腺病毒载体介导XIAP在初级听皮层高表达抑制老年性聋听力下降及机制研究

发布时间：2018-04-26 19:36

  本文选题：XIAP + GABA能神经元　； 参考：《第三军医大学》2012年硕士论文

【摘要】：研究背景及目的： 老年性聋疾病是老年人群中的常见病。随着中国进入老龄化社会，老年性聋患者逐渐增多，老年性聋疾病严重影响了老年人的生活质量。 老年性聋疾病的发生发展是一个缓慢的自然过程，缺乏逆转性疗法。临床治疗老年性聋疾病的过程中，医生常建议患者早期配戴助听器以辅助日常生活交流。助听器的广泛应用虽然解决了老年性聋患者高频听力下降的困扰，但是助听器本身无选择性全频率声信号放大的物理特性也给患者带来了中低频声信号杂音的干扰。对患者而言，助听器始终不能代替听觉通路进行听觉信号的传导和分析。 患者的普遍性和病理的不确定性使老年性聋疾病成为临床治疗的难点，引起相关科学家的关注。现阶段研究发现，老年性聋疾病的发生与听觉通路局部组织氧自由基形成、代谢失衡、线粒体DNA突变、糖原聚集、微循环障碍和细胞凋亡相关。老年性聋疾病的特征性表现提示大脑听皮层可能出现病变。进一步对老年性聋动物模型听皮层的实验研究证实老年性聋疾病与初级听皮层神经细胞丢失密切相关，丢失的主要方式为神经细胞凋亡。生理状态下，哺乳动物脑部神经元可大致分为兴奋性神经元和抑制性神经元，兴奋性神经元数目多于抑制性神经元，前者主要为谷氨酸能神经元，后者主要由GABA能神经元组成。由此启示我们，老年性聋发病过程中可能存在初级听皮层GABA能神经元凋亡。寻找特异性抑制初级听皮层GABA能神经元凋亡途径的靶向分子、缓解GABA能神经元凋亡，可能有助于缓解老年性聋高频听力下降的过程。 X染色体凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)是细胞凋亡抑制蛋白家族中最重要的成员，能通过caspase-3途径、NF-κB/bcl-2途径、TAB1-TAK1-JNK1途径、MEK1途径和PI3K-Akt途径抑制细胞凋亡。XIAP抗凋亡效应在肿瘤治疗领域和细胞凋亡相关疾病领域中得到证实，但在老年性聋疾病听觉中枢系统中的研究尚未见报道。 鉴于XIAP抗细胞凋亡功能以及GABA能神经元凋亡对老年性聋疾病的重要影响，本实验在国家自然科学基金的支持下，，我们首先观察生理状态下老年性聋模型小鼠初级听皮层GABA能神经元凋亡数目多于青年组小鼠；然后通过构建携带XIAP的重组腺病毒载体，验证外源性XIAP在体外培养皮层神经细胞中高表达对神经细胞活性的影响；再次通过立体定向注射技术构建初级听皮层神经细胞内XIAP高表达的老年性聋动物模型，观察XIAP治疗后对高频听阈和初级听皮层GABA能神经元的保护作用，检测初级听皮层凋亡相关因子与GABA能神经元凋亡的相关性，明确XIAP高表达对老年性聋高频听力的保护作用并阐述其分子机制，为老年性聋疾病特异性临床治疗提供实验研究基础。 研究方法: 第一部分：构建Ad-XIAP感染体外培养老年性聋模型小鼠皮层神经细胞的实验研究 1、C57BL/6j老年性聋模型小鼠皮层神经细胞体外原代培养及鉴定； 2、构建携带XIAP的重组腺病毒载体感染体外原代培养的皮层神经细胞； 3、RT-PCR和Wb方法检测Ad-XIAP感染皮层神经细胞效率； 4、MTT方法检测Ad-XIAP感染皮层神经细胞不同时相点对皮层神经细胞活性的保护作用。 第二部分：外源性XIAP高表达抑制听皮层GABA能神经细胞凋亡对老年性聋的保护作用 实验一：听皮层XIAP高表达对老年性聋高频听力的保护作用 1、立体定向注射Ad-XIAP构建初级听皮层XIAP高表达的老年性聋动物模型； 2、观察Ad-XIAP感染初级听皮层神经细胞效率，Wb方法检测初级听皮层XIAP蛋白表达水平； 3、Ad-XIAP感染初级听皮层不同时相点对老年性聋模型小鼠高频听力的保护作用，筛选最有效的Ad-XIAP感染时间； 实验二：XIAP高表达抑制初级听皮层GABA能神经元凋亡及机制研究 1、免疫荧光化学染色方法检测老年性聋动物模型初级听皮层神经细胞XIAP高表 达对caspase-3表达的抑制作用和对GABA能神经元的保护作用； 2、TUNEL实验明确老年组小鼠初级听皮层存在GABA能神经元凋亡现象； 3、TUNEL法和免疫荧光化学方法检测老年性聋动物模型初级听皮层神经细胞XIAP高表达对GABA能神经元凋亡的抑制作用。 