摘要
目的观察Smac/DIABLO反义寡脱氧核苷酸(asODN)对H_2O_2引起的大鼠心肌细胞凋亡的影响。方法将原代培养的新生Wistar大鼠心肌细胞随机分为正常对照组(不行转染)、对照组(单纯用脂质体处理)、错义寡脱氧核苷酸(scrODN)组(转染Smac/DIABLO scrODN)、asODN组(转染Smac/DIABLO asODN)、H_2O_2组(仅受H_2O_2刺激)、scrODN+H_2O_2组(同scrODN组处理后受H_2O_2刺激)、asODN+H_2O_2组(同asODN组处理后受H2O_2刺激),Smac/DIABLO scrODN或asODN浓度均为4μmoL/L,H_2O_2为0．5 mmol/L。采用逆转录聚合酶链反应和蛋白质印迹法观察Smac/DIABLO mRNA和蛋白质表达水平的变化；采用hoechst 33258荧光染色,计算凋亡核百分率；抽提DNA行琼脂糖凝胶电泳,观察是否出现DNA“梯状”条带；采用半胱氨酸天冬氨酸蛋白酶(caspases)活性检测试剂盒观察caspase-3、caspase-9的活化情况。结果asODN组细胞Smac/DIABLO mRNA的表达明显受抑制,其蛋白质水平较正常对照组下降约80%；对照组、scrODN组、正常对照组此2项指标相近(P＞0．05)。正常对照组、scrODN组及asODN组凋亡核百分率低,且caspase-3、caspase-9的活性也处于低表达水平。H_2O_2组、scrODN+H_2O_2组凋亡核百分率分别为(52±3)%、(55±5)%,均见典型DNA“梯状”条带,caspase-3、caspase-9活性均明显高于正常对照组(P＜0．01)；asODN+H_2O_2组凋亡核百分率降至(19±5)%,且caspase-3、caspase-9活性明显低于scrODN+H_2O_2组(P＜0．01),DNA“梯状”条带亦明显减少。结论Smac/DIABLO asODN能抑制H_2O_2所致大鼠心肌细胞的凋亡。
Objective To investigate the effects of antisense phosphorothioate oligonucleotides against Smac/DIABLO（asODN） on hydrogen peroxide （H2O2）induced myocardial apoptosis in neonatal rats. Methods Primary myocardial cells from neonatal rats were cultured in vitro, and randomly divided into A （normal control, without transfection） , B （ with treatment of single liposome） , C （ with transfection of scrODN） ,D（with transfection of asODN ）, E（ with H2O2 stimulation）, F（ with H2O2 stimulation after scrODN transfection）, and G（ with H2O2 stimulation after asODN transfection） groups. The expression of asODN mRNA and protein were determined with RT-PCR and Western blotting, respectivly. The changes in cellular nuclear morphology were observed with 33258 fluorescent staining, and the percentage of nuclear apoptosis was calculated. DNA fragmentation was observed by agarose gel electrophoresis. Activation of caspase-3 and caspase-9 were evaluated by caspase colorimetric analysis kit. Results The expression of Smac/DIABLO mRNA and protein was obviously inhibited by asODN, which was about 80% percent lower than the protein level in A,B and C groups, but there was no difference noted among A,B and C groups（ P 〉 0.05）. Not only the nuclear apoptotic percentage, but also the activity of caspase-3 and caspase-9 in A, C and D groups were in very low levels. But these indices in G group 24 hours after H2O2 stimulation were obviously lower than that in E and F groups[ the nuclear apoptotic percentage were （ 19 ±5）% , （52 ±3）% , （55 ± 5 ）% ,respectively, P 〈 0.01 ） ]. The DNA ladders in G group were also decreased markedly. Conclusion Myocardial apoptosis induced by H2O2 can be inhibited by asODN in rat.
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2006年第3期175-179,共5页
Chinese Journal of Burns
