C8-神经酰胺对小鼠肺泡上皮屏障功能的影响

发布时间：2018-02-17 02:24

本文关键词： 肺泡II型上皮细胞神经酰胺紧密连接凋亡　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的探讨外源性C8-神经酰胺对小鼠肺泡上皮屏障功能的影响,包括肺泡II型上皮细胞(AEC II)单层通透性和紧密连接蛋白的改变,以及C8-神经酰胺诱导AEC II凋亡的作用。方法将孕18天的ICR小鼠剖杀,取出胎鼠肺组织,经胰蛋白酶消化法分离出AECII,并运用差速贴壁法进行纯化;应用碱性磷酸酶染色法和电镜对得到的AECII进行鉴定;于Transwell构建单层AECII,待细胞融合后给予不同浓度的C8-神经酰胺(0、25、50、100μmol/L)继续培养24 h,应用伏欧计测定跨上皮电阻值(TEER)并用辣根过氧化物酶(HRP)标记法检测吸光度值(OD470);分别提取AECII的细胞总蛋白,采用Western blot法检测不同浓度的C8-神经酰胺对AECII紧密连接蛋白(ZO-1,Occludin,Claudin-4)蛋白水平的影响;提取AECII的细胞总RNA,采用实时定量聚合酶链式反应方法检测不同浓度的C8-神经酰胺对AECII紧密连接蛋白(ZO-1,Occludin,Claudin-4)m RNA水平的影响;采用Annexin V-FITC/PI双染法检测各组AECII的凋亡率。结果细胞鉴定结果证明,本次实验提取的细胞为胎鼠肺泡II型上皮细胞,具有肺泡II型上皮细胞的特征性结构。随着C8-神经酰胺浓度的增加,TER值逐渐下降,各组间TER值差异有统计学意义(F=38.780,P0.001)。不同浓度组与对照组相比,差异均具有统计学意义(P0.05)。HRP测定结果显示,随着浓度的增加,OD470呈上升趋势,各组间OD470差异有统计学意义(F=19.780,P0.001),神经酰胺50μmol/L组和100μmol/L组与对照组相比,差异均有统计学意义(P0.05)。C8-神经酰胺100μmol/L剂量组与对照组相比,ZO-1的表达显著下调(P0.05),与25μmol/L剂量组相比,ZO-1的表达亦显著下调(P0.05);各剂量组Occludin的表达差异有统计学意义(F=7.703,P=0.002);神经酰胺100μmol/L组与对照组和25μmol/L剂量组相比,Occludin的表达均下调;各剂量组Claudin-4的蛋白表达水平差异无统计学意义(P0.05)。C8-神经酰胺各剂量组Claudin-4 m RNA水平差异无统计学意义(P0.05);各组间ZO-1及Occludin m RNA表达水平的差异均有统计学意义(ZO-1:F=4.887,P=0.014;Occludin:F=3.637,P=0.048);C8-神经酰胺100μmol/L组与对照组相比,ZO-1及Occludin m RNA表达水平的差异有统计学意义(P0.05)。同时神经酰胺各剂量组与对照组相比,细胞凋亡率的差异均有统计学意义(均P0.01);不同剂量组间细胞凋亡率的差异仍有统计学意义(均P0.05)结论 C8-神经酰胺可能通过下调紧密连接蛋白(ZO-1和Occludin)的表达增加AEC II单层细胞的通透性,同时诱导AEC II细胞凋亡进一步破坏肺泡上皮屏障,为进一步研究神经酰胺引起肺上皮屏障功能受损的分子机制提供了基础,同时为ALI提供新的诊断思路及治疗靶点。
[Abstract]:Objective to investigate the effects of exogenous C8-ceramide on alveolar epithelial barrier function in mice, including the changes of monolayer permeability and tight junction protein in alveolar type II epithelial cells. Methods 18 days pregnant ICR mice were dissected, the fetal lung tissues were isolated by trypsin digestion, and purified by differential adherent method. The AECII was identified by alkaline phosphatase staining and electron microscope. The monolayer AECII was constructed from Transwell and cultured for 24 hours with different concentrations of C8-ceramide 250 渭 mol / L after cell fusion. The transdermal resistance value was measured by voltammeter and the absorbance value was detected by horseradish peroxidase (HRP) labeling method, and the absorbance value of OD470 was detected by horseradish peroxidase (HRP) labeling method, and the absorbance value was determined by the method of horseradish peroxidase (horseradish peroxidase). The total cellular protein of AECII, The effect of different concentrations of C8-ceramide on the protein level of AECII tight junction protein ZO-1 Occludinia Claudin-4 was detected by Western blot assay. The effects of different concentrations of C8-ceramide on the level of Claudin-4m RNA of AECII tight junction protein ZO-1 Occludinia were detected by real-time quantitative polymerase chain reaction (RQPCR). The apoptosis rate of AECII in each group was detected by Annexin V-FITC / Pi double staining. Results the results of cell identification showed that the cells extracted in this experiment were fetal alveolar type II epithelial cells. With the increase of C8-ceramide concentration, the value of ter decreased, and the difference of TER value among the three groups was statistically significant. Compared with the control group, the concentration of C8-ceramide in the different concentration groups was higher than that in the control group. The difference was statistically significant (P 0.05). The results of HRP showed that with the increase of the concentration of OD470, the difference of OD470 was statistically significant (P 0.001). Compared with the control group, the ceramide 50 渭 mol/L group and the 100 渭 mol/L group were different from the control group. There were significant differences in the expression of P0.05N. C8-ceramide 100 渭 mol/L compared with the control group. Compared with the control group, the expression of ZO-1 was significantly down-regulated, and the expression of P0.05-1 was also significantly down-regulated compared with the dose of 25 渭 mol/L. The expression of Occludin in each dose group was significantly lower than that in the control group (F7.703P0.002), and the expression of Occludin in each dose group was significantly lower than that in the control group. Compared with the control group and the 25 渭 mol/L group, the expression of Occludin was down-regulated in the amine-100 渭 mol/L group. There was no significant difference in the protein expression of Claudin-4 in different dose groups. There was no significant difference in the level of Claudin-4 m RNA in each dose of ceramide. There was no significant difference in the expression of ZO-1 and Occludin m RNA among the groups. There were significant differences in the expression of ZO-1 and Occludin m RNA among the three groups, and there were significant differences in the expression of ZO-1 and Occludin m RNA among the groups. There was significant difference in the expression of ZO-1 and Occludin m RNA between the 100 渭 mol/L group and the control group (P 0.05). There were significant differences in apoptosis rate between different dose groups (all P 0.01). Conclusion C8-ceramide may increase the expression of ZO-1 and Occludinin by down-regulating the expression of tight junction protein ZO-1 and Occludinin. Permeability of AEC II monolayer cells, At the same time, inducing apoptosis of AEC II cells further destroyed the alveolar epithelial barrier, which provided the basis for further study of the molecular mechanism of the damage of pulmonary epithelial barrier function induced by ceramide, and provided a new diagnostic and therapeutic target for ALI.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R563.8

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