结果： 1、无血清培养法体外培养皮层神经细胞，经β-Tubulin Ⅲ免疫荧光化学染色方法鉴定细胞纯度80%。 2、Ad-XIAP感染体外培养皮层神经细胞后细胞内XIAP基因相对表达量升高； 3、Ad-XIAP感染体外培养皮层神经细胞72小时细胞内XIAP基因相对表达量达到峰值(p0.05)，此时细胞活性高于正常组细胞(p0.05)。 4、成功构建了初级听皮层神经细胞内XIAP高表达的老年性聋动物模型。 5、Ad-XIAP感染初级听皮层组织2周时组织中XIAP蛋白表达量最高(p0.05)，老年性聋模型小鼠高频听阈值改善最明显(p0.05)。 6、生理状态下，老年组小鼠初级听皮层GABA能神经元数目少于青年组，GABA能神经元凋亡数多于青年组。 7、Ad-XIAP感染初级听皮层组织2周时，局部组织中capase-3表达、GABA神经元凋亡低于对照组(p0.05)。 结论: 1、重组腺病毒载体能有效感染皮层神经细胞，介导细胞内XIAP高表达并维持细胞活性。 2、XIAP在初级听皮层神经细胞中高表达能有效缓解老年性聋高频听力下降； 3、老年性聋初级听皮层存在GABA能神经元凋亡现象； 4、XIAP能够抑制老年性聋初级听皮层组织中caspase-3表达和GABA能神经元凋亡。 5、上述研究提示XIAP通过caspase-3途径抑制初级听皮层GABA能神经元凋亡可能作为老年性聋疾病新的治疗靶点。
[Abstract]:Research background and purpose:
Senile deafness is a common disease among the elderly. As China enters an aging society, the elderly deafness is gradually increasing, and senile deafness seriously affects the quality of life of the elderly.
The development of senile deafness is a slow natural process and a lack of reverse therapy. In the process of treating senile deafness, doctors often suggest that patients wear hearing aids early to assist in daily life communication. The wide application of hearing aids has solved the problem of hearing loss in the high frequency hearing loss of the elderly, but the hearing aids The physical characteristics of the full frequency sound signal amplification without selectivity also bring the patient with the noise of the middle and low frequency sound signal. For the patient, the hearing aids can never replace the auditory pathway for the conduction and analysis of the auditory signal.
