本文選題：XIAP + GABA能神經(jīng)元��； 參考：《第三軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：研究背景及目的： 老年性聾疾病是老年人群中的常見(jiàn)病。隨著中國(guó)進(jìn)入老齡化社會(huì)，老年性聾患者逐漸增多，老年性聾疾病嚴(yán)重影響了老年人的生活質(zhì)量。 老年性聾疾病的發(fā)生發(fā)展是一個(gè)緩慢的自然過(guò)程，缺乏逆轉(zhuǎn)性療法。臨床治療老年性聾疾病的過(guò)程中，醫(yī)生常建議患者早期配戴助聽(tīng)器以輔助日常生活交流。助聽(tīng)器的廣泛應(yīng)用雖然解決了老年性聾患者高頻聽(tīng)力下降的困擾，但是助聽(tīng)器本身無(wú)選擇性全頻率聲信號(hào)放大的物理特性也給患者帶來(lái)了中低頻聲信號(hào)雜音的干擾。對(duì)患者而言，助聽(tīng)器始終不能代替聽(tīng)覺(jué)通路進(jìn)行聽(tīng)覺(jué)信號(hào)的傳導(dǎo)和分析。 患者的普遍性和病理的不確定性使老年性聾疾病成為臨床治療的難點(diǎn)，引起相關(guān)科學(xué)家的關(guān)注。現(xiàn)階段研究發(fā)現(xiàn)，老年性聾疾病的發(fā)生與聽(tīng)覺(jué)通路局部組織氧自由基形成、代謝失衡、線粒體DNA突變、糖原聚集、微循環(huán)障礙和細(xì)胞凋亡相關(guān)。老年性聾疾病的特征性表現(xiàn)提示大腦聽(tīng)皮層可能出現(xiàn)病變。進(jìn)一步對(duì)老年性聾動(dòng)物模型聽(tīng)皮層的實(shí)驗(yàn)研究證實(shí)老年性聾疾病與初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞丟失密切相關(guān)，丟失的主要方式為神經(jīng)細(xì)胞凋亡。生理狀態(tài)下，哺乳動(dòng)物腦部神經(jīng)元可大致分為興奮性神經(jīng)元和抑制性神經(jīng)元，興奮性神經(jīng)元數(shù)目多于抑制性神經(jīng)元，前者主要為谷氨酸能神經(jīng)元，后者主要由GABA能神經(jīng)元組成。由此啟示我們，老年性聾發(fā)病過(guò)程中可能存在初級(jí)聽(tīng)皮層GABA能神經(jīng)元凋亡。尋找特異性抑制初級(jí)聽(tīng)皮層GABA能神經(jīng)元凋亡途徑的靶向分子、緩解GABA能神經(jīng)元凋亡，可能有助于緩解老年性聾高頻聽(tīng)力下降的過(guò)程。 X染色體凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)是細(xì)胞凋亡抑制蛋白家族中最重要的成員，能通過(guò)caspase-3途徑、NF-κB/bcl-2途徑、TAB1-TAK1-JNK1途徑、MEK1途徑和PI3K-Akt途徑抑制細(xì)胞凋亡。XIAP抗凋亡效應(yīng)在腫瘤治療領(lǐng)域和細(xì)胞凋亡相關(guān)疾病領(lǐng)域中得到證實(shí)，但在老年性聾疾病聽(tīng)覺(jué)中樞系統(tǒng)中的研究尚未見(jiàn)報(bào)道。 鑒于XIAP抗細(xì)胞凋亡功能以及GABA能神經(jīng)元凋亡對(duì)老年性聾疾病的重要影響，本實(shí)驗(yàn)在國(guó)家自然科學(xué)基金的支持下，，我們首先觀察生理狀態(tài)下老年性聾模型小鼠初級(jí)聽(tīng)皮層GABA能神經(jīng)元凋亡數(shù)目多于青年組小鼠；然后通過(guò)構(gòu)建攜帶XIAP的重組腺病毒載體，驗(yàn)證外源性XIAP在體外培養(yǎng)皮層神經(jīng)細(xì)胞中高表達(dá)對(duì)神經(jīng)細(xì)胞活性的影響；再次通過(guò)立體定向注射技術(shù)構(gòu)建初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞內(nèi)XIAP高表達(dá)的老年性聾動(dòng)物模型，觀察XIAP治療后對(duì)高頻聽(tīng)閾和初級(jí)聽(tīng)皮層GABA能神經(jīng)元的保護(hù)作用，檢測(cè)初級(jí)聽(tīng)皮層凋亡相關(guān)因子與GABA能神經(jīng)元凋亡的相關(guān)性，明確XIAP高表達(dá)對(duì)老年性聾高頻聽(tīng)力的保護(hù)作用并闡述其分子機(jī)制，為老年性聾疾病特異性臨床治療提供實(shí)驗(yàn)研究基礎(chǔ)。 研究方法: 第一部分：構(gòu)建Ad-XIAP感染體外培養(yǎng)老年性聾模型小鼠皮層神經(jīng)細(xì)胞的實(shí)驗(yàn)研究 1、C57BL/6j老年性聾模型小鼠皮層神經(jīng)細(xì)胞體外原代培養(yǎng)及鑒定； 2、構(gòu)建攜帶XIAP的重組腺病毒載體感染體外原代培養(yǎng)的皮層神經(jīng)細(xì)胞； 3、RT-PCR和Wb方法檢測(cè)Ad-XIAP感染皮層神經(jīng)細(xì)胞效率； 4、MTT方法檢測(cè)Ad-XIAP感染皮層神經(jīng)細(xì)胞不同時(shí)相點(diǎn)對(duì)皮層神經(jīng)細(xì)胞活性的保護(hù)作用。 第二部分：外源性XIAP高表達(dá)抑制聽(tīng)皮層GABA能神經(jīng)細(xì)胞凋