Liang Peng-fei, Huang Xiao-yuan, Long Jian-hong, Xiao Mu-zhang, Yang Xing-hua, Zhang Pi-hong
Departnent of Burns and Plastic Surgery, Xiangya Hospital, Central Southern University, Changsha 410008, PR China.
Zhonghua Shao Shang Za Zhi. 2006 Jun;22(3):175-9.
To investigate the effects of antisense phosphorothioate oligonucleotides against Smac/DIABLO (asODN) on hydrogen peroxide (H2O2) induced myocardial apoptosis in neonatal rats.
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, respectively. 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.
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.
Myocardial apoptosis induced by H2O2 can be inhibited by asODN in rat.
探讨抗Smac/DIABLO硫代磷酸反义寡核苷酸（asODN）对过氧化氢（H2O2）诱导的新生大鼠心肌细胞凋亡的影响。
体外培养新生大鼠原代心肌细胞，随机分为A组（正常对照组，未转染）、B组（单纯脂质体处理组）、C组（scrODN转染组）、D组（asODN转染组）、E组（H2O2刺激组）、F组（scrODN转染后H2O2刺激组）和G组（asODN转染后H2O2刺激组）。分别采用RT-PCR和Western印迹法检测asODN mRNA和蛋白的表达。用33258荧光染色观察细胞核形态变化，并计算核凋亡率。通过琼脂糖凝胶电泳观察DNA片段化情况。采用caspase比色分析试剂盒评估caspase-3和caspase-9的激活情况。
asODN明显抑制Smac/DIABLO mRNA和蛋白的表达，其蛋白水平比A、B、C组降低约80%，但A、B、C组之间无差异（P>0.05）。A、C、D组的核凋亡率以及caspase-3和caspase-9的活性均处于很低水平。但H2O2刺激24小时后，G组的这些指标明显低于E组和F组[核凋亡率分别为（19±5）%、（52±3）%、（55±5）%，P<0.01]。G组的DNA梯形条带也明显减少。
asODN可抑制大鼠H2O2诱导的心肌细胞凋亡。
