【作者】 高金枝；

【导师】 罗小平；

【作者基本信息】 华中科技大学 ， 儿科学， 2013， 博士

【摘要】 第一部分赖氨酸致原代大鼠纹状体神经元神经毒性的体外研究目的分离培养高纯度的原代大鼠纹状体神经元,探讨赖氨酸对体外培养纹状体神经元的神经毒性作用。方法分离出生24h内SD乳鼠的纹状体,应用胰酶消化、无血清培养基培养法培养纹状体神经元。利用微管相关蛋白(microtubule-associated protein MAP2)染色免疫荧光及流式细胞方法鉴定神经元纯度。不同浓度的赖氨酸(0、5、10、15、20mmol/L)干预神经元24h后,MTT法测定细胞活性,探讨不同浓度赖氨酸对原代纹状体神经元的神经毒性作用。结果MAP2免疫荧光法显示培养细胞神经元纯度为92.4±1.6%,MAP2染色流式细胞计数显示神经元纯度为94.3±2.5%。MTT结果显示小于10mmol/L赖氨酸干预24h对神经元存活无影响。15mmol/L赖氨酸干预24h神经元存活率为84.63%,20mmol/L赖氨酸干预24h神经元存活率降为45.60%。结论胰酶消化无血清培养基培养法可成功培养高纯度的大鼠纹状体神经元。赖氨酸干预24h,浓度不超过10mmol/L时对神经元无毒性,浓度达到15mmol/L以上时,赖氨酸神经毒性呈浓度依赖型,浓度越高,神经元存活率越低。结合正常人类给予高赖氨酸饮食并不致病考虑,后续实验选取5mmol/L、10mmol/L赖氨酸干预GCDH基因沉默神经元,探讨神经损伤机制。第二部分慢病毒介导的GCDH基因沉默和赖氨酸过量摄取对纹状体神经元的神经毒性作用目的利用慢病毒载体携带shRNA转染纹状体神经元细胞内并稳定表达,靶向抑制GCDH基因表达,大量赖氨酸饲养模拟细胞高代谢状态,探讨大量赖氨酸对GCDH基因沉默神经元的神经毒性作用。方法利用已知的靶向沉默大鼠GCDH基因的siRNA序列构建慢病毒载体,并利用病毒载体携带的绿色荧光蛋白(green fluorescent protein, GFP)的表达确定最佳感染复数。Real-time PCR和Western blot测定GCDH基因沉默效率。MTT检测赖氨酸对GCDH基因沉默神经元细胞活性影响。Hoechest33342检测细胞核形态变化。结果慢病毒携带shRNA载体成功转染原代大鼠纹状体神经元,转染效率高达96.5%。该载体能有效抑制GCDH基因表达,蛋白表达下降80.78%。慢病毒介导的GCDH基因沉默降低纹状体神经元细胞活性至68.62%,5mmol/L、10mmol/L赖氨酸饲养则进一步降低细胞活性,且呈浓度依赖性。阴性对照组空载体慢病毒对神经元活性无影响。5mmol/L赖氨酸对阴性对照组神经元细胞活性无影响,但10mmol/L赖氨酸降低阴性对照组神经元细胞活性。Hoechest33342染色显示细胞活性降低组神经元的细胞核呈凋亡表现。结论慢病毒携带shRNA载体能有效且稳定的抑制神经元GCDH基因表达。5mmol/L赖氨酸对正常纹状体神经元活性无影响,对GCDH基因沉默神经元有明显的致凋亡作用。5mmol/L赖氨酸作用于GCDH基因沉默神经元可模拟GA1患儿高代谢状态,成功建立新的GA1研究模型,为研究GA1神经损伤机制提供实验基础。第三部分赖氨酸对GCDH基因沉默纹状体神经元神经毒性作用的可能机制探讨目的探讨赖氨酸对GCDH基因沉默纹状体神经元神经毒性作用的可能机制。方法TMRM (tetramethylrhodamine methyl ester)染色、共聚焦显微镜和流式检测细胞线粒体膜电位变化。Western blot检测各处理组神经元caspase3,8,9蛋白表达情况。在用慢病毒感染神经元之前,先用caspase抑制剂Z-VAD-FMK (benzyloxy-carbonyl-Val-Ala-Asp(OMe)-fluoromethylketone)100μmol/L预孵育1h。然后再用Annexin V-PE/7-AAD流式定量分析各组神经元凋亡情况。结果TMRM染色、共聚焦显微镜和流式均显示GCDH基因沉默神经元线粒体膜电位明显降低,5mmol/L赖氨酸饲养更进一步促进线粒体膜电位降低。GCDH基因沉默神经元内caspase3,9蛋白表达明显增加,caspase8蛋白表达无明显变化。5mmol/L赖氨酸饲养促进GCDH基因沉默神经元内caspase3,8,9蛋白表达增加。Z-VAD-FMK预处理后,流式显示GCDH基因沉默神经元及赖氨酸饲养GCDH基因沉默神经元的细胞凋亡比例均明显下降。结论线粒体膜电位变化参与赖氨酸对GCDH基因沉默纹状体神经元神经毒性作用机制,同时该神经毒性是caspase途径依赖型。更多还原

