【作者】 蔡添才；

【导师】 张健鹏； 樊毫军；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 硕士

【摘要】 目的:探讨全氟化碳原液(PFC)和新型全氟化碳乳剂(NPE)对A549细胞生长的影响;观察PFC和NPE对TNF-α攻击的A549细胞钠离子通道(ENaC)表达的影响。方法:1.PFC对A549细胞生长影响及对TNF-α攻击的A549细胞ENaC表达影响的研究。分为对照组、TNF-α组、PFC组和治疗组共四组。TNF-α以100ng/ml的浓度攻击细胞,PFC以1/10细胞培养液体积的比例与细胞共同培养。正常组细胞不做任何处理,实验组细胞分别用不同因素处理2、4、6h,用MTT法测各组细胞活力变化;提取细胞RNA和蛋白,分别用real-time PCR法检测细胞ENaCα、β、γ亚单位表达水平变化;ENaC-α蛋白表达水平采用Western-blot法检测。2. NPE对A549细胞生长影响及对TNF-α攻击的A549细胞ENaC表达影响的研究。分为对照组、TNF-α组、NPE组和治疗组共四组。TNF-α以100ng/ml的浓度攻击细胞,NPE以1/10细胞培养液体积的比例与细胞共同培养。正常组细胞不做任何处理,实验组细胞分别用不同因素处理2、4、6h,用MTT法测各组细胞活力变化;提取细胞RNA和蛋白,分别用real-time PCR法检测ENaCα、β、γ亚单位表达水平变化;ENaC-α蛋白表达水平采用Western-blot法检测。结果:1. PFC对A549细胞生长影响:2h组:与对照组细胞相比较:TNF-α组、PFC组和治疗组细胞基本形态无明显变化。4h组:TNF-α组细胞细胞生长状态不良,死亡细胞较多。PFC组和治疗组细胞一般情况较TNF-α组好,死亡细胞数较少。6h各组细胞一般情况与4h组无明显差别。2. PFC对TNF-α攻击的A549细胞活力的影响:2h组:TNF-α组、PFC组、治疗组细胞吸光度值与对照组比较,差异均无统计学意义(P﹥0.05)。4h组:PFC组细胞吸光度值为与对照组比较,差异无统计学意义(P﹥0.05);TNF-α组、治疗组细胞吸光度值较对照组降低,治疗组吸光度值较TNF-α组增高,差异具有统计学意义(P﹤0.05)。6h各组吸光度值比较结果与4h组相似。3. real-time PCR结果显示:2h组:与对照组相比,各组ENaC-α、β、γ亚单位mRNA水平变化不明显;处理4h后,与对照组相比,TNF-α组细胞ENaC各亚单位mRNA水平均有不同程度的降低,PFC组细胞则与对照组无明显差异,治疗组细胞ENaC各亚单位mRNA水平也有不同程度降低,与TNF-α组同时相点相比,差异无统计学意义。6h组情况与4h组相同。4. Western-blot结果显示:2h组:与对照组相比,各组ENaC-α蛋白表达变化不明显;4h组和6h组中,有TNF-α组细胞ENaC-α蛋白表达明显下调,其他各组细胞ENaC-α蛋白表达与对照组并无差别。5.NPE对A549细胞生长影响:2h组:与对照组细胞相比较:TNF-α组、NPE组、治疗组细胞基本形态无明显变化。4h组:TNF-α致伤组细胞细胞生长状态不良,死亡细胞较多。NPE组和治疗组细胞一般情况较TNF-α组好,细胞形态变化不明显,死亡细胞数较少。6h组各组细胞一般情况与4h组无明显区别。6. NPE对TNF-α攻击的A549细胞活力的影响: 2h组:NPE组、TNF-α组、治疗组细胞吸光度值与对照组比较,无明显差异(P﹥0.05)。4h组:NPE组细胞吸光度值与对照组比较,无明显差异(P﹥0.05);TNF-α组、治疗组细胞吸光度值较对照组明显降低,治疗组吸光度值较T组增高,差异具有统计学意义(P﹤0.05)。6h各组吸光度值比较结果与4h组相似。7. real-time PCR结果显示:2h组:与对照组相比,各组ENaC-α、β、γ亚单位mRNA水平变化不明显;处理4h后,与对照组相比,TNF-α组细胞ENaC各亚单位mRNA水平均有不同程度的降低,NPE组细胞则与对照组无明显差别,治疗组细胞ENaC各亚单位mRNA水平也有不同程度降低,但较TNF-α组同时相点相比有明显升高,差异具有统计学意义。6h组情况与4h组相同。8. Western-blot结果显示:2h组:与对照组相比,各组ENaC-α蛋白表达变化不明显;4h组和6h组中, TNF-α组细胞ENaC-α蛋白表达明显下调,治疗组细胞ENaC-α蛋白表达与TNF-α组相比有明显增高,差异具有统计学意义(P﹤0.05),NPE组细胞ENaC-α蛋白表达与对照组无明显差异。结论:1. TNF-α可对A549细胞生长及活力产生抑制作用,且可以下调细胞ENaC-α、β、γ亚单位及ENaC-α蛋白表达水平。2.PFC可能通过物理屏障作用对TNF-α攻击的A549细胞产生一定的保护作用,对细胞ENaC-α、β、γ亚单位及ENaC-α蛋白表达的下调无改善作用。3.NPE可改善TNF-α对A549细胞生长的抑制作用,同时可以改善TNF-α对细胞ENaC各基因及ENaC-α蛋白表达的下调。更多还原

