颗粒蛋白前体在新生儿坏死性小肠结肠炎动物实验模型中的保护作用探究

Protective effects of progranulin in animal model of neonatal necrotizing enterocolitis

目的:探讨颗粒蛋白前体(progranulin,PGRN)在新生儿坏死性小肠结肠炎(neonatal necrotizing enterocolitis,NEC)新生小鼠的保护作用机制。方法:将60只10日龄的野生型(wild type,WT)C57小鼠分为WT+Control组和WT+NEC组;将60只10日龄C57B/L背景的PGRN基因敲除鼠(PGRN^(-/-))分为PGRN^(-/-)+Control组和PGRN^(-/-)+NEC组。QPCR和ELISA检测小鼠肠道PGRN mRNA和蛋白水平变化;记录小鼠体质量改变,HE染色检测小鼠肠组织损伤情况并进行病理损伤评分;ELISA检测小鼠肠道炎症相关因子[肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、转录生长因子β1(transforming growth factor-β,TGF-β1)、CC趋化因子配体2(C-C motif chemokine ligand 2,CCL2)]的水平改变;免疫荧光染色和流式细胞术检测小鼠肠道的巨噬细胞浸润改变。结果:WT+NEC组小鼠肠组织中PGRN的mRNA和蛋白表达量较WT+Control组小鼠明显提高(P<0.05)。与WT+Control组相比,WT+NEC组小鼠肠组织病理损伤评分升高,PGRN^(-/-)+NEC组小鼠肠道病理损伤评分较WT+NEC组更高(P<0.05)。相比较WT+Control组,WT+NEC组小鼠肠组织促炎因子CCL2,TNF-α水平升高,抗炎因子TGF-β1水平降低;PGRN^(-/-)+NEC组小鼠肠组织较WT+NEC组有更高的CCL2,TNF-α水平,更低的TGF-β1水平(P<0.05)。与WT+Control组相比,WT+NEC组小鼠肠黏膜固有层浸润的巨噬细胞数量增加,PGRN^(-/-)+NEC组小鼠肠组织中浸润的巨噬细胞数量较WT+NEC组更多(P<0.05)。结论:PGRN在NEC小鼠肠组织中高表达,PGRN基因敲除加重了小鼠NEC肠道损伤,其作用可能与PGRN缺失促进肠道巨噬细胞浸润,加剧炎症反应相关。

Objective:To investigate the protective role of progranulin(PGRN)in necrotizing enterocolitis(NEC)in neonatal mice.Methods:Sixty 10-day-old wild-type C57 mice were divided into WT+Control group and WT+NEC group.Sixty 10-day-old PGRNdeficient(PGRN^(-/-))C57B/L mice were divided into PGRN^(-/-)+Control group and PGRN^(-/-)+NEC group.We measured PGRN mRNA and protein levels in the mouse intestine using quantitative PCR and enzyme-linked immunosorbent assay(ELISA);recorded changes in the body weight of the mice;observed and scored the histological damage of the intestinal tissue with HE staining;measured the levels of inflammatory factors(C-C motif chemokine ligand 2[CCL2],tumor necrosis factor-alpha[TNF-α],and transforming growth factorbeta 1[TGF-β1])in the mouse intestine;and assessed macrophage infiltration in the mouse intestine by immunofluorescence staining and flow cytometry.Results:The mRNA and protein expression levels of PGRN in the WT+NEC group were significantly higher than those in the WT+Control group(P<0.05).Compared with the WT+Control group,the pathology score of the intestinal tissue was signifi⁃cantly higher in the WT+NEC group;and the PGRN^(-/-)+NEC group mice had a higher pathology score than the WT+NEC group(P<0.05).Compared with the WT+Control group,the WT+NEC group showed significantly higher levels of pro-inflammatory CCL2 and TNF-αand a significantly lower level of anti-inflammatory TGF-β1;and compared with the WT+NEC group,the PGRN^(-/-)+NEC group showed significantly higher CCL2 and TNF-αlevels and a significantly lower TGF-β1 level(P<0.05).Compared with the WT+Control group,the WT+NEC group had a significantly larger number of macrophages infiltrating in the lamina propria of the intestinal mucosa;and the PGRN^(-/-)+NEC group had significantly more infiltrating macrophages than the WT+NEC group(P<0.05).Conclusion:PGRN is highly expressed in the intestinal tissue of NEC mice,and PGRN knockout exacerbates intestinal damage in NEC mice.PGRN deficiency may increase macrophage infiltration and inflammatory response.