多系分化持续应激细胞保护肠上皮细胞间紧密连接蛋白研究  

Protective effect of tight junction proteins among intestinal epithelial cells by multilineage-differentiating stress enduring cells

作　　者：孙东 杨柳[1] 曹欢 吴龙龙 宋红丽[3] Sun Dong;Yang Liu;Cao Huan;Wu Longlong;Song Hongli(Tianjin First Central Hospital Clinic Institute,Tianjin Medical University,Tianjin 300070,China;School of Medicine,Nankai University,Tianjin 300071,China;Department of Organ Transplantation,Tianjin First Central Hospital,Tianjin Key Laboratory of Organ Transplantation,Key Laboratory of Transplant Medicine,Chinese Academy of Medical Sciences,Tianjin 300192,China)

机构地区：[1]天津医科大学一中心临床学院,天津300192 [2]南开大学医学院,天津300192 [3]天津市第一中心医院器官移植中心,天津市器官移植临床医学研究中心,中国医学科学院移植医学重点实验室,天津300192

出　　处：《中华实验外科杂志》2023年第3期452-455,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(81670574、81270528、81441022)。

摘　　要：目的观察大鼠多系分化持续应激(Muse)细胞对肠上皮细胞间紧密连接结构的保护作用。方法贴壁筛选法自大鼠(6只,购自中国食品药品检定院)股骨内分离大骨髓间充质干细胞(BMMSCs),长时间胰蛋白酶孵育法(LTi)自BMMSCs中分离大鼠Muse细胞,流式细胞术标记如白细胞分化抗原(CD)29、CD34、CD90等及特异阶段胚胎抗原-3(SSEA-3)抗体;免疫组织化学法标记Nanog同框蛋白(NANOG)等抗体,定向诱导分化后标记α-甲胎蛋白(AFP)等鉴定其多能分化能力;重组肿瘤坏死因子-α(TNF-α)建立体外肠上皮细胞损伤损伤模型;与Muse细胞共培养后检测细胞增殖能力及桥接蛋白-1(ZO-1)的表达水平及形态,组间比较采用t检验。结果LTi后,大鼠BMMSCs中(2.57±0.57)%能够形成特征性的Muse细胞簇;流式细胞术结果表明,Muse细胞保留了BMMSCs表面分子特征,SSEA-3的阳性细胞比例[(73.2±1.4)%]显著高于BMMSCs[(2.3±0.3)%];免疫荧光实验表明,培养的Muse细胞阳性表达NANOG等多能分化标记,定向诱导分化后分别能够表达AFP等分化标记;Muse细胞与TNF-α炎症损伤的肠上皮细胞共培养后,其增殖细胞比例[(40.06±1.04)%]高于损伤[(23.10±2.05)%]组的细胞(t=12.79,P<0.05)及ZO-1蛋白的相对表达水平(0.55±0.03、0.27±0.01,t=13.03,P<0.05)。结论大鼠Muse细胞具有更加理想的保护炎症损伤肠上皮细胞紧密连接结构的作用。Objective To investigate the presence of multilineage-differentiating stress enduring(Muse)cells in rat bone marrow mesenchymal stem cells(BMMSCs),and protective effects on inflammatory-damaged intestinal epithelial cells with ideal tight junction structure.Methods The adherent screening method was used to isolate BMMSCs from the femurs of six rats,and long-term trypsin incubation(LTi)was used to isolate rat Muse cells from BMMSCs.Flow cytometry markers were used to sign cells,such as Cluster of differentiation(CD)29,CD34,CD90 and specific stage embryonic antigen-3(SSEA-3),etc.Immunohistochemistry was used to label pluripotent differentiation markers such as Nanog homeobox(NANOG),and alpha-fetoprotein(AFP)after induced-differentiation to identify its pluripotent differentiation ability.Inflammatory-damaged intestinal epithelial cells were established by recombinant tumor necrosis factor-α(TNF-α)in vitro.After co-culture with BMMSCs or Muse cells,the cell proliferation ability and the expression level and morphology of zonula occludens-1(ZO-1)were detected.The t-test analysis was performed on the count data.Results There were(2.57±0.57)%of rat BMMSCs which could form Muse cell clusters after LTi.Flow cytometry showed that Muse cells retained the surface molecular characteristics of BMMSCs,but high expression of SSEA-3[(73.2±1.4)%],significantly higher than BMMSCs[(2.3±0.3)%].The positive expression of pluripotent markers such as NANOG and differentiation markers such as AFP after induced differentiation was detected by immunofluorescence.After co-culture with inflammatory-damaged intestinal epithelial cells,Muse cells showed significantly protective effect on cell proliferation ability[(40.06±1.04)%vs.(23.10±2.05)%,t=12.79,P<0.05]and ZO-1 protein expression level(0.55±0.03 vs.0.27±0.01,t=13.03,P<0.05),which was better than BMMSCs.Conclusion There were a modicum of Muse cells in rat BMMSCs,and it plays a more desirable role in protecting the tight junction structure among intestinal epithelial cells.

关 键 词：骨髓间充质干细胞 多系分化持续应激细胞 肠上皮细胞 紧密连接 闭锁小带蛋白-1 

分 类 号：R574[医药卫生—消化系统]