盘状结构域受体1对实验性结肠炎小鼠肠道炎症及纤维化的影响

Effects of discoidin domain receptor 1 on intestinal inflammation and fibrosis in experimental colitis mice

目的探究盘状结构域受体1(discoidin domain receptors,DDR1)在慢性结肠炎小鼠肠道炎症及肠纤维化中的功能。方法针对DDR1基因exon4设计并合成gRNA序列,与编码Cas9的mRNA混合显微注射入C57BL/6小鼠受精卵内,构建DDR1基因突变小鼠。选择F4代基因敲除纯合子小鼠(DDR1^(-/-))和野生型C57BL/6小鼠,通过葡聚糖硫酸钠(DSS)诱导慢性结肠炎模型。比较两组小鼠疾病活动指数(DAI)、体质量、结肠长度及组织病理学变化。ELISA法检测外周血细胞因子的含量,Western blot法检测小鼠结肠纤维化相关蛋白表达。结果PCR法检测结果显示利用CRISPR/Cas9技术成功构建DDR1^(-/-)小鼠;循环饮用DSS后,与野生型小鼠相比,DDR1^(-/-)小鼠体质量下降减缓,DAI评分及肠组织病理学评分降低,结肠短缩减轻(5.81±0.11 cm vs 5.19±0.05 cm,P<0.05);血清IL-1β、TNF-α及TGF-β水平下调(P<0.01);Masson染色胶原容积评分显示肠纤维化减轻(DSS-WT vs DSS-DDR1,41.43±0.08 cm vs 21.37±0.07 cm,P<0.01);肠组织TGF-β、α-SMA和COL1A1的蛋白表达下降。结论DDR1参与慢性结肠炎的发生,敲除DDR1基因可减轻慢性结肠炎肠道炎症及纤维化的症状和疾病进程。

Objective To explore the function of discoidin domain receptor 1(DDR1)in intestinal inflammation and fibrosis in mice with chronic colitis.Methods A 20 bp gRNA sequence targeting exon4 of DDR1 gene was designed and synthesized,and subsequently microinjected into the fertilized eggs of C57BL/6 mice after being mixed with Cas9 mRNA in order to generate DDR1 mutant mice.The F4-generation homozygous mice(DDR1^(-/-))as well as wild-type C57BL/6 mice(WT)were used to induce chronic colitis model by being given 2.5%dextran sodium sulfate(DSS)in drinking water.Disease activity index(DAI),body weight,colon length and histopathological changes were compared between the 2 groups.The contents of pro-inflammatory cytokines in peripheral blood were detected by ELISA.The severity of intestinal fibrosis and the expression of fibrosis related proteins were observed by Masson staining and Western blotting,respectively.Results PCR results identified that DDR1^(-/-)mutant mice were successfully generated using CRISPR/Cas9 technology.As compared with the WT group,the DDR1^(-/-)mice exhibited slower body weight loss,lower DAI scores and intestinal histopathology scores,and mild colon shortening(5.81±0.11 vs 5.19±0.05 cm,P<0.05)after DSS drinking.The serum levels of IL-1β,TNF-αand TGF-βwere decreased(P<0.01),with down-regulated expression of TGF-β,α-SMA and COL1A1 proteins in the colon tissues of DDR1^(-/-)mice.Masson staining displayed that collagen volume fraction was higher in the WT group than the DDR1^(-/-)mice(41.43±0.08 vs 21.37±0.07 cm,P<0.01).Conclusion DDR1 is probably involved in the development of chronic colitis,and DDR1 deletion may mitigate the symptoms and disease progression of chronic intestinal inflammation and fibrosis.