Caspase-3反义寡核苷酸抗凋亡作用的研究

Study on the in the anti-apoptosis effect of caspase-3 antisense oligonucleotide

目的研究caspase-3反义寡核苷酸对6-OHDA诱导大鼠中脑多巴胺神经元凋亡的保护作用。方法向四组SD大鼠的中脑黑质部位注入反义、错义、正义寡核苷酸及NS,然后再注入6-OHDA,取中脑做连续切片,原位杂交及免疫组化检测黑质caspase-3的表达及TH的表达,原位末端标记法(Tunel)检测黑质细胞的凋亡。结果反义组Tunel阳性细胞数为82±8.6,方差分析显示反义组阳性细胞数显著性低于其他各组(p<0.05);反义组手术侧TH阳性细胞数为168.6±11.4,与对侧阳性细胞比值为63%±11.3%,显著性高于其余各组(P<0.05)。结论有效阻断caspase-3的表达可减轻6-OHDA诱导的多巴胺神经元凋亡,caspase-3可作为保护性治疗帕金森病的靶点。

Objective To investigate the inhibition of 6-OHDA-induced apoptosis of dopaminergic neurons by caspase-3 antisense oligonucleotides （ODN）. Methods Male Sprague-Dawley （SD） rats were randomly distributed into 4 groups （antisense ODN, missense ODN, sense ODN and NS）, n= 10 for each group. Rats were placed in a Kopf stereotaxic apparatus and given an infusion of 3 IxL ODN or 3ul NS in the mesencephalic substantia nigra for 3 d, and on the 4a day, 18 μg/3 μL 6-OHDA solution was injected in the same region. Then all the rats were sacrificed and their midbrains were taken out. The removed brain tissues were then fixed, dehydrated and sliced into serial sections. In situ hybridization （ISH） and immunohistochemical staining were performed to detect the expression of caspase-3 in the substantia nigra and TdT-mediated dUTP-biotin nick end labeling （TUNEL） was undertaken to detect nigral neuron apoptosis. Data were expressed as Mean±SD. Statistical analysis was performed using one-way analysis of variance （ANOVA）. Results The number of TUNEL positive cells in the antisense group is 82±8.6 ,which was remarkably lower than those in other groups （P〈0.05）; the number of TH positive cells in antisense group was 168.6± 11.4, and its ratio with that of contralateral positive cells was 63%±11.3%, which was significantly higher than those of other groups （P〈0.05）. Conclusion Apoptosis of dopaminergic neurons induced by 6-OHDA can be inhibited by effectively blocking the expression of caspase-3, which may be regarded as a target for protective treatment of Parkinson＇s disease.