本文關鍵詞： 肺泡II型上皮細胞 神經(jīng)酰胺 緊密連接 凋亡　出處：《安徽醫(yī)科大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的 探討外源性C8-神經(jīng)酰胺對小鼠肺泡上皮屏障功能的影響,包括肺泡II型上皮細胞(AEC II)單層通透性和緊密連接蛋白的改變,以及C8-神經(jīng)酰胺誘導AEC II凋亡的作用。方法 將孕18天的ICR小鼠剖殺,取出胎鼠肺組織,經(jīng)胰蛋白酶消化法分離出AECII,并運用差速貼壁法進行純化;應用堿性磷酸酶染色法和電鏡對得到的AECII進行鑒定;于Transwell構建單層AECII,待細胞融合后給予不同濃度的C8-神經(jīng)酰胺(0、25、50、100μmol/L)繼續(xù)培養(yǎng)24 h,應用伏歐計測定跨上皮電阻值(TEER)并用辣根過氧化物酶(HRP)標記法檢測吸光度值(OD470);分別提取AECII的細胞總蛋白,采用Western blot法檢測不同濃度的C8-神經(jīng)酰胺對AECII緊密連接蛋白(ZO-1,Occludin,Claudin-4)蛋白水平的影響;提取AECII的細胞總RNA,采用實時定量聚合酶鏈式反應方法檢測不同濃度的C8-神經(jīng)酰胺對AECII緊密連接蛋白(ZO-1,Occludin,Claudin-4)m RNA水平的影響;采用Annexin V-FITC/PI雙染法檢測各組AECII的凋亡率。結果 細胞鑒定結果證明,本次實驗提取的細胞為胎鼠肺泡II型上皮細胞,具有肺泡II型上皮細胞的特征性結構。隨著C8-神經(jīng)酰胺濃度的增加,TER值逐漸下降,各組間TER值差異有統(tǒng)計學意義(F=38.780,P0.001)。不同濃度組與對照組相比,差異均具有統(tǒng)計學意義(P0.05)。HRP測定結果顯示,隨著濃度的增加,OD470呈上升趨勢,各組間OD470差異有統(tǒng)計學意義(F=19.780,P0.001),神經(jīng)酰胺50μmol/L組和100μmol/L組與對照組相比,差異均有統(tǒng)計學意義(P0.05)。C8-神經(jīng)酰胺100μmol/L劑量組與對照組相比,ZO-1的表達顯著下調(P0.05),與25μmol/L劑量組相比,ZO-1的表達亦顯著下調(P0.05);各劑量組Occludin的表達差異有統(tǒng)計學意義(F=7.703,P=0.002);神經(jīng)酰胺100μmol/L組與對照組和25μmol/L劑量組相比,Occludin的表達均下調;各劑量組Claudin-4的蛋白表達水平差異無統(tǒng)計學意義(P0.05)。C8-神經(jīng)酰胺各劑量組Claudin-4 m RNA水平差異無統(tǒng)計學意義(P0.05);各組間ZO-1及Occludin m RNA表達水平的差異均有統(tǒng)計學意義(ZO-1:F=4.887,P=0.014;Occludin:F=3.637,P=0.048);C8-神經(jīng)酰胺100μmol/L組與對照組相比,ZO-1及Occludin m RNA表達水平的差異有統(tǒng)計學意義(P0.05)。同時神經(jīng)酰胺各劑量組與對照組相比,細胞凋亡率的差異均有統(tǒng)計學意義(均P0.01);不同劑量組間細胞凋亡率的差異仍有統(tǒng)計學意義(均P0.05)結論 C8-神經(jīng)酰胺可能通過下調緊密連接蛋白(ZO-1和Occludin)的表達增加AEC II單層細胞的通透性,同時誘導AEC II細胞凋亡進一步破壞肺泡上皮屏障,為進一步研究神經(jīng)酰胺引起肺上皮屏障功能受損的分子機制提供了基礎,同時為ALI提供新的診斷思路及治療靶點。
[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.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R563.8

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