【Abstract】 Part I The in vitro study of neurotoxicity induced by lysine in primary rat striatal neuronsObjective To culture and identify primary rat striatal neurons. To investigate the neurotoxic effect of lysine on rat striatal neurons in vitro.Methods Striatum tissues were separated from SD rats born within24h. Trypsin digestion and serum free cultivation method were used to culture primary striatal neurons. The purity of neurons was identified by using immunohistochemical method of neuron specific enolase (NSE), using immunofluorescence and flow cytometry methods of microtubule associated protein (MAP2).24h after the intervention of different concentrations of lysine (0,5,10,15,20mmol/L) with neurons, the cell viability was determined by MTT method. Discussed the neurotoxicity induced by different concentrations of lysine in primary rat striatal neurons.Results This method cuccessfully cultured rat striatal neurons. MAP2immunofluorescence showed that the purity of neurons was92.4±1.6%. MAP2staining flow cytometry showed that the purity of neurons was94.3±2.5%. NSE immunohistochemistry showed the purity of neurons was96.2±3.3%. The neurotoxicity induced by lysine in primary rat striatal neurons was concentration dependent. After incubated with lysine for24h, the results of MTT showed the concentration less than10mmol/L had no effect on neuron viability,15mmol/L and20mmol/L lysine reduced the viability of neurons to84.63%and45.60%respectively.Conclusion The rat striatal neurons with high purity were successfully cultured by using trypsin digestion and serum free cultivation method. Lysine intervention for24h, the concentration of not more than10mmol/L had no neurotoxic effect on neurons. When the concentration reached more than15mmol/L, the neurotoxicity was concentration dependent. In view of that high lysine diet does not promote neurotoxicity in normal human, the concentrations of5mmol/L and10mmol/L were used in the following experiments. Part II The neurotoxicity of targeted suppression of GCDH by lentivirus-mediated shRNA and excessive intake of lysine on rat striatal neuronsObjective To investigate the neurotoxicity induced by excessive lysine in GCDH silencing neurons by using lentivirus-mediated shRNA and excessive intake of lysine.Methods Lentiviral vector was constructed by using a known siRNA sequence targeted suppression of GCDH gene expression. The appropriate multiplicity of infection was determined by using GFP carried by virus vector. The interference efficiency of lentivirus was determined by using Real-time PCR and Western blotting. The viability of GCDH silencing neurons incubated with lysine or not was determined by using MTT. The nuclear morphological changes in rat neurons were detected by using Hoechst33342staining. The quantity of neural apoptosis was analysed by using Annexin V-PE/7-AAD and flow cytometry.Results shRNA was transfected into primary rat striatal neurons successfully by using lentiviral vector, and the transfection efficiency was as high as96.5%. The vector can effectively suppressed the GCDH gene expression. The expression of protein was reduced as much as80.78%. The targeted suppression of GCDH by lentivirus-mediated shRNA reduced the neuron viability to68.62%,5mmol/L and10mmol/L lysine enhanced this effect markedly, and the neurotoxicity was concentration dependent. Hoechst33342 staining showed nuclear apoptosis. Annexin V-PE/7-AAD flow quantitative analysis further verified the results of MTT analysis.Conclusion Lentivirus-mediated shRNA vector suppressed the expression of GCDH gene effectively and stably.5mmol/L lysine had no effect on neuron viability, but induced GCDH silencing neurons apoptosis. GCDH silencing neurons incubated with addition of5mmol/L lysine can simulate the hypermetabolic condition in GA1patients. A novel model of GA1was established. PartⅢ the mechanism of the neurotoxicity induced by lysine in GCDH silencing neurons was investigatedObjective To investigate the the mechanism of the neurotoxicity induced by lysine in GCDH silencing neurons.Methods Mitochondrial membrane potential was monitored using TMRM staining, confocal microscope and flow cytometry. The levels of caspase3,8,9, AIF and Hsp70expression were determined by Western bloting.100μmol/L Z-VAD-FMK was added to the medium1h prior to lentiviral infection, and then the quantity of neural apoptosis was analysed by using Annexin V-PE/7-AAD and flow cytometry.Results TMRM staining, confocal microscope and flow cytometry all showed suppression the expression of GCDH gene significantly decreased the neuron mitochondrial membrane potential, and5mmol/L lysine enhanced this decrease. The protein levels of caspases3and9were significantly upregulated by lentivirus. The combination of lysine and lentivirus intensified the upregulation of caspases3and9. Neither lentivirus nor5mmol/L lysine alone changed the level of caspase8expression, but exposure to both increased the protein level of caspase8. With Z-VAD-FMK pretreatment, the apoptotic cell fraction in cells infected with lentivirus decreased to21.87%in cells not exposed to lysine and41.66%in cells exposed to5mmol/L lysine.Conclusion Mitochondrial membrane potential changes may be involved in the the mechanism of the neurotoxicity induced by lysine in GCDH silencing neurons. And the neurotoxicity is caspase pathway dependent.更多还原