| BACKGROUND: Serving a critical function as the defensive front line between organism and outside antigens, intestinal epithelial cells (IECs) are continually exposed to Gram-negative bacteria and lipopolysaccharide (LPS), but could not be activated. So there should be relevant mechanism by which IECs tolerate lipopolysaccharide. Furthermore, findings of recent researches suggested that intestinal mucosal barrier function's decreasing and intestine-derived bacteria and lipopolysaccharide's translocation were correlated with severe surgical patients' complications such as sepsis and multiple organ dysfunction syndromes (MODS). So it may be helpful for endotoxemia's preventing and treating to understand the function of intestinal epithelium barrier completely and to clarify the molecular mechanism by which IECs tolerate lipopolysaccharide. At present, no research about it is reported in our country, and because there is a lack of normal huamn IEC line, foreign correlative researches came from cultured animal IECs and human cancer cell lines of colon, so their results may be diferent from those of normal human. The present study is to isolate noraml human IEC model, to observe the responsiveness of human IECs to LPS stimulation and to explore the molecular mechanism by which IECs tolerate lipopolysaccharide.OBJECTIVE: ①To establish the methods of isolation, culture and identification of human intestinal epithelial cells (IECs) . ②To observe the responsiveness of human IECs to LPS stimulation, and to explore the molecular mechanism by which IECs tolerate lipopolysaccharide.METHODS: ①Primary culture of normal human IECs: Specimens of small intestines of fetuses from 4 to 6 months of gestation were obtained after legal abortion. After incubating at 37℃ for 2h in the presence of thermolysin, IECs were incubated in DMEM which contained several nutrient components (such as human epidermal growth factor and heparin) that facilitated proliferation and growth of IECs. IECs, which were purified by the different anchoring time between IECs and fibroblasts, were identified by theirmorphologic character, ultrastructure and epithelial membrane antigens. ②Observation of the responsiveness of human IECs to LPS stimulation: IL-8 in supernatants was measured by ELISA at 18h following LPS stimulation. The activation of NF-κB in IECs was detected by EMSA at 1h. ③Detection of mRNA expressions of LPS receptors on IECs: Total RNA was extracted from human normal IECs with and without LPS stimulation, human intestinal cancer epithelial cells (HIC), and THP1 cells, respectively. TLR4, TLR2, CD14 and MD2 mRNA were detected by RT-PCR and RPA. ④NF-κB and IL-8 in HICs transfected with TLR4, CD14 and MD2 plasmids were analysed.RESULTS: ①Primary culture of normal human IECs was successful with positive staining with the epithelial membrane antigens. ②NF-κB and IL-8 was not detectable after IECs were treated with LPS, suggesting no responsiveness of IECs to LPS stimulation. ③ The expressions of TLR4, TLR2, CD14 and MD2 mRNA of human normal IECs were low both before and after LPS stimulation. HICs also did not express TLR4, CD14 and MD2 mRNA.④The activation of NF-κB and the secretion of IL-8 were detectable in HICs when simultaneously transfected with TLR4, CD14 and MD2 plasmids in response to LPS, while the activation of NF-κB was weaker and IL-8 was not detectable in HICs when cotransfected with TLR4 and CD14 plasmids. The activation of NF-κB and the secretion of IL-8 were not detectable in HICs which were not transfected any plasmids. CONCLUSIONS: ①It is the best way using thermolysin to isolate IECs. ②By detecting NF-κB and IL-8, the present study, for the first time, examined the responsiveness for human small IECs to LPS stimulation. It was found that there was no response of human IECs to LPS stimulation, which was different from that of monocyte/macrophages. ③The expressions of TLR4, TLR2, CD14 and MD2 mRNA were shown to be low, and even after LPS stimulation. ④The low or no expressions of TLR4, CD14 a...
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