摘要
目的研究Aβ-结合性酒精脱氢酶蛋白（ABAD）在缺氧性细胞中的表达及其对细胞缺氧性损害的保护作用，以探讨急性缺氧性疾病的治疗新方法。方法①观测缺氧状态下细胞Aβ-结合性酒精脱氢酶蛋白表达水平的变化：体外培养的COS-1细胞置于缺氧箱诱导缺氧1～16h（氧浓度控制1％～2％），收集细胞，裂解，离心得到蛋白提取液，同时用TRlzol方法提取总RNA；Northern blot法检测正常和缺氧状态下细胞ABADmRNA的表达水平；15μg总RNA于10％甲醛变性琼脂糖凝胶电泳分离并转移至尼龙膜，采用QuikHyb快速杂交系统与α-^32P标记的全长ABAD cDNA探针杂交，杂交膜于-80℃放射自显影18h；Western blot检测正常和缺氧状态下细胞ABAD蛋白的表达水平：20μg蛋白样品于SDS-NuPAGE胶电泳分离，电转移使样品印迹至尼龙膜上，后者依次与鼠抗人ABAD单克隆抗体（一抗）和兔抗鼠抗体（二抗）孵育，用化学发光法显示印迹条带；②观测上调ABAD表达对细胞缺氧性损害的影响：采用逆转录PCR（RT—PCR）方法，扩增ABAD全长编码序列，PCR产物直接克隆至pcDNA3表达质粒中，构建ABAD-pcDNA3表达质粒；将1μgpcDNA3-ABAD表达质粒DNA用Lipofectmine2000转染剂转染至COS-1细胞株，培养36h。正常对照组则以1μg pcDNA3空载体转染；转染后细胞按上述方法进行缺氧处理，收集细胞，裂解，采用DNA片段化检测试剂盒定量检测ABAD正常表达和高表达条件下缺氧诱导的细胞死亡事件。结果与正常表达组相比，ABADmRNA及蛋白水平均于缺氧后1h开始下降，4～8h后显著降低，至16h表达几乎完全消失；提高ABAD的表达可明显降低缺氧所致的DNA片段化水平（P〈0．05）。结论ABAD是缺氧高度敏感性蛋白，同时又是一个对缺氧性损害有明显保护作用的蛋白。因此，它可望成为急性缺血缺氧性疾病临床监测的一个新指标，并具有潜在治疗应
Objective To explore the alteration of the ABAD expression in response to hypoxia and the role of ABAD in the protection against hypoxic cellular damage, and, on this basis, to seek novel therapeutic targets for acute hypoxic disorders. Methods （1） Observing the expression of ABAD in response to hypoxic induction： cultured COS-1 cells were exposed to hypoxic conditions （PO2 maintained at 1%-2%） in an environmental chamber for 1 to 16 hours. After the treatment, the cells were harvested, lysed and spinned to obtained the protein extract. Meanwhile, total RNA was extracted utilizing TRIzol reagent. To examine the ABAD mRNA level, 15 μg of total RNA was loaded and separated on a 10% formalin denatured gel and subsequently transferred to a Nilon membrane. The α-^32P-labeled full-length ABAD cDNA probe was used for hybridization with the QuikHyb System. The film yeas subjected to autoradiography at --80℃ for 18 hours. Western blot was performed to measure the protein level. 20μg of protein was loaded and run in a 12% Bis-Tris SDS-NuPAGE gel and transferred to a Nilon membrane, which was then incubated sequentially with monoclonal anti-human ABAD antibody （primary Ab） and rabbit anti-mouse IgG （secondary Ab）. Banding of ABAD protein was obtained with chemoluminescence. （2）Examining the effect of over-expression of ABAD on the cellular hypoxic damage： RT- PCR was performed to amplify the full-length ABAD coding sequence, followed by direct cloning into pcDNA3 expression vector to generate the pcDNA3-ABAD expression construct. To over-express ABAD,1μg of pcDNA3-ABAD construct or pcDNA3 control vector was transfected into COS-1 cells with Lipofectamine 2000 and cultured for 36 hours. The transfected cells were then subjected to hypoxic exposure for 1-8 h. Finally, the cells were harvested and lysed and DNA fragmentation assay was performed to compare cell death level between the ABAD-overexpressed and ABAD-normal cells. Result Both the mRNA and protein levels of ABAD started to diminish after
出处
《江西医学院学报》
CAS
2006年第6期5-8,共4页
Acta Academiae Medicinae Jiangxi
