【作者】 张玲玲；

【导师】 范晓云；

【作者基本信息】 安徽医科大学 ， 老年医学呼吸内科专业， 2017， 硕士

【摘要】 目的:研究多聚左旋精氨酸(Poly-L-arginine,PLA)诱导气道上皮细胞NCI-H292细胞凋亡的通路机制以及对细胞内Bcl-2/Bax、Caspase-3表达的影响,从而揭示PLA参与哮喘气道上皮损伤的部分机制。方法:1、细胞培养常规培养基(RPMI1640+10%胎牛血清)培养NCI-H292细胞,于37℃、5%二氧化碳环境的培养箱内孵育。每2~3天换液一次,细胞分裂铺展至培养瓶70%~80%时用0.25%胰酶消化传代,用于后续实验。2、透射电镜制片观察NCI-H292细胞凋亡NCI-H292细胞分为对照组、PLA(60 mg/L)组两组,PLA作用24 h后,收集细胞,固定后脱水,包埋干燥,切片浸染后用透射电镜摄片观察两组细胞的超微结构。3、Annexin V-FITC/PI双染色法检测细胞凋亡率按加入PLA浓度不同将细胞分为空白对照组、10 mg/L PLA组、20 mg/L组PLA、40 mg/L PLA组、60 mg/L PLA组,按是否加入ERK1/2信号通路抑制剂PD98059分为空白对照组、PD组、PLA组、PLA+PD组。收集各组细胞后,运用流式细胞仪检测凋亡率,flowjo软件分析凋亡结果。4、Western Blot法检测凋亡相关蛋白Bax、Bcl-2、Casepase-3及信号通路ERK1/2的表达采用Western Blot法分析在不同浓度PLA(0、10、20、40、60 mg/L)刺激下Bax、Bcl-2、Casepase-3和ERK1/2的蛋白含量,并比较ERK信号通路抑制剂PD98059(20μmol/L)作用前后对Bcl-2/Bax、Casepase-3和ERK1/2磷酸化的影响。5、统计学处理采用统计学软件SPSS16.0进行处理分析,数据以(?)x±s表示,数据均进行正态性检验,多组间比较采用单因素方差分析(One-Way ANOVA);两两间比较采用LSD-t检验。所有实验均重复不少于3次。P<0.05为差异有统计学意义。结果:1、电镜下可见PLA刺激后的NCI-H292细胞核膜皱缩外突,染色质在核膜下浓聚;线粒体肿胀变圆、呈空泡状,线粒体嵴排列杂乱多不规则,数量明显减少,甚至部分消失,呈明显凋亡改变。2、Annexin V-FITC/PI双染法凋亡检测显示,对照组、10 mg/L PLA组、20 mg/L PLA组、40 mg/L PLA组、60 mg/L PLA组NCI-H292细胞凋亡率分别为(4.97±0.17)%,(7.82±0.21)%,(11.99±0.21)%,(29.52±0.55)%,(55.23±0.67)%,与对照组相比,差异有统计学意义(P<0.001),并呈浓度依赖性。3、Western Blot检测10 mg/L PLA组、20 mg/L PLA组、40 mg/L PLA组、60 mg/L PLA组的Bcl-2/Bax比值相对于空白对照组皆显示降低趋势,差异都有统计学意义(P均<0.01);在活性Caspase-3蛋白表达水平的检测中,10 mg/L PLA组、20 mg/L PLA组、40 mg/L PLA组、60 mg/L PLA组的活性Caspase-3蛋白表达量比之于对照组都有所增加,有统计学差异(P均<0.001)。4、与空白对照组相比较,10 mg/L PLA组、20 mg/L PLA组、40 mg/L PLA组、60mg/L PLA组ERK磷酸化水平均增加(P均<0.01),且在PLA终浓度为20mg/L时,P-ERK/ERK的表达量达到高峰(P<0.01)。5、空白对照组、PD组、PLA组、PLA+PD组NCI-H292细胞凋亡率分别为(5.04±0.26)%、(4.99±0.19)%(P=0.801)、(20.72±1.37)%(P<0.001)、(8.67±0.18)%(P<0.001),且PLA+PD组凋亡率明显低于PLA组,其差异统计学意义(P<0.001)。6、PLA+PD组相比PLA组Bcl-2/Bax明显升高,活性Caspase-3明显降低,差异均有统计学意义(P<0.001)。与空白对照组相较,PLA组活性P-ERK/ERK表达水平明显升高(P<0.001);PLA+PD组相比PLA组P-ERK/ERK明显降低(P<0.001)。结论:1、PLA可诱导气道上皮细胞凋亡;2、PLA可能通过激活Bax、Bcl-2、Caspase-3等凋亡相关蛋白诱导气道上皮细胞凋亡;3、PLA可能通过调控ERK信号通路激活Bax、Bcl-2、Caspase-3等凋亡相关蛋白的表达诱导气道上皮细胞凋亡。更多还原

