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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 replicate M-like cells in vitro and identify M-like cells in terms of function and gene level, and to observe the effects of culture supernatants of different lymphoid activated lymphocytes on the transformation of Caco2 cells to M-like cells. Methods The lymphocytes of Peyer’s knot (PP), mesenteric lymph node (MLN) and spleen (Sp) were stimulated with 3μg / ml concanavalin A (Con A) for 3 days. The supernatant of the cell culture was collected, Caco2 cells were co-cultured. The microspheres were collected in the upper chamber of TranswellTM and the culture medium in the lower chamber was collected. The number of fluorescent microspheres was analyzed by flow cytometry. The expression of CCL20, CCL20, (CLDN4), tumor necrosis factor receptor superfamily member 9 (TNFRSF9) and Ets family transcription factor Spi-B mRNA, and to observe the effects of different lymphoid tissue culture supernatants on the induction rate of Caco2 cells to M-like cells . Results Compared with the blank control group, the number of microspheres and the mRNA levels of CCL20, CLDN4, TNFRSF9 and Spi-B in Caco2 cells induced by PP, MLN and Sp lymphocyte culture supernatants were significantly increased compared with the control group; Compared with the stimulated group, the number of microspheres and the mRNA levels of CCL20, CLDN4, TNFRSF9 and Spi-B in Caco2 cells induced by Con A were significantly increased. Conclusion Both Con A-stimulated and unstimulated lymphocyte culture supernatants can induce Caco2 cells to transdifferentiate into M-like cells.