Preliminary Assessment Of Innate Immune Function Of Mouse Mammary Epithelial Cells Cultured In Vitro

In order to confirm whether TLR2or TLR4contribute to the innate immuneresponse of mouse mammary epithelial cells(MECs). Improved collagenase/trypsindigestion and Two-step enzyme digestion methods were used to culture mouse MECs,and the cell growth was evidenced by revealing the morphological characteristicsunder inverted microscope in this experiment and detection of Keratin18, a specificmarker for epithelial cell, by Confocal Lasers Scanning Miccruscope. cultured mouseMECs were treated with10μg/mL LPS to verify whether the cytokines can begenerated, then detected the content of TNF-α and IL-8of supernatant by ELISA.Meanwhile, Detection of TLR2,4protein in mouse MECs by indirectImmunocytochemistry. Finally, preincubated with TLR4and TLR2antagonists toinhibit receptors activity, so as to verify whether the cytokines can be generated.The results showed that plenty of pure MECs can be harvested by usingcollagenase/trypsin digestion method and continuously cultured for4generations. Acertain amount of cells can be harvested with Two-step enzyme digestion method, butcells were subcultured difficultly, and hard to purify. The cultured cell can be used forthe next experiments with improved collagenase/trypsin digestion. Meanwhile, mouseMECs can response to the presence of LPS and significantly upregulate secretion ofIL-8and TNF-a protein(P＞0.05), and there are TLR2and TLR4protein expressionin the cytomembrance, Moreover, interaction of LPS and TLR4leads to theupregulation of IL-8and TNF-a in cultured mouse MECs(P＜0.01), however, TLR2poorly response to LPS stimulation(P＞0.05).The above results suggest that improved collagenase/trypsin digestion can bepreferably used to culture mouse MECs. and LPS-challenged mouse MECs play apivotal role in innate immunity by secreting IL-8and TNF-a. Furthermore, there areTLR2and TLR4protein expression in the cytomembrance. Finally, mouse MECs cansecrete IL-8and TNF-a, mainly via interaction of LPS and TLR4, which contributes to the innate immune response of mouse MECs. These data lay a solid foundation forfurther study of cell function and the impact of traditional Chinese medicine on themouse MECs.