Objective:To investigate the influence of edaravone on the expression of growth arrest and DNA damage-inducible protein 34(GADD34).Methods:A total of 108 healthy male Sprague-Dawlcy rats were randomly divided into sha...Objective:To investigate the influence of edaravone on the expression of growth arrest and DNA damage-inducible protein 34(GADD34).Methods:A total of 108 healthy male Sprague-Dawlcy rats were randomly divided into sham operation group,model group and edaravone.group(36 cases for each group).Transient focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 h followed by reperfusion in Sprague-Dawlev rats.Then.GAOD34 expression was measured with immunohistochemistry at different time-points after reperfusion in the peri-infarct regions of all rats.Results:The GADD34 expression was detected in the peri-infaret regions of rats 1 h after reperfusion,which reached its peak 24 h after reperfusion.And edaravone could significantly down-regulate the GAOD34 expression.Conclusions:Edaravon could down-regulate GADD34 expression,which suggests that edaravone may exert an important function in inhibiting endoplasmic reticulum stress reaction by scavenging free radicals in the upper stream.展开更多
目的:探讨下调生长阻滞和DNA损伤诱导蛋白45β(growth arrest and DNA damage inducible protein 45β,GADD45β)表达对PC9肺腺癌细胞及吉非替尼敏感性的影响。方法:设计并合成GADD45β基因小干扰RNA(GADD45β-small interfering RNA,GA...目的:探讨下调生长阻滞和DNA损伤诱导蛋白45β(growth arrest and DNA damage inducible protein 45β,GADD45β)表达对PC9肺腺癌细胞及吉非替尼敏感性的影响。方法:设计并合成GADD45β基因小干扰RNA(GADD45β-small interfering RNA,GADD45β-siRNA)序列,通过慢病毒介导将GADD45β-siRNA转入PC9肺腺癌细胞中,通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Western印迹检测转染前后PC9肺腺癌细胞GADD45β的mRNA及蛋白水平,采用膜联蛋白V(annexin V)-别藻蓝蛋白(allophycocyanin,APC)双染流式细胞法检测转染后细胞凋亡水平;通过流式细胞术检测转染后细胞内DNA含量,计算转染后细胞各周期时相百分率,分析转染对细胞生长周期的影响;通过计数克隆形成数检测RNA干扰对细胞成瘤能力的影响;采用MTT法检测PC9肺腺癌细胞的吉非替尼半数抑制浓度IC50。结果:筛选出5'-AAATCCACTTCACGCTCAT-3'为GADD45β基因RNA干扰的有效序列。转染GADD45β-siRNA 48 h后,qRT-PCR和Western印迹结果显示PC9肺腺癌细胞GADD45β的mRNA和蛋白表达水平明显下调(均P<0.05),细胞凋亡率明显增加(P<0.05),且成瘤克隆数明显减少(P<0.05);PC9肺腺癌细胞位于S期及G2/M期细胞增多(P<0.05),吉非替尼的IC50明显下降(P<0.05)。结论:PC9肺腺癌细胞转染GADD45β-siRNA后,能成功下调GADD45β基因的mRNA和蛋白表达;下调GADD45β表达可降低PC9肺腺癌细胞的克隆形成能力,促进细胞凋亡;下调GADD45β表达可明显提高PC9肺腺癌细胞对吉非替尼的敏感性。展开更多
观察生长抑制和DNA损伤基因45(Growth arrest and DNA damage gene 45,GADD45)X射线照射后表达的改变,并研究其与照射剂量之间的关系。外周血给予0-5 GyX线照射后分离出单个核细胞并培养,在不同时间点上用反转录聚合酶链反应(Reverse tr...观察生长抑制和DNA损伤基因45(Growth arrest and DNA damage gene 45,GADD45)X射线照射后表达的改变,并研究其与照射剂量之间的关系。外周血给予0-5 GyX线照射后分离出单个核细胞并培养,在不同时间点上用反转录聚合酶链反应(Reverse transcriptase-Polymerase clain reaction,RT-PCR)的方法测定 GADD45基因的相对表达量,分析该基因的剂量、效应关系。照射后GADD45基因在转录水平表达呈剂量依赖性增加,照射后4h达峰值,以后开始下降,但在24h仍未恢复到初始水平。展开更多