The prevalence and pathological uncertainty of the patients make senile deafness diseases a difficult point in clinical treatment. The current study shows that the occurrence of senile deafness is related to the formation of oxygen free radicals in the auditory pathway, metabolic imbalance, mitochondrial DNA process, glycogen aggregation, microcirculation disorder and cell apoptosis. The characteristic manifestations of senile deafness suggest that the cerebral auditory cortex may occur. Further research on the auditory cortex of the senile deafness animal model confirms that the senile deafness disease is closely related to the loss of the primary auditory cortex, the main way of the loss is the apoptosis of the nerve cells. In physiological state, the neuron of the mammalian brain can be found. It is roughly divided into excitatory neurons and inhibitory neurons. The number of excitatory neurons is more than that of inhibitory neurons. The former is mainly glutamatergic neurons, and the latter mainly consists of GABA energy neurons. Thus, we suggest that the primary auditory cortex GABA may be apoptotic in the process of senile deafness. The targeted molecules in the auditory cortex of GABA neurons can alleviate the apoptosis of GABA neurons, which may help to alleviate the hearing loss process in senile deafness.
X-linked inhibitor of apoptosis protein (XIAP) is the most important member of the apoptosis inhibitory protein family, which can be used in the field of cancer treatment and apoptosis through caspase-3 pathway, NF- kappa B/bcl-2 pathway, TAB1-TAK1-JNK1 pathway, MEK1 pathway and inhibition pathway to inhibit apoptosis. It has been confirmed in the field of disease, but the study of auditory central nervous system in senile deafness has not been reported.
In view of the anti apoptosis function of XIAP and the important effect of GABA energy neuron apoptosis on senile deafness disease, under the support of National Natural Science Foundation, we first observed that the number of GABA neurons apoptosis in the primary auditory cortex of the aged deaf model mice was more than that of the young mice, and then the XIAP was constructed. The recombinant adenovirus vector was used to verify the effect of high expression of exogenous XIAP on the activity of neural cells in cultured cortical neurons in vitro, and again by stereotactic injection technique, the XIAP high expression of senile deafness animal model in the primary auditory cortex nerve cells was constructed, and the high frequency hearing threshold and the primary auditory cortex of GABA were observed after XIAP treatment. The relationship between the apoptosis related factors of primary auditory cortex and the apoptosis of GABA neurons was detected by the protective effect of the primary auditory cortex, and the protective effect of high expression of XIAP on high frequency hearing of senile deafness was defined and its molecular mechanism was expounded, which provided an experimental basis for the specific clinical treatment of senile deafness disease.
Research methods:
Part one: Construction of Ad-XIAP infection in vitro culture of cortical neurons in mice with senile deafness.
1. In vitro culture and identification of cortical neurons in C57BL/6j model of senile deafness.
2, a recombinant adenovirus vector carrying XIAP was constructed to infect primary cultured cortical neurons in vitro.
3, RT-PCR and Wb methods were used to detect the efficiency of Ad-XIAP infection in cortical neurons.
4, the MTT method was used to detect the protective effect of Ad-XIAP on different cortical neurons.
The second part: the protective effect of overexpression of exogenous XIAP on hearing loss of GABA neurons in auditory cortex.
Experiment 1: high expression of XIAP in auditory cortex protects against hearing loss in senile deafness.
1, stereotactic injection of Ad-XIAP was used to construct an animal model of advanced hearing loss with high XIAP expression in the primary auditory cortex.
2, observe the efficiency of Ad-XIAP infection in the primary auditory cortex, and detect the expression level of XIAP protein in the primary auditory cortex by Wb.
3, the protective effect of Ad-XIAP infection on the hearing loss in the auditory cortex of different age groups was observed, and the most effective Ad-XIAP infection time was screened.
Experiment two: high expression of XIAP inhibits apoptosis of GABA neurons in primary auditory cortex and its mechanism.
1, immunofluorescence staining was used to detect the high XIAP of neurons in the primary auditory cortex of the animal models of senile deafness.
It can inhibit the expression of Caspase-3 and protect the GABA neurons.
2, TUNEL test showed that GABA neurons apoptosis existed in the primary auditory cortex of the elderly group.
3, TUNEL method and immunofluorescence method were used to detect the inhibitory effect of XIAP overexpression on the neuronal apoptosis of GABA neurons in the primary auditory cortex of the animal models of senile deafness.
Result锛

本文编号：1807405