摘要
目的探讨脂肪酸合成酶(Fatty acid synthase, Fas)基因沉默对帕金森病大鼠脑纹状体多巴胺能神经元凋亡的影响及机制。方法取40只大鼠,30只大鼠脑纹状体注射4μL 6-羟基多巴胺(6-Hydroxydopamine Hydrobromide, 6-OHDA)建立帕金森病大鼠模型,剩余10只为假手术组,脑纹状体注射等量2 g/L抗坏血酸的生理盐水;取建模成功的27只大鼠并随机分为模型组、阴性对照组和沉默组,每组各9只;阴性对照组和沉默组分别向大鼠纹状体内注射3μL含无义序列的短片断干扰RNA(Small interfer RNA,siRNA)和Fas siRNA,模型组和假手术组分别向大鼠纹状体内注射等量生理盐水;干预1周后实时荧光定量反转录-聚合酶链反应(Real-time quantitative polymerase chain reaction, RT-qPCR)和Western blot检测大鼠纹状体多巴胺能神经元Fas mRNA和蛋白相对表达水平,苏木精-伊红(hematoxylin eosin, HE)染色观察大鼠纹状体多巴胺能神经元损伤情况,原位末端标记法(TdT-mediated dUTP nick and labeling, TUNEL)染色检测大鼠纹状体多巴胺能神经元凋亡,RT-qPCR,Western blot检测纹状体多巴胺能神经元Fas配体(Fas Ligand, Fasl)、Fas相关死亡结构域蛋白(Fas-associateddeathdomain, FADD)、半胱氨酸天冬氨酸蛋白酶-8(Cysteine containing aspartate-specific proteases-8,Caspase-8)mRNA、蛋白相对表达水平。结果与假手术组比较,模型组、阴性对照组和沉默组Fas mRNA和蛋白相对表达水平升高(P<0.05);与模型组和阴性对照组比较,沉默组Fas mRNA和蛋白相对表达水平降低(P<0.05);HE染色显示,Fas基因沉默后神经元排列紊乱程度及胞质肿胀程度减轻,间质较为清晰,空泡皱缩坏死减少;与假手术组比较,模型组、阴性对照组和沉默组神经元凋亡率升高(P<0.05);与模型组和阴性对照组比较,沉默组神经元凋亡率降低(P<0.05);与假手术组比较,模型组、阴性对照组和沉默组Fasl, FADD,Caspase-8 mRNA及蛋白相对表达水平升高(P<0.05);与模型组比较,沉默组Fasl, FADD,Caspase-8 mRNA及蛋白相对表达水平降低(P<0.05)。结论 Fas基因沉默能抑制帕金森病大鼠脑纹状体多巴胺能神经元凋亡,且可能通过下调Fas, Fasl, FADD,Caspase-8 mRNA及蛋白表达水平来发挥调控作用。
Objective To investigate the effect and mechanism of fatty acid synthase(Fas) gene silencing on apoptosis of dopaminergic neurons in striatum of Parkinson’s disease rat model.Methods Forty rats were selected, thirty of which were injected with 4 μL 6-hydroxydopamine(6-OHDA) into the striatum to establish Parkinson’s disease model, while the remained ten rats were recognized as sham-operated control group and were injected with the same amount of 2 g/L ascorbic acid saline into the striatum. Twenty-seven successful models were randomly divided into model group, negative control group and silence group, with 9 rats in each group. The negative control group and silence group were injected with 3 μL small interference RNA(siRNA) containing nonsense sequence and 3 μL Fas siRNA into the striatum, respectively. The model group and sham-operated control group were injected with the same amount of normal saline. After one week of intervention, the relative expressions of Fas mRNA and protein was detected by RT-qPCR and western blot. and hematoxylin eosin(HE) staining was used to observe the damage of dopaminergic neurons in the striatum of rats. TUNEL staining was used to detect the apoptosis of dopaminergic neurons in striatum. RT-qPCR and western blot were used to detect the mRNA and protein levels of Fas ligand(Fasl), fas related death domain protein(FADD) and Caspase-8 in striatal dopaminergic neurons.Results Compared with the sham-operated control group, the relative expressions of Fas mRNA and protein in the model group, negative control group and silence group increased(P<0.05);Compared with the model group and negative control group, the relative expressions of Fas mRNA and protein in the silencing group decreased(P<0.05). HE staining showed that the disruption of neuron arrangement and cytoplasmic swelling were rescued after silencing fas gene, accompanied by clearer interstitial and decreased vacuole shrinkage and necrosis. Compared with sham-operated control group, the apoptosis rate of neurons in model group, negative control group and silence group increased(P<0.05);Compared with the model group and negative control group, the apoptosis rate of neurons in the silence group decreased(P<0.05). Compared with the sham group, the relative levels of Fasl, FADD and Caspase-8 mRNA and protein in model group, negative control group and silence group increased;Compared with the model group, the mRNA and protein expressions of FasL, FADD and Caspase-8 in the silencing group decreased(P<0.05).Conclusion Fas Gene silencing of Fas can inhibit the apoptosis of dopaminergic neurons in the striatum of Parkinson’s disease model via down-regulation of Fas, Fasl, FADD and Caspase-8.
作者
刘会星
马建法
王阳
吴少璞
Liu Huixing;Ma Jianfa;Wang Yang(Department of Neurology,The third people’s Hospital of Henan Province,Zhengzhou 450006)
出处
《卒中与神经疾病》
2021年第3期268-273,共6页
Stroke and Nervous Diseases
基金
2017年河南省科技研发项目(162102310283)。