【Abstract】 Objective:To explore the impaction of perfluorocarbon and new perfluorocarbon emulsion on A549 cells; To investigate the protection and mechanism of PFC and NPE on TNF-αattacked A549 cells.Methods:1. The exploration of PFC: A549 cells were divided into four groups: Control group untreated as blank control; TNF-αgroup was incubated with 100ng/ml TNF-αand PFC group with PFC at a 1/10 volume; we used the same dose of TNF-αand PFC in treat group. After treatment for 2、4、6 hours,the activity of cells were determined by MTT assay. The mRNA levels of ENaC-α、β、γsubunits and protein levels of ENaC-αwere detected by real-time PCR and western-blot, respectively.2. The exploration of NPE: A549 cells were divided into four groups: Control group untreated as blank control; TNF-αgroup was incubated with 100ng/ml TNF-αand NPE group with NPE at a 1/10 volume; we used the same dose of TNF-αand NPE in treat group. After treatment for 2、4、6 hours,the activity of cells were determined by MTT assay. The mRNA levels of ENaC-α、β、γsubunits and protein levels of ENaC-αwere detected by real-time PCR and western-blot, respectively. Results:1. Compared with control group, the cell morphology of other groups had no significant change after treated for 2 hours; however, when it came to 4 and 6 hours, the cells in TNF-αgroup grew alas, and many cells died and float. Meanwhile, the cells in PFC group and treat group had no difference with control group (P﹥0.05).2. The impaction of cell activity: after treatment for 2 hours, the optical densities of cells had no significant change among each group, compared with control group (P﹥0.05). As treated for 4 and 6 hours, the optical densities of cells in TNF-αgroup and treat group decreased, however, treat group has higher densities than TNF-αgroup.(P﹤0.05).3.The mRNA levels of ENaC-α、β、γsubunits: after treated for 2 hours,the mRNA levels of any group had no difference(P﹥0.05). Afeter treated for 4 and 6 hours,the mRNA levels of TNF-αgroup and treat group significant decreased compared with control group (P﹤0.05), and there was no difference between TNF-αgroup and treat group(P﹥0.05). There was no significant change in PFC group compared with control group after treated for 4 and 6 hours( P﹥0.05).4. The protein levels of ENaC-α: the protein levels of ENaC-αhad no significant change among each groups after treated for 2 hours. When treated for 4 and 6 hours, the protein levels of ENaC-αin TNF-αgroup and treat group decreased compared with control group, there was no difference between treat group and TNF-αgroup(P﹥0.05). The mRNA levels of PFC group had no difference with group C(P﹥0.05).5. The cell morphology of each group had no significant difference after treated for 2 hours; however, when incubated for 4 and 6 hours, the cells in TNF-αgroup grew bad, and many cells died and float. Meanwhile, the cells in NPE group and treat group had no difference with control group (P﹥0.05).6. The impaction of cell activity: after treatment for 2 hours, the optical densities of cells had no significant change among each group, compared with control group (P﹥0.05). As treated for 4 and 6 hours, the optical densities of cells in TNF-αgroup and treat group decreased, however, treat group has higher densities than TNF-αgroup.(P﹤0.05).7. The mRNA levels of ENaC-α、β、γsubunits: after treated for 2 hours,the mRNA levels of any group had no difference(P﹥0.05). Afeter treated for 4 and 6 hours,the mRNA levels of TNF-αgroup and treat group significant decreased compared with control group (P﹤0.05), and the treat group had a higer level than TNF-αgroup (P﹤0.05). There was no significant change in NPE group compared with control group after treated for 4 and 6 hours( P﹥0.05).8. The protein levels of ENaC-α: the protein levels of ENaC-αhad no significant change among each groups after treated for 2 hours. When treated for 4 and 6 hours, the protein levels of ENaC-αin TNF-αgroup and treat group decreased compared with control group, and the level of treat group was higer than TNF-αgroup (P﹤0.05). But the NPE group has the same level as control group (P﹥0.05).Conclusion:1. Incubated with TNF-αimpacted the morphology and cell activity of A549 cell.When the incubation last to 4 and 6 hours, the mRNA expression of ENaC-α、β、γsubunits and protein level of ENaC-αwere decreased.2. PFC protected A549 cells attacted with TNF-αby physical barriers, but has no ameliorations in the mRNA and protein expression.3. NPE released the depression of TNF-α, and aliveiated the downregulation of TNF-αto the mRNA and protein expression of ENaC in A549 cells.更多还原