fundamental research of the cause of inflammatory bowel disease on Caco-2 cells mucosal model

在威尔斯孔上形成Caco-2细胞（人结肠癌细胞）的粘液瘤模型。制备4个组并孵育48小时和72小时。一种是不加细胞因子的黏膜模型，另一种是加IL-1、IL-6、TNF-α的黏膜模型。提取各组大鼠胃粘膜RNA含量。用北方印迹法检测各组细胞内磷脂酶A2 mRNA的表达，并与β-actin的相对表达量进行比较。48 h时IL-6组细胞内磷脂酶A2 mRNA表达较其他各组明显增加。72 h时TNF-α组细胞内磷脂酶A2 mRNA表达明显高于其他各组。在transwell上建立有/无细胞因子的粘膜上皮细胞模型，测定细胞因子作用后6、12、24、72 h的跨上皮电阻（TEER），IL-6组TEER显著高于对照组（P < 0. 05），IL-6组TEER显著高于对照组（P < 0. 05），IL-6组TEER显著高于对照组（P < 0. 05）。 关于我们 OL组分别于6、12、24 h处死。72 h时IL-1和IL-6组的TEER较对照组显著降低。促炎细胞因子IL-6可增加细胞内和培养液中sPLA 2（磷脂酰胆碱水解物）的表达。使用薄层色谱法分析培养基和细胞裂解物的PL组成。两组间培养基中的PL组成没有显示出任何差异。细胞内磷脂总含量也无变化，但IL-6可导致磷脂组成的显著变化，包括磷脂酰乙醇胺（PE）和鞘磷脂（SM）的增加以及磷脂酰胆碱（PC）和溶血磷脂酰胆碱（LPC）的减少（p<0.05）。PE和SM均为炎症信号因子，与IBD的发病密切相关，本研究提示细胞因子可改变炎症介质的黏膜通透性，但这种改变可能不是磷脂酶A2分解磷脂形成溶血磷脂的作用。少

Mucosal model of Caco-2 cells, human colon cancer cells, was formed on wells. The 4 groups were made and incubated for 48 hours and 72 hours. One is that mucosal model was incubated without cytokine, another is that mucosal model was incubated with IL-1, the other with IL-6, the forth with TNF-α. The content of RNA was extracted from mucosa of each group. The expression of intracellular phospholipase A2 mRNA of each group was measured with Northern Blot, compared to the relative expression of β-actin. The expression of intracellular phospholipase A2 mRNA of IL-6 group was significantly increased than the others at 48 hours. At 72 hours, the expression of intracellular phospholipase A2 mRNA of TNF-α group was significantly increased than the others. Mucosal models were made on transwells with/without cytokine, to measure the transepithelial electrical resistance (TEER) at 6, 12, 24, 72 hours after the addition of cytokine.The TEER of IL-6 group was significantly higher compared to contr … More ol group at 6,12,24 hours. At 72 hours, the TEER of IL-1 and IL-6 group was significantly decreased than the control group. The proinflammatory cytokine IL-6 increased the expression of sPLA2 (a hydrolyzer of phosphatidylcholine) of both intracellular and media.Mucosal models were incubated for 72 h with IL-6 or media alone (control). Both media and cell lysate were analyzed for PL composition using thin-layer chromatography. The PL composition in the media did not show any differences between the two groups. Total intracellular PL contents were also unchanged ; however, IL-6 led to significant changes in PL composition including an increase in phosphatidylethanolamine(PE) and sphingomyelin(SM) and a decrease in phosphatidylcholine(PC) and lysophosphatidylcho-line(LPC) (p<0.05). Both PE and SM are known as inflammatory signaling factors involved in human IBD.Our study suggests that cytokine may change the mucosal permeability, however this change may not be the effect of lysophospholipids made from phospholipids by phospholipase A2. Less