中文摘要：

目的利用乏氧与辐射联合诱导Smac过表达，探讨其对A549细胞凋亡的作用及相关分子机制。方法氯化钴（CoCl2）乏氧，细胞经x射线照射后分别利用膜联蛋白V（AnnexinV）-异硫氰酸荧光素（FITC）试剂盒检测凋亡率，采用实时定量聚合酶链反应（Real—timePCR）检测细胞色素C（Cytc）和半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3mRNA和Westernblot检测相应蛋白的表达。实验分为正常对照组（Contr01）、pcDNA3．1-Egr-1-Smac（pE—Smae）组、peDNA3．1-HRE／Egr-1-Smac（pH／E—Smac）组、2Gy、pE-Smac＋2Gy和pH／E-Smac＋2Gy组，按照常氧与乏氧处理。结果与Control组比较，常氧处理后，2Gy、pE．Smac＋2Gy和pH／E．Smac＋2Gy组细胞凋亡率、CytC和Caspase-3mRNA表达显著增加（P〈0．05），凋亡率增加到4．72、7．33和7．67倍，CytC和Caspase-3mRNA表达分别增加到1．69、7．21、7．39倍和5．63、32．03、31．08倍；乏氧处理后，2Gy、pE-Smac＋2Gy和pI-I／E—Smae＋2Gy组细胞凋亡率、CytC和Caspase．3mRNA表达以及pH／E—Smac组凋亡率均显著增加（P〈0．05），凋亡率增加到1．76、2．54、2．89和5．83倍，CytC和Caspase-3nLRNA表达增加到2．35、6．20、13．69倍和3．93、13．67、19．08倍。与常氧处理比较，乏氧各组（Control和pE-Smac组CytCmRNA表达除外）凋亡率、CytC和Caspase-3mRNA均显著增加（P〈0．05）。常氧与乏氧处理时，各组Caspase．3前体无明显变化，而2Gy、pE．Smac＋2Gy和pH／E—Smac＋2Gy组CytC和断裂Caspase-3表达增加，且乏氧较常氧处理的CytC和断裂的Caspase-3表达增加。结论乏氧和辐射联合诱导的Smac过表达能诱导A549细胞凋亡率增加，其调控可能涉及到CytC—Caspase-3的分子机制。

英文摘要：

Objective To study the effects of~Smac ovcrexpression induced by hypoxia and radia- tion on apoptosis of A549 cells and the molecular mechanism. Methods CoC12 was used to mimic hypoxi- a. After cells were irradiated by X -rays, cell apoptosis was measured with Annexin V -fluoresceine iso- thiocyanate （FITC） kits. The expression of cytochrome C （ Cyt C） and cysteinyl aspartate - specific protc- ase （Caspase） -3 mRNA and protein was detected by real -time quantitative polymerase chain reaction （Real -time PCR） and Western blotting, respectively. Folowing groups were set up： control, pcDNA3.1 - Egr - 1 - Smae （ pE -Smac）, pcDNA3. 1 - HRE/Egr - 1 - Smac （ pH/E - Smac ）, 2 Gy, pE -Smac ＋ 2 Gy, and pH/E - Smac ＋ 2 Gy. Cells were performed under normoxic or hypoxic conditions. Results As compared with control group, apoptosis rate, Cyt C and Caspase - 3 mRNA in 2 Gy, pE - Smac ＋ 2 Gy and pH/E - Smac ＋ 2 Gy groups under normoxia were significantly increased （ P 〈 0. 05 ）. Apoptosis rate reached 4. 72, 7.33 and 7.67 folds, and Cyt C and Caspase -3 mRNA expression reached 1.69, 7.21, 7. 39 folds and 5.63, 32.03, 31.08 folds, respectively. Under hypoxia, apoptosis rate, Cyt C and Caspase - 3 mRNA in 2 Gy, pE - Smac ＋ 2 Gy and pH/E - Smac ＋ 2 Gy groups, and apoptosis rate in pH/E group were all significantly increased （ P 〈 0.05 ）. The apoptosis rate reached 1.76, 2. 54, 2. 89 and 5.83 folds, and Cyt C and Caspase - 3 mRNA expression reached 2. 35,6. 20, 13.69 folds and 3.93, 13.67, 19.08 folds, respectively. As compared with normoxic treatment, apoptosis rate, Cyt C and Caspase - 3 mRNA expression （ except Cyt C mRNA in control group and pE - Smac group） in the rest groups under hypxia were all significantly increased （ P 〈 O. 05 ）. With normoxic and hypoxic treatment, Caspase -3 precursor had no obvious changes in each group, but Cyt C and Caspase -3 cleavage in 2 Gy, pE -Smac ＋2 Gy and pH/E -Smac ＋2 Gy groups were increased, more significantly in pH/E -Smac ?