摘要
目的探讨供者T细胞Stat3基因敲除对急性肠道移植物抗宿主病(GI-aGVHD)的影响及其机制。方法对BALB/c小鼠进行清髓剂量辐照,经尾静脉输注BALB/c小鼠(同基因对照组)、C57BL/6小鼠(野生型T细胞组,WT组)及C57BL/6J-Stat3 em1cyagen小鼠(Stat3基因敲除T细胞组,Stat3-KO组)的骨髓和脾脏细胞构建aGVHD模型。监测小鼠生存率、体重变化及临床评分,流式微球阵列术检测血清细胞因子浓度,分离组织中的淋巴细胞进行流式细胞术分析,HE染色后观察肠道病理学变化,FITC-葡聚糖检测肠道通透性,免疫组化评估Ki67和Muc2的表达,实时荧光定量逆转录PCR(qRT-PCR)分析小肠Olfm4、Lysozyme和Muc2的基因表达水平,代谢组学检测血清和肠道代谢物。同时,构建小肠类器官与T细胞的共培养体系,体外模拟GI-aGVHD模型,观察类器官数量与面积变化。此外,通过接种荧光素酶转染的急性淋巴细胞白血病(ALL/Luc)细胞和生物发光成像来评估移植物抗白血病(GVL)效应。结果与WT组相比,Stat3-KO组小鼠临床症状(体重下降、弓背、腹泻)较轻,生存率较高(P<0.05),IL-2、IL-6、IFN-γ、TNF-α、IL-17A以及IL-10血清浓度较低(均P<0.05),肠道炎症细胞浸润及肠黏膜通透性减弱(均P<0.05)。此外,Stat3-KO组小肠Muc2和Ki67表达显著上调(均P<0.05),Olfm4、Lysozyme和Muc2基因的表达水平亦明显上调(均P<0.05)。体外实验显示,Stat3-KO组的类器官发育优于WT组。代谢组学分析提示,敲除供者T细胞Stat3基因减轻GI-aGVHD可能与调节胆汁酸及不饱和脂肪酸代谢相关。在GVL小鼠模型中,回输去除T细胞的骨髓细胞(TCD-BM组)的ALL/Luc细胞迅速生长,而Stat3-KO组未观察到肿瘤生长,80%小鼠的无肿瘤存活期超过100 d(P<0.05)。结论敲除供者T细胞Stat3基因可减轻T细胞对肠道干细胞的损伤,从而缓解GI-aGVHD的肠道损伤,同时保留稳定的GVL效应。
ObjectiveTo investigate the effects and underlying mechanisms of Stat3 knockout in donor T cells on acute gastrointestinal graft-versus-host disease(GI-aGVHD).MethodsBALB/c mice were exposed to lethal irradiation and transplanted with bone marrow and spleen cells from BALB/c mice(syngeneic control group),C57BL/6 mice(wild-type T cell group,WT group),or C57BL/6J-Stat3 em1cyagen mice(Stat3 gene knockout T cell group,Stat3-KO group)via tail vein injection to establish the aGVHD model.The survival rate,body weight changes,and clinical scores of mice were monitored.Cytometric bead array(CBA)was used to detect the concentrations of serum cytokines.Lymphocytes were isolated from tissues for flow cytometric analysis.H&E staining was performed to observe intestinal pathological changes.FITC-dextran assay was conducted to assess intestinal permeability.Immunohistochemistry was used to evaluate the expression of Ki67 and Muc2.Real-Time Quantitative Reverse Transcription PCR(qRT-PCR)was employed to analyze the gene expression levels of Olfm4,Lysozyme,and Muc2 in the small intestine.Metabolomics was conducted to detect metabolites in serum and intestinal tissues.An in vitro GI-aGVHD organoid model was established by coculturing intestinal organoids with allogeneic T cells,where the number and area of small intestinal organoids were recorded.The GVL effect was assessed using luciferase-transfected ALL cells(ALL/Luc)and bioluminescent imaging.ResultsCompared with the WT group,Stat3 knockout T cells alleviated body weight loss,reduced symptoms—such as hunchback and diarrhea—in mice,improved survival rate(P<0.05),and reduced serum interleukin(IL)-2,IL-6,interferon-γ,tumor necrosis factor-α,IL-17A,and IL-10 levels(all P<0.05),intestinal inflammatory cell infiltration(P<0.05),and intestinal mucosal permeability.Further,Muc2 and Ki67 expression levels in the small intestine of the Stat3 knockout group were markedly increased,and Olfm4,Lysozyme,and Muc2 gene expression levels were significantly increased(all P<0.05).In vitro,the Stat3 knockout group demonstrated better organoid development than the WT group.Metabolomic analyses indicated that Stat3 knockout in T cells may affect the pathways associated with bile acid secretion and unsaturated fatty acids.ALL/Luc cells in the GVL mouse model proliferated rapidly in the TCD-BM group;however,80%of the mice in the Stat3-KO group survived tumor-free for>100 days(P<0.05).ConclusionKnocking out Stat3 in graft T cells reduces T cell damage to intestinal stem cells,thereby ultimately alleviating GI-aGVHD while maintaining a stable GVL effect.
作者
许玉溪
王筱淇
杨世杰
宋清晓
魏锦
张曦
Xu Yuxi;Wang Xiaoqi;Yang Shijie;Song Qingxiao;Wei Jin;Zhang Xi(Department of Hematology,Affiliated Hospital of North Sichuan Medical College,Nanchong 637002,China;Medical Center of Hematology,Xinqiao Hospital of Army Medical University,Blood Ecology and Smart Cell Science Innovation Center,State Key Laboratory of Trauma and Chemical Poisoning,Chongqing Key Laboratory of Hematology and Microenvironment,Chongqing 400037,China;Jinfeng Laboratory,Chongqing 401329,China)
出处
《中华血液学杂志》
北大核心
2025年第4期302-313,共12页
Chinese Journal of Hematology
基金
国家自然科学基金青年项目(82100235、82100226)
国家自然科学基金国际(地区)合作与交流项目(82020108004)
重庆市自然科学基金面上项目(CSTB2024NSCQ-MSX0410、CSTB2022NSCQ-MSX1060)
陆军军医大学第二附属医院学科人才建设专项项目(2024XKRC009、2022XKRC001)。
