下注射100 μL 50%TCE(TCE、橄榄油、丙酮体积比为5:2:3)和FCA(50 μL)混合物进行第一次刺激。第4、7、10天,在背部的剃毛区域涂上100 μL 50%TCE,继续致敏。在第17天和第19天,使用100 μL 30%TCE(TCE、橄榄油、丙酮体积比为3:2:5)涂抹两次激发背部皮肤。TCE+GdCl3组遵循与TCE组相同的方案,除了在第17天和第19天腹腔注射GdCl3(40 mg/kg)[11]。溶剂对照组小鼠用不含TCE、相同比例和剂量的橄榄油和丙酮混合液进行类似处理。空白对照组未接受任何处理。用滤纸覆盖每只小鼠的剃毛区域,并在喘气后用无刺激性胶带密封24 h。在第20天(末次激发24 h后),小鼠背部的皮肤反应以4分量表评分:0,无反应;1,散在轻度发红;2,中度和弥漫性发红;3,强烈的红斑和肿胀。任何动物的皮肤反应评分≥ 1,则将其判断为致敏反应并归类为致敏阳性组(阳性组),否则为致敏阴性组(阴性组),计算TCE组和TCE+GdCl3组的致敏率(发红数或肿胀数/小鼠数)。在第22天(72 h时间点)通过颈椎脱位处死小鼠后,根据实验要求无菌操作收集血液,肝脏和其他样品。

    从眼球静脉丛收集血液,在4℃下以3 000 r/min(离心半径87 mm)离心15 min。按照试剂盒说明进行血清ALT和AST检测,通过全自动生化分析仪测定血清中ALT和AST的活性。

    新鲜小鼠肝脏置于多聚甲醛中固定后石蜡包埋,制成5 μm厚的切片后,二甲苯脱蜡、乙醇梯度脱水、PBS清洗玻片,苏木素染色液缸染2 min,纯水清洗2 min,伊红染色液缸染10 s,清洗后烤片20 min,中性树脂封片,凝固后镜下观察拍照。

    新鲜肝脏样本置于多聚甲醛中固定后石蜡包埋,制成5 μm厚的切片后,二甲苯脱蜡、乙醇梯度脱水,内源性过氧化物酶处理,0.01 mol/L柠檬酸钠缓冲液在微波炉中抗原修复,冷却至室温后山羊血清处理15 min后,将切片分别与F4/80和TNF-α抗体在4℃孵育过夜。第2天,依次与二抗和辣根酶反应,DAB显色后镜下观察拍照。

    新鲜肝脏样本置于多聚甲醛中固定后包埋在石蜡中,然后制成5 μm厚的切片,用二甲苯脱蜡,乙醇梯度脱水,在微波炉中用0.01 mol/L柠檬酸钠缓冲液进行抗原修复。冷却至室温后,使用0.3%Triton-X100处理30 min,血清封闭2 h,滴加CD11c一抗过夜。第二天,二抗室温孵育2 h,使用DAPI对细胞核染色15 min,在显微镜下观察(从孵育二抗开始完全避光,直至实验结束)。

    使用SPSS 13.0统计软件进行分析。皮肤致敏率采用Fisher确切概率法分析,单因素方差分析(ANOVA)用于分析多个组之间的差异,所有计量数据均以均数±标准差表示,分析前进行正态性和方差齐性检验,检验水准α=0.05。

    TCE组的致敏率为45.45%(5/11),TCE+GdCl3组的致敏率为33.33%(5/15),各组之间差异无统计学意义(χ2=0.394,P=0.530)。见表 1

    表  1 

    小鼠皮肤致敏评分和致敏率

    Table  1. 

