Polarization state and significance of macrophage in acute rejection after intestinal transplantation
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摘要:
目的 探究小肠移植术后发生急性排斥反应（AR）时巨噬细胞极化状态的改变。 方法 将6只Brown Norway（BN）大鼠和24只Lewis大鼠分为假手术组（6只Lewis大鼠）、同基因组（Lewis→Lewis，供受体各6只）和异基因组（BN→Lewis，供受体各6只）。对各组大鼠术后7 d的移植肠组织进行苏木素-伊红（HE）染色和脱氧核糖核酸末端转移酶介导的 dUTP 缺口末端标记（TUNEL）法检测，观察其病理学表现和细胞凋亡情况；采用酶联免疫吸附试验（ELISA）检测血清中M1和M2型巨噬细胞极化相关细胞因子表达水平；利用免疫荧光技术检测各组移植肠组织中M1和M2型巨噬细胞表面标志物并进行共定位计数分析。 结果 HE染色和TUNEL检测结果显示假手术组与同基因组肠上皮形态结构正常，未见明显凋亡小体；异基因组大鼠术后7 d移植肠组织上皮层绒毛结构破坏严重，隐窝数量减少，凋亡小体增多，炎症细胞浸润肠壁全层，呈现中-重度AR。ELISA结果显示异基因组受体鼠血清中M1型巨噬细胞极化相关细胞因子肿瘤坏死因子（TNF）-α、干扰素（IFN）-γ和白细胞介素（IL）-12表达水平高于假手术组和同基因组，同基因组中M2型巨噬细胞极化相关细胞因子IL-10和转化生长因子（TGF）-β表达水平高于假手术组和异基因组，差异均有统计学意义（均为P<0.05）。免疫荧光结果显示异基因组移植肠组织中M1型巨噬细胞计数多于假手术组和同基因组，同基因组M2型巨噬细胞计数多于假手术组和异基因组，差异均有统计学意义（均为P<0.05）。 结论 小肠移植术后发生AR的移植物中，大量巨噬细胞浸润肠壁全层，以M1型为主并分泌大量促炎因子，调控巨噬细胞极化方向是治疗小肠移植术后AR的潜在方法。 Abstract:Objective To investigate the changes of macrophage polarization during acute rejection (AR) after intestinal transplantation. Methods Six Brown Norway (BN) rats and 24 Lewis rats were divided into the sham operation group (6 Lewis rats), syngeneic transplantation group (Lewis→Lewis, 6 donors and 6 recipients) and allogeneic transplantation group (BN→Lewis, 6 donors and 6 recipients). At postoperative 7 d, the intestinal graft tissues in all groups were collected for hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Pathological manifestations and cell apoptosis were observed. The expression levels of serum cytokines related to M1 and M2 macrophage polarization were determined by enzyme-linked immunosorbent assay (ELISA). Surface markers of M1 and M2 macrophages of intestinal graft tissues in each group were co-localized and counted by immunofluorescence staining. Results HE staining and TUNEL assay showed that the intestinal epithelial morphology and structure were normal and no evident apoptotic bodies were found in the sham operation and syngeneic transplantation groups. At 7 d after transplantation, the epithelial villi structure of intestinal graft tissues was severely damaged, the number of crypts was decreased, the number of apoptotic bodies was increased, and inflammatory cells infiltrated into the whole intestinal wall, manifested with moderate to severe AR in the allogeneic transplantation group. ELISA revealed that the expression levels of serum cytokines related to M1 macrophage polarization, such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-12, of the recipient rats in the allogeneic transplantation group were higher than those in the sham operation and syngeneic transplantation groups. The expression levels of serum cytokines related to M2 macrophage polarization, such as IL-10 and transforming growth factor (TGF)-β, in the syngeneic transplantation group were higher compared with those in the sham operation and allogeneic transplantation group, and the differences were statistically significant (all P<0.05). Immunofluorescence staining showed that the number of M1 macrophages in the allogeneic transplantation group was higher than those in the sham operation and syngeneic transplantation groups, and the number of M2 macrophages in the syngeneic transplantation group was higher than those in the sham operation and allogeneic transplantation groups, and the differences were statistically significant (all P<0.05). Conclusions Among the allografts with AR after intestinal transplantation, a large number of macrophages, mainly M1 macrophages secreting a large number of pro-inflammatory cytokines, infiltrate into the whole intestinal wall. Regulating the direction of macrophage polarization is a potential treatment for AR after intestinal transplantation. -
图 1 各组移植肠组织病理学表现（HE，×100）
Figure 1. Pathological findings of intestinal grafts in each group
图 2 各组移植肠组织荧光染色及凋亡细胞计数
注：A图为凋亡细胞荧光染色（×100）；B图为凋亡细胞计数，与假手术组比较，aP<0.05，与同基因组比较，bP<0.05。
Figure 2. Fluorescence staining and apoptotic cell count in intestinal grafts of each group
图 3 各组移植肠组织M1、M2型巨噬细胞免疫荧光共定位（免疫荧光，×100）
注：A图为免疫荧光双标定位检测M1型巨噬细胞表面标志物F4/80、CD11b；B图为免疫荧光双标定位检测M2型巨噬细胞表面标志物F4/80、CD206。
Figure 3. Immunofluorescence co-localization of M1 and M2 type macrophages in intestinal grafts of each group
图 4 各组移植肠组织M1型和M2型巨噬细胞计数
注：图为各组移植肠组织中M1型和M2型巨噬细胞在100倍视野下计数；与假手术组比较，aP<0.05，与同基因组比较，bP<0.05。
Figure 4. Counts of M1 and M2 type macrophages in intestinal grafts of each group
表 1 各组血清中M1型与M2型巨噬细胞极化相关细胞因子表达水平（$\bar {\boldsymbol{x}} $±s，pg/mL）
Table 1. Expression levels of M1 and M2 macrophage polarization related cytokines in serum of each group
组别 n TNF-α IFN-γ IL-12 IL-10 TGF-β 假手术组 6 89.3±2.5 47.4±5.0 10.3±0.6 23. 8±1.0 97.7±4.8 同基因组 6 97.9±6.1 50.0±2.1 11.1±1.2 45.8±1.9a 147.1±19.2a 异基因组 6 213.8±4.1a,b 102.4±3.3 a,b 22.1±1.3 a,b 36.6±2.1b 74.6±3.6b F值 1 456.16 432.27 234.97 246.95 60.99 注：与假手术组比较，aP<0.05，与同基因组比较，bP<0.05。 
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