简介:目的研究重组腺病毒Ad—Ku70shRNA对C6胶质瘤细胞系Ku70蛋白表达的影响,探讨放射治疗过程中基因增敏的新途径。方法以穿梭质粒为基础,构建重组腺病毒Ad—Ku70shRNA载体,扩增并鉴定其病毒滴度。然后转染至C6鼠脑胶质瘤细胞中,应用WestemBlot及免疫荧光观察其Ku70蛋白的表达情况,研究重组腺病毒Ad—Ku70shRNA对C6细胞Ku70表达的抑制效果。结果成功构建并扩增Ad—Ku70shRNA重组腺病毒,感染C6胶质瘤细胞后Ku70的表达明显降低。结论Ad—Ku70shRNA重组腺病毒载体可以明显降低C6胶质瘤细胞Ku70的表达。该结果为放射治疗基因增敏研究提供了有利的工具。
简介:目的探讨热休克蛋白(HSP70)在人胶质瘤细胞BT-325p38MAPK信号通路中的作用.方法用脂质体介导法将hsp70基因导入人胶质瘤细胞BT-325中,倒置显微镜观察转染细胞的形态学及粘附性变化,紫外线照射30min后,采用免疫组化和Western-blot方法测定转染前后HSP70的表达水平及照射前后p38MAPK表达情况.结果免疫组化和Western-blot证实hsp70基因成功转染入BT-325中,转染细胞受到紫外线照射后p38MAPK表达减弱.结论体外转染hsp70基因可抑制紫外线照射后BT-325细胞p38MAPK的表达.
简介:Heatshockprotein70(HSP70)maintainsCa~(2+)homeostasisinPC12cells,whichmayprotectagainstapoptosis;however,themechanismsofneuroprotectionareunclear.Therefore,inthisstudy,weexaminedCa~(2+)levelsinPC12cellstransfectedwithanexogenouslentiviralHSP70geneexpressionconstruct,andwesubsequentlysubjectedthecellstoischemia-hypoxia/reoxygenationinjury.HSP70overexpressionincreasedneuronalviabilityandATPaseactivity,anditdecreasedcellularreactiveoxygenspecieslevelsandintracellularCa~(2+)concentrationafterhypoxia/reoxygenation.HSP70overexpressionenhancedtheproteinandmRNAexpressionlevelsofsarcoplasmic/endoplasmicreticulumCa~(2+)-ATPase(SERCA),butitdecreasedtheproteinandmRNAlevelsofinositol1,4,5-trisphosphatereceptor(IP3R),therebyleadingtodecreasedintracellularCa~(2+)concentrationafterischemia-hypoxia/reoxygenation.TheseresultssuggestthatexogenousHSP70protectsagainstischemia-hypoxia/reoxygenationinjury,atleastinpart,bymaintainingcellularCa~(2+)homeostasis,byupregulatingSERCAexpressionandbydownregulatingIP_3Rexpression.