摘要
目的:探讨人羊膜上皮干细胞(hAECs)对小鼠肝脏缺血再灌注损伤(IRI)的保护作用及机制。方法:将10只C57BL/6小鼠随机分为对照组(IRI＋PBS组)和实验组(IRI＋hAECs组)，每组各5只。IRI＋PBS组经阴茎背静脉注射PBS(100 μL)，IRI＋hAECs组经阴茎背静脉注射hAECs(1×105个)，注射后0.5 h进行常温下缺血1.5 h、再灌注6 h。检测小鼠血清ALT、AST和乳酸脱氢酶(LDH)水平，HE染色观察小鼠肝组织病理变化，免疫组织化学染色观察巨噬细胞(F4/80)以及M1(CD68)、M2 (CD206)型巨噬细胞浸润情况。比较两组凋亡相关因子B淋巴细胞瘤-2(BCL-2)、Bcl-2相关X蛋白(BAX)、半胱天冬氨酸蛋白酶(Caspase)-3、Caspase-7以及炎症因子IL-6、IL-23、TNF-α和IL-β mRNA相对表达量。符合正态分布的连续变量资料采用独立样本t检验比较。P<0.05为差异具有统计学意义。结果:与IRI＋PBS组相比，IRI＋hAECs组小鼠肝组织病理损伤明显改善。IRI＋hAECs组小鼠血清ALT、AST和LDH分别为(1 456±20)、(1 375±49)和(2 435±147)U/L，IRI＋PBS组分别为(3 919±48)、(3 142±145)和(3 499±147) U/L，差异均有统计学意义(t＝47.13、11.53和4.53，P均<0.05)。IRI＋hAECs组BCL-2、BAX、Caspase-3和Caspase-7 mRNA表达水平分别为(0.20±0.05)、(0.19±0.09)、(0.46±0.13)和(0.27±0.03)，均低于IRI＋PBS组的(1.01±0.16)、(1.02±0.28)、(1.01±0.17)和(1.00±0.11)，差异均有统计学意义(t＝8.45、4.88、4.39和10.79，P均<0.05)。免疫组化染色结果显示，巨噬细胞在IRI＋hAECs组浸润更低。IRI＋hAECs组IL-6、IL-23、TNF-α和IL-β mRNA水平分别为(0.38±0.04)、(0.33±0.05)、(0.43±0.08)和(0.12±0.04)，均低于IRI＋PBS组的(1.10±0.11)、(1.00±0.05)、(1.00±0.02)和(1.10±0.06)，差异均有统计学意义(t＝11.27、16.37、12.96和22.34，P均<0.05)。M1型巨噬细胞在IRI＋hAECs组表达明显降低，M2型巨噬细胞比例升高。结论:hAECs通过调节M1/M2型巨噬细胞极化，减轻炎症反应，从而在小鼠肝脏IRI中发挥保护作用。
Abstract
Objective:To investigate the protective effect and mechanism of human amnion epithelial stem cells (hAECs) against liver ischemia-reperfusion injury (IRI) in mice.Methods:Ten C57BL/6 mice were randomly divided into the control group (IRI+ PBS group) and the experimental group (IRI+ hAECs group), with 5 mice in each group. The IRI+ PBS group was injected with PBS (100 μL) via the dorsal vein of the penis, and the IRI+ hAECs group was injected with hAECs (1×105 pieces) via the dorsal vein of the penis. At 0.5 h after injection, ischemia at normal temperature for 1.5 h and reperfusion for 6 h. HE staining was used to observe the pathological changes of mouse liver tissue. Biochemical analysis was used to measure serum ALT, AST and lactate dehydrogenase (LDH) levels. Immunohistochemical staining was used to observe the infiltration of macrophages (F4/80) and M1 (CD68) and M2 (CD206) macrophages. At the same time, the apoptosis-related factors B-lymphoma-2 (BCL-2), BCL-2-related X protein (BAX), cysteinyl aspartate-specific protease (Caspase)-3 and Caspase-7, and the inflammatory factors IL-6, IL-23, TNF-α and IL-β mRNA relative expressions were also compared. Data of continuous variables conforming to normal distribution were compared by independent sample t test. P<0.05 was considered statistically significant.Results:Compared with the IRI + PBS group, the pathological damage of the liver tissue was significantly improved in the IRI + hAECs group. The expression levels of serum ALT, AST and LDH in the IRI+ hAECs group were (1 456±20), (1 375±49) and (2 435±147) U/L, respectively, and those in the IRI+ PBS group were (3 919±48), (3 142±145) and (3 499±147) U/L, the differences were statistically significant (t=47.13, 11.53 and 4.53, all P<0.05). The mRNA expression levels of BCL-2, BAX, Caspase-3 and Caspase-7 in the IRI + hAECs group were (0.20±0.05), (0.19±0.09), (0.46±0.13) and (0.27±0.03), respectively; they were all lower than those in the IRI + PBS group (1.01±0.16), (1.02±0.28), (1.01±0.17) and (1.00±0.11), and the differences were statistically significant (t=8.45, 4.88, 4.39 and 10.79, all P<0.05). Immunohistochemical staining showed that macrophage infiltration was lower in the IRI+ hAECs group. The mRNA levels of IL-6, IL-23, TNF-α and IL-β in the IRI+ hAECs group were (0.38±0.04), (0.33±0.05), (0.43±0.08) and (0.12±0.04), respectively; they were all lower than those in the IRI+ PBS group (1.10±0.11), (1.00±0.05), (1.00±0.02) and (1.10±0.06), and the differences were statistically significant (t=11.27, 16.37, 12.96 and 22.34, all P<0.05). The expression of M1 macrophages cells was significantly decreased in the IRI+ hAECs group, and the proportion of M2 macrophages cells was increased.Conclusion:hAECs protect mice from liver IRI by attenuating inflammatory responses by modulating the polarization of M1/M2 macrophages.
万方数据