目的:研究人生长阻滞和DNA损伤45α(growth arrest and DNA damage 45 alpha,Gadd45α)基因对滋养细胞HTR8/SVneo增殖、凋亡、迁移和侵袭等生物学功能的影响,探讨其在子痫前期(preeclampsia,PE)发生发展中的可能作用。方法:构建Gadd45...目的:研究人生长阻滞和DNA损伤45α(growth arrest and DNA damage 45 alpha,Gadd45α)基因对滋养细胞HTR8/SVneo增殖、凋亡、迁移和侵袭等生物学功能的影响,探讨其在子痫前期(preeclampsia,PE)发生发展中的可能作用。方法:构建Gadd45α短发夹干扰RNA,以阴性对照组作为参照,转染人滋养细胞HTR8/SVneo,即分为实验组(si-Gadd45α)和阴性对照组(si-NC)进行实验;应用流式细胞仪检测转染效率,并应用q RT-PCR和Western blot检测转染后各组细胞中Gadd45αm RNA和蛋白表达水平;采用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)显色法检测转染后各组细胞增殖能力;应用流式细胞仪对转染后各组细胞进行细胞凋亡分析;采用Transwell法检测转染后各组细胞迁移和侵袭能力;采用早孕绒毛外植体培养模型观察敲除Gadd45α基因后对绒毛外滋养细胞外生性迁移能力的影响;收集各组细胞培养上清液,明胶酶谱法检测基质金属蛋白酶(matrix metalloproteinases,MMPs)的表达,蛋白免疫印迹检测基质金属蛋白酶组织抑制因子(tissue inhibitors of MMPs,TIMPs)的表达。结果:流式细胞仪检测转染效率约为90%;转染后实验组较对照组Gadd45αm RNA的表达量约降低80%,蛋白表达水平约下降70%,差异均有统计学意义(P<0.01),证明干扰Gadd45α表达成功。MTT法和流式细胞仪检测细胞增殖和凋亡,结果显示实验组和对照组细胞增殖和凋亡均无统计学差异(P>0.05)。Transwell实验表明干扰Gadd45α后HTR8/SVneo侵袭能力明显增加,约为对照组的1.65倍;迁移能力也明显增加,约为对照组的2倍,均有统计学差异(P<0.01)。早孕绒毛外植体培养结果显示:与阴性对照组的绒毛相比,Gadd45α干扰片段处理的绒毛外滋养细胞外生性迁移的距离明显增加,差异有统计学意义(P=0.005)。明胶酶谱实验结果显示干扰Gadd45α后基质金属蛋白酶-2(matrix metalloproteinases,MMP-2)和MMP-9的活性增强,而Western blot检测发现其抑制因子TIMP-1和TIMP-2相应地下降(P<0.01)。结论:推测Gadd45α可能是通过调控蛋白酶的活性来抑制滋养细胞的迁移和侵袭,从而参与PE的发生发展。展开更多
基金Supported by Clinical Special Funds of Chinese University Medical Journals.China(Grant No:11321937)
文摘Objective:To investigate the influence of edaravone on the expression of growth arrest and DNA damage-inducible protein 34(GADD34).Methods:A total of 108 healthy male Sprague-Dawlcy rats were randomly divided into sham operation group,model group and edaravone.group(36 cases for each group).Transient focal cerebral ischemia was induced by middle cerebral artery occlusion for 2 h followed by reperfusion in Sprague-Dawlev rats.Then.GAOD34 expression was measured with immunohistochemistry at different time-points after reperfusion in the peri-infarct regions of all rats.Results:The GADD34 expression was detected in the peri-infaret regions of rats 1 h after reperfusion,which reached its peak 24 h after reperfusion.And edaravone could significantly down-regulate the GAOD34 expression.Conclusions:Edaravon could down-regulate GADD34 expression,which suggests that edaravone may exert an important function in inhibiting endoplasmic reticulum stress reaction by scavenging free radicals in the upper stream.
文摘目的:探讨下调生长阻滞和DNA损伤诱导蛋白45β(growth arrest and DNA damage inducible protein 45β,GADD45β)表达对PC9肺腺癌细胞及吉非替尼敏感性的影响。方法:设计并合成GADD45β基因小干扰RNA(GADD45β-small interfering RNA,GADD45β-siRNA)序列,通过慢病毒介导将GADD45β-siRNA转入PC9肺腺癌细胞中,通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Western印迹检测转染前后PC9肺腺癌细胞GADD45β的mRNA及蛋白水平,采用膜联蛋白V(annexin V)-别藻蓝蛋白(allophycocyanin,APC)双染流式细胞法检测转染后细胞凋亡水平;通过流式细胞术检测转染后细胞内DNA含量,计算转染后细胞各周期时相百分率,分析转染对细胞生长周期的影响;通过计数克隆形成数检测RNA干扰对细胞成瘤能力的影响;采用MTT法检测PC9肺腺癌细胞的吉非替尼半数抑制浓度IC50。结果:筛选出5'-AAATCCACTTCACGCTCAT-3'为GADD45β基因RNA干扰的有效序列。转染GADD45β-siRNA 48 h后,qRT-PCR和Western印迹结果显示PC9肺腺癌细胞GADD45β的mRNA和蛋白表达水平明显下调(均P<0.05),细胞凋亡率明显增加(P<0.05),且成瘤克隆数明显减少(P<0.05);PC9肺腺癌细胞位于S期及G2/M期细胞增多(P<0.05),吉非替尼的IC50明显下降(P<0.05)。结论:PC9肺腺癌细胞转染GADD45β-siRNA后,能成功下调GADD45β基因的mRNA和蛋白表达;下调GADD45β表达可降低PC9肺腺癌细胞的克隆形成能力,促进细胞凋亡;下调GADD45β表达可明显提高PC9肺腺癌细胞对吉非替尼的敏感性。