    Skin sensitization score and sensitization rate of mice

    组别
    Group
    小鼠数(n
    Number of mice
    评分结果 致敏率(%)
    Sensitization rate
    0 1 2 3
    空白对照(Blank control) 5 5 0 0 0 0.00
    溶剂对照(Vehicle control) 5 5 0 0 0 0.00
    TCE 11 6 2 2 1 45.45
    TCE+GdCl3 15 10 3 2 0 33.33
    下载: 导出CSV 
    | 显示表格

    空白对照组与溶剂对照组血清ALT和AST水平的差异无统计学意义(P > 0.05)。TCE阳性组ALT水平及AST水平分别为(115.05±13.74)U/L和(224.68± 30.98)U/L,TCE+GdCl3阳性组ALT及AST水平分别为(97.59±9.16)U/L和(124.69±11.26)U/L,经统计学分析发现,与溶剂对照组和TCE阴性组相比,TCE阳性组血清ALT和AST水平升高(P < 0.05)。TCE+GdCl3阳性组血清ALT和AST结果低于TCE阳性组,差异有统计学意义(P < 0.05)。见图 1

    图  1 

    小鼠血清丙氨酸氨基转移酶(A)与天冬氨酸氨基转移酶水平(B)

    [注]a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。
    Figure  1. 

    The serum ALT (A) and AST (B) levels of mice

    [Note]a: Compared with the vehicle control group, P < 0.05; b: Comparedwith the TCE negative group, P < 0.05; c: Compared with the TCEpositive group, P < 0.05.

    肝脏HE染色结果显示,空白对照组、溶剂对照组、阴性组小鼠肝脏细胞形态正常,染色均匀,未见水肿。TCE阳性组小鼠肝脏部分区域可见细胞空泡样变性,胞质疏松,细胞形态异常,而TCE+GdCl3阳性组小鼠肝脏出现细胞水肿。见图 2

    图  2 

    小鼠肝脏病理染色结果(HE染色)

    [注]A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组;G:图D所指区域放大部分。图中箭头所指为细胞形态异常。
    Figure  2. 

    Results of liver pathological staining in mice (HE staining)

    [Note]A: Blank control group; B: Vehicle control group; C: TCE negativegroup; D: TCE positive group; E: TCE+GdCl3 negative group; F: TCE+GdCl3 positive group; G: The enlarged area indicated in FigureD. The arrows indicate abnormal cell morphology.

    免疫组化结果显示,空白对照组和溶剂对照组的F4/80表达水平较低,TCE阳性组肝脏F4/80大量沉积,TCE+GdCl3阳性组肝脏中F4/80表达水平下降。见图 3

    图  3 

    小鼠肝脏F4/80表达结果

    [注]上图:免疫组化结果;下图:F4/80表达变化。A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组;G:图D所指区域放大部分。a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。图中箭头所指为F4/80阳性细胞。
    Figure  3. 

    Mouse liver F4/80 expression

    [Note]Above: Immunohistochemical results; Below: F4/80 expressionchanges. A: Blank control group; B: Vehicle control group; C: TCEnegative group; D: TCE positive group; E: TCE+GdCl3 negativegroup; F: TCE+GdCl3 positive group; G: The enlarged area indicatedin Figure D. a: Compared with the vehicle control group, P < 0.05; b: Compared with the TCE negative group, P < 0.05; c: Compared withthe TCE positive group, P < 0.05. The arrow refers to F4/80 positivecells.

    免疫荧光技术检测结果显示,空白对照组和溶剂对照组CD11c表达较少,TCE阳性组表达较高,TCE+GdCl3阳性组肝脏中CD11c表达水平下降。见图 4

    图  4 

    小鼠肝脏CD11c表达结果

    [注]上图:免疫荧光图(红色:CD11c;蓝色:DAPI);下图:CD11c表达变化。A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组。a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。
    Figure  4. 

    Mouse liver CD11c expression

    [Note]Above: Immunofluorescence (red: CD11c; blue: DAPI); Below: CD11c expression changes. A: Blank control group; B: Vehiclecontrol group; C: TCE negative group; D: TCE positive group; E: TCE+GdCl3 negative group; F: TCE+GdCl3 positive group. a: Compared with the vehicle control group, P < 0.05; b: Comparedwith the TCE negative group, P < 0.05; c: Compared with the TCEpositive group, P < 0.05.

    免疫组化结果显示,空白对照组和溶剂对照组TNF-α表达水平极低,TCE阳性组肝脏TNF-α大量沉积,TCE+GdCl3阳性组肝脏中TNF-α表达水平下降。见图 5

    图  5 

    小鼠肝脏TNF-α表达结果

    [注]上图:免疫组化结果;下图:TNF-α变化。A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组;G:图D所指区域放大部分。a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。图中箭头所指为TNF-α阳性细胞。
    Figure  5. 

