不同淋巴组织淋巴细胞培养上清诱导Caco2细胞向微皱褶样细胞转分化

Culture supernatants of lymphocytes from different lymphoid tissues induce transdifferentiation of Caco2 cells into M-like cells

目的复制微皱褶(M)样细胞体外诱导模型,从功能和基因水平两方面鉴定M样细胞,同时观察不同淋巴组织活化的淋巴细胞的培养上清对Caco2细胞向M样细胞转化的影响。方法用3μg/m L伴刀豆球蛋白A(Con A)刺激Peyer结(PP)、肠系膜淋巴结(MLN)和脾脏(Sp)的淋巴细胞3 d,收集细胞培养上清后,将其并与Caco2细胞共培养。在TranswellTM上室加入微球,收集下室的培养液,流式细胞术分析荧光微球数量;反转录PCR检测M样细胞相关基因CC趋化因子配体20(CCL20)、密封蛋白基因4(CLDN4)、肿瘤坏死因子受体超家族成员9(TNFRSF9)、Ets家族转录因子Spi-B mRNA的水平,观察不同淋巴组织淋巴细胞培养上清对Caco2细胞向M样细胞转化的诱导率的影响。结果与空白对照组相比,PP、MLN和Sp淋巴细胞培养上清诱导后,诱导后的Caco2细胞转运的微球数量和CCL20、CLDN4、TNFRSF9、Spi-B的mRNA水平均显著增加;与未刺激组相比,Con A刺激后,诱导后的Caco2细胞转运的微球数量和CCL20、CLDN4、TNFRSF9、Spi-B的mRNA水平均显著增加。结论 Con A刺激和未刺激的淋巴细胞培养上清,均可以诱导Caco2细胞向M样细胞转分化。

Objective To establish the microfold （M） -like cell model in vitro and identity M-like cells through detecting the capacity of transporting fluorescent beads and the levels of the associated genes, and to observe the effects of lymphocyte culture supematants stimulated by concanavalin A （ Con A） from different lymphoid tissues on the differentiation of Caco2 cells into M-like cells. Methods The isolated lymphocytes of Peyer＇ s patch （ PP）, mesenteric lymph node （MLN） and spleen （Sp） were incubated with 3 μg/mL Con A for 3 days. The culture supematants were collected and co-cultured with Caco2 cells. The fluorescent bead suspension was added into the upper compartment of the TranswellTM inserts, and then basolateral solutions were then sampled and analyzed. The number of transported fluorescent beads was measured by flow cytometry. The expressions of M-like cells-associated genes, such as chemokine （C-C motif） ligand 20 （CCL20）, claudin4 （CLDN4）, tumor necrosis factor receptor superfamUy member 9 （TNFRSFg）, and Spi-B were detected by reverse transcription PCR. Results Compared with blank control group, the number of fluorescent beads transported by induced Caco2 cells and the levels of CCL20, CLDN4, TNFRSF9 and Spi-B mRNAs significantly increased in induced Caco2 cells treated with the culture supernatants of lymphocytes from PP, MLN and Sp. After Con A stimulation, the number of fluorescent beads transported by induced Caco2 cells and the levels of CC1_20, CLDN4, TNFRSF9 and Spi-B mRNAs were higher than those in the unstimulated group. Conclusion The lymphocyte culture supernatants stimulated or unstimulated by Con A can induce the transdifferentiation of Caco2 cells into M-like cells.