【Abstract】 ObjectiveHerein,we aimed to study the signaling mechanism whereby Poly-L-arginine(PLA)induces the apoptosis of airway epithelial cell and the expression on Bcl-2/Bax and Caspase-3 in NCI-H292 cells to reveal the mechanism that PLA plays an important role in airway injury.Methods 1.Cell cultureNCI-H292 cells were cultured and propagated in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2/95% air at 37°C.The cells were subcultured every 3 to 4 days according to their growth status.Optimal growth state of the cells were selected and seeded into 6-well culture plates for subsequent experiments.2.The apoptosis of NCI-H292 cells was observed by transmission electron microscopy NCI-H292 cells were grouped into control group and PLA(60 mg/L)group.The cellswere digested by trypsin and collected.After gradient dewatering,the embedding tissuewas sliced to thin sections.After dipped in the citrate for 15 min,thin sections wereobservated by ultrastructural electron microscopy.3.The apoptosis rate of NCI-H292 cells was detected by Annexin V-FITC/PI The cells were grouped into control,10 mg/L,20 mg/L,40 mg/L and 60 mg/L group.The others were grouped into control,PD,PLA and PLA+PD group.The apoptotic rate was measured by flow cytometry.Flowering software was used to analyze the apoptotic results.4.The expression of the Bax,Bcl-2,Casepase-3 and signal pathway ERK1/2 was observed by Western BlotNCI-H292 cells were grouped into control,10 mg/L,20 mg/L,40 mg/L and 60 mg/L group.The others were grouped into control,PD,PLA and PLA + PD group.The levels of Bax,Bcl-2,Casepase-3 and ERK1/2 in different concentrations of PLA and the effects of ERK signal pathway inhibitor PD98059(20 μmol/L)on Bcl-2/Bax,Casepase-3 and ERK1/2 phosphorylation were detected by chemiluminescence substrate of Western Blot.5.Statistical processingAll the experiments were repeated for three times.Statistical analyses were perfored using SPSS version 16.0.All values were displayed as means ± standard error of the mean.One-way Analysis of Variance(ANOVA)was applied for comparisons of more than two groups,and LSD was used when equal variance was assumed between groups.P-values less than 0.05 were considered to denote statistically significant differences.Result1.The NCI-H292 cells in PLA group changed in apoptosis significantly.The nuclear membrane shrinked and the chromatin on the nuclear membrane concentrated.The arrangement of the cristae of mitochondria was irregular,and the number was reduced significantly,even disappeared in some cases.2.Annexin V-FITC/PI double staining for apoptosis detection showed that in the control group,10 mg/L group,20 mg/L group,40 mg/L group,60 mg/L group,the apoptosis rate of NCI-H292 cells were(4.97±0.17)%,(7.82±0.21)%,(11.99± 0.21)%,(29.52±0.55)% and(55.23±0.67)%.Compared with the control group,the difference was statistically significant(P<0.001).3.The expression level of Bcl-2/Bax in the 10 mg/L group,20 mg/L group,40 mg/L group and 60 mg/L group showed decreased compared with the control group(P<0.01).The activity of protein expression of actived Caspase-3 in the 10 mg/L group,20 mg/L group,40 mg/L group and 60 mg/L group were increased compared with the control group(P<0.001).4.Compared with the control group,the phosphorylation of ERK increased statistically in the 10 mg/L group,20 mg/L group,40 mg/L group and 60 mg/L group.And the peak of P-ERK/ERK was advanced in the 20 mg/L group(P<0.01).5.The apoptosis rate of NCI-H292 cells in the control group,PD group,PLA group,PLA+PD group were(5.04±0.26)%,(4.99±0.19)%(P=0.801),(20.72±1.37)%(P<0.001),(8.67±0.18)%(P<0.001).The apoptosis rate of PLA+PD group was significantly lower than that in the PLA group(P<0.001).6.Compared with group PLA,the activity of Bcl-2/Bax increased significantly in the PLA+PD group and the activity of Caspase-3 was lower significantly(P<0.001).Compared with the control group,the expression of P-ERK/ERK in the PLA group increased significantly(P<0.001).Compared with PLA group,the activity of P-ERK/ERK decreased significantly in PLA+PD group(P<0.001).Conclusion 1.PLA can induce apoptosis of airway epithelial cells in asthma.2.PLA induced apoptosis of airway epithelial cells by activating Bax,Bcl-2 and Caspase-3.3.PLA can regulate the expression of Bax,Bcl-2 and Caspase-3 to induce apoptosis of airway epithelial cells by ERK signaling pathway.更多还原