    Mouse liver TNF-α expression

    [Note]Above: Immunohistochemical results; Below: TNF-α changes. A: Blank control group; B: Vehicle control group; C: TCE negativegroup; D: TCE positive group; E: TCE+GdCl3 negative group; F: TCE+GdCl3 positive group; G: The enlarged area indicated in Figure D. a: Compared with the vehicle control group, P < 0.05; b: Comparedwith the TCE negative group, P < 0.05; c: Compared with the TCEpositive group, P < 0.05. The arrow refers to TNF-α positive cells.

    TCE在我国生产行业中应用广泛,且使用量逐年上升,使用地区主要集中在广东沿海地区,我国目前至少仍有2万名工人职业暴露于TCE,且OMLDT新发病例仍时有报道[13-14]。职业环境中,工人可经呼吸道和皮肤等途径暴露于TCE,导致多系统和器官损害。在本课题组前期研究中发现,TCE致敏小鼠的肝脏中内存在CD68的表达水平增高,表明TCE可能会导致小鼠肝脏KCs激活[15],KCs的激活会导致肝细胞的炎症性损伤。本实验通过腹腔注射GdCl3来抑制KCs的活性,GdCl3选择性地降低KCs摄取细胞物质的能力并减少响应肝损伤的细胞因子和自由基产生,防止细胞凋亡,减少炎性介质的释放并改善肝功能,通过GdCl3预处理抑制KCs功能已经显示出改善甚至防止化学诱导的大鼠或小鼠的肝损伤 [11, 16]。本次研究发现,通过TCE皮肤接触使BALB/c小鼠致敏,在TCE阳性组小鼠肝脏中,F4/80表达升高,而使用GdCl3阻断KCs活性的同时会降低F4/80的表达,对照组和TCE阴性组ALT和AST水平没有明显差异,而TCE阳性组ALT和AST的却出现明显升高,同时发现GdCl3在一定程度上降低了AST和ALT的含量。肝脏病理学检测与肝功能的变化趋势基本相符,TCE阳性组肝功能表达升高,同时病理损伤严重,KCs发生极化改变,向M1型巨噬细胞极化,而使用GdCl3后,KCs的功能得到有效抑制;致敏阴性组中未出现肝损伤,也未发生肝功能指标变化。

    KCs是机体单核巨噬细胞系统的主要组成部分,通过模式识别受体识别体内不同的外源性和内源性危险信号,当巨噬细胞非抗原特异性地激活辅助型Th1细胞时,这种活化状态形成的KCs被称为M1型巨噬细胞,M1型巨噬细胞高表达诱导型一氧化氮合酶;而Th2细胞分泌的IL-4与IL-13可以选择性地激活巨噬细胞,提高其主要组织相容性复合物Ⅱ抗原的表达,这种活化状态形成的则是M2型巨噬细胞,M2型巨噬细胞高表达甘露糖受体CD206和精氨酸酶1[17-18],KCs的正常极化与自身稳态有关,其异常极化会导致多种疾病的产生[19-20]。KCs的M1亚型极化会导致特异性的细胞因子TNF-α分泌增加,TNF-α与其表面受体(即TNFR1和TNFR2)结合,将损伤信号从细胞膜传至细胞核,与相应靶基因的启动子结合,从而介导免疫性肝脏损伤[21]。但目前关于TCE致敏小鼠肝脏损伤与KCs极化机制的研究却十分缺乏。作为M1型巨噬细胞的特异性标志物和分泌的主要促炎细胞因子,本研究结果发现CD11c在TCE阳性组中大量沉积,GdCl3预处理抑制了KCs的活性,从而减弱了肝组织中CD11c表达;并且,阻断KCs会影响细胞因子的分泌,即这些变化伴随着TNF-α表达的变化。

    综上所述,M1型KCs极化在TCE致敏小鼠免疫性肝损伤中发挥重要作用,但M1型KCs极化介导肝脏损伤的具体机制尚未澄清,值得进一步研究。

  • 图  1  

    小鼠血清丙氨酸氨基转移酶(A)与天冬氨酸氨基转移酶水平(B)

    [注]a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。

    Figure  1.  

