Lewis肺腺癌对小鼠肠道干细胞及其微环境的影响

Eftect of Lewis lung cancer on mouse intestinal stem cells and their microenvironment

目的探讨Lewis肺腺癌(Lewis lung cancer,LLC)对小鼠肠道干细胞及其微环境的影响。方法比较不同接种方式(皮下接种和静脉移植)在C57BL/6J小鼠肺部形成肺腺癌的能力差异。通过HE、BrdU免疫染色和qPCR检测LLC对肠道形态、增殖动力学、肠道干细胞及其微环境相关基因、肠上皮细胞紧密连接及炎症因子表达的影响。使用EdU染色评价LLC条件培养基(LLC-CM)对IEC-6细胞增殖的影响。使用LLC细胞和LLC-CM观察肺腺癌对肠类器官生长的影响。结果静脉移植LLC细胞较皮下接种荷瘤更易在小鼠肺部形成肺腺癌。肺腺癌导致绒毛和隐窝长度较对照组显著增加(P<0.01),BrdU阳性细胞数量和Ki67基因mRNA水平显著高于对照组(P<0.05)。LLC-CM可增加EdU阳性IEC-6细胞数量(P<0.05)。肺腺癌小鼠肠组织中肠道干细胞标志物基因(Lgr5、Olfm4、Axin2)表达水平显著下降,微环境相关基因(Wnt3)表达水平降低(P<0.05)。肺腺癌引起肠上皮ZO-1免疫染色以及ZO-1和OCLN mRNA表达显著降低,IL-1β和TNF-α的mRNA含量显著增加(P<0.05)。LLC细胞和LLC-CM均抑制肠类器官生长。结论Lewis肺腺癌能够破坏肠道干细胞及其微环境,为肺癌与肠道交互作用提供了新的实验依据。

Objective To investigate the changes in intestinal stem cells(ISCs)and their microenvironment in a mouse model bearing Lewis lung cancer(LLC).Methods We compared the tumorigenicity of LLC cells in mice following subcutaneous transplantation and tail vein injection of the cells.HE staining,immunostaining and qPCR were used to analyze how LLC affected the intestinal morphology,cell proliferation dynamics,ISCs and their niche-related genes,intestinal epithelial cell tight junctions,and the expression of inflammatory cytokines.We also evaluated the effect of LLC conditioned medium(LLC-CM)on the proliferation of cultured IEC-6 cells using EdU staining,and observed the growth of intestinal enteroids co-cultured with LLC cells or in the presence of LLC-CM.Results Tail vein injection more efficiently induced the formation of LLC in mice than subcutaneous transplantation of LLC cells.The mice bearing LLC exhibited significantly increased length of the mucosal villi and depth of the crypts(P<0.01),and had more BrdU-positive cells and a higher level of Ki67 mRNA expression in the intestines as compared with the control mice(P<0.05).LLC-CM obviously promoted the incorporation of EdU in IEC-6 cells.The tumor-bearing mice showed significantly decreased expression of ISC markers and microenvironment-related genes(including Lgr5,Olfm4,Axin2,and Wnt3)in the intestines(P<0.05),where the staining signals of ZO-1 and the mRNA expression of TJP1 and OCLN was significantly decreased and the mRNA levels of IL-1βand TNF-αwere markedly increased(P<0.05).Both LLC and LLC-CM significantly inhibited the growth of cultured mouse intestinal organoids Conclusions LLC disrupts the ISCs and their niche in mice,which provides new evidence of the interaction between lung cancer and the gastrointestinal tract.