细胞固定技术在两种细胞共同培养研究中的应用

目的探讨细胞固定对鉴别支气管上皮细胞和嗜酸粒细胞两种细胞共同培养过程中细胞代谢产物来源的作用及其机制。方法用1％多聚甲醛固定嗜酸粒细胞和支气管上皮细胞系BEAS-2B细胞；通过锥虫蓝、伊红细胞染色，观察多聚甲醛固定对细胞形态、结构、活力和与正常细胞接触培养过程中与正常细胞黏附能力的影响；用酶联免疫吸附测定（ELISA）方法定量分析固定细胞与正常细胞共同培养过程中对白细胞介素（IL）-6释放的影响。结果多聚甲醛固定后支气管上皮细胞和嗜酸粒细胞均变为死亡细胞，但其形态、结构及嗜酸粒细胞中颗粒着色无明显变化；多聚甲醛固定的BEAS-2B细胞与正常及固定的嗜酸粒细胞接触培养仅有极少量细胞发生黏附，而多聚甲醛固定的嗜酸粒细胞与正常BEAS-2B细胞接触培养则有较大数量的细胞发生黏附，但黏附细胞数量少于两种正常细胞的共同培养；固定后的嗜酸粒细胞与正常BEAS-2B细胞共同培养过程中可诱导BEAS-2B细胞释放IL-6。但与嗜酸粒细胞单独培养比较，固定后BEAS-2B细胞与正常嗜酸粒细胞共同培养对细胞培养上清液中IL-6浓度无明显影响。结论经多聚甲醛固定后，细胞死亡并丧失其代谢功能，但其表面仍保留可诱导其他细胞活化的结构成分，此技术可用于两种或多种细胞共同培养过程中代谢产物来源的鉴别。

Application of cell fixation in co-culture of two different types of cells

Objective To investigate the interaction of eosinophils and epithelial cells (BEAS-2B cells) fixed with paraformaldehyde on their mortality, cellular structure,IL-6 release and elucidate a method to identify the source of releasing products in co-culture of two types of cells.Methods Eosinophils and BEAS-2B cells were fixed with 1 % paraformaldehyde for 1h separately. The cells were stained with trypan blue and eosin to assess the mortality and structure of cells. IL-6 concentrations in cell culture supernatant were analyzed by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results BEAS-2B cells and eosinophils fixed with paraformaldehyde showed no significant change in structure. Only a few fixed or normal eosinophils adhered to fixed BEAS-2B cell, but a lot of fixed or normal eosinophils could adhere to normal BEAS-2B cells in co-culture. Fixed eosinophils could significantly induce BEAS-2B cells to release IL-6, but fixed BEAS-2B cells did not show any significant effect on eosinophils during co-culture.ConclusionsAlthough fixed cells with paraformaldehyde could induce cell death and the lose of metabolic functions, they could still remain the cell surface structure to interact and activate other type of cells. The above results demonstrated a useful method that can be applied to investigate the interaction between different cells in co-culture.