    The serum ALT (A) and AST (B) levels of mice

    [Note]a: Compared with the vehicle control group, P < 0.05; b: Comparedwith the TCE negative group, P < 0.05; c: Compared with the TCEpositive group, P < 0.05.

    图  2  

    小鼠肝脏病理染色结果(HE染色)

    [注]A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组;G:图D所指区域放大部分。图中箭头所指为细胞形态异常。

    Figure  2.  

    Results of liver pathological staining in mice (HE staining)

    [Note]A: Blank control group; B: Vehicle control group; C: TCE negativegroup; D: TCE positive group; E: TCE+GdCl3 negative group; F: TCE+GdCl3 positive group; G: The enlarged area indicated in FigureD. The arrows indicate abnormal cell morphology.

    图  3  

    小鼠肝脏F4/80表达结果

    [注]上图:免疫组化结果;下图:F4/80表达变化。A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组;G:图D所指区域放大部分。a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。图中箭头所指为F4/80阳性细胞。

    Figure  3.  

    Mouse liver F4/80 expression

    [Note]Above: Immunohistochemical results; Below: F4/80 expressionchanges. A: Blank control group; B: Vehicle control group; C: TCEnegative group; D: TCE positive group; E: TCE+GdCl3 negativegroup; F: TCE+GdCl3 positive group; G: The enlarged area indicatedin Figure D. a: Compared with the vehicle control group, P < 0.05; b: Compared with the TCE negative group, P < 0.05; c: Compared withthe TCE positive group, P < 0.05. The arrow refers to F4/80 positivecells.

    图  4  

    小鼠肝脏CD11c表达结果

    [注]上图:免疫荧光图(红色:CD11c;蓝色:DAPI);下图:CD11c表达变化。A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组。a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。

    Figure  4.  

    Mouse liver CD11c expression

    [Note]Above: Immunofluorescence (red: CD11c; blue: DAPI); Below: CD11c expression changes. A: Blank control group; B: Vehiclecontrol group; C: TCE negative group; D: TCE positive group; E: TCE+GdCl3 negative group; F: TCE+GdCl3 positive group. a: Compared with the vehicle control group, P < 0.05; b: Comparedwith the TCE negative group, P < 0.05; c: Compared with the TCEpositive group, P < 0.05.

    图  5  

    小鼠肝脏TNF-α表达结果

    [注]上图:免疫组化结果;下图:TNF-α变化。A:空白对照组;B:溶剂对照组;C:TCE阴性组;D:TCE阳性组;E:TCE+GdCl3阴性组;F:TCE+GdCl3阳性组;G:图D所指区域放大部分。a:与溶剂对照组相比,P < 0.05;b:与TCE阴性组相比,P < 0.05;c:与TCE阳性组相比,P < 0.05。图中箭头所指为TNF-α阳性细胞。

    Figure  5.  

    Mouse liver TNF-α expression

    [Note]Above: Immunohistochemical results; Below: TNF-α changes. A: Blank control group; B: Vehicle control group; C: TCE negativegroup; D: TCE positive group; E: TCE+GdCl3 negative group; F: TCE+GdCl3 positive group; G: The enlarged area indicated in Figure D. a: Compared with the vehicle control group, P < 0.05; b: Comparedwith the TCE negative group, P < 0.05; c: Compared with the TCEpositive group, P < 0.05. The arrow refers to TNF-α positive cells.

    表  1  

    小鼠皮肤致敏评分和致敏率

    Table  1  

    Skin sensitization score and sensitization rate of mice

    组别
    Group
    小鼠数(n
    Number of mice
    评分结果 致敏率(%)
    Sensitization rate
    0 1 2 3
    空白对照(Blank control) 5 5 0 0 0 0.00
    溶剂对照(Vehicle control) 5 5 0 0 0 0.00
    TCE 11 6 2 2 1 45.45
    TCE+GdCl3 15 10 3 2 0 33.33
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  • 收稿日期:  2019-06-03
  • 录用日期:  2019-08-07
  • 发布日期:  2019-10-24
  • 刊出日期:  2019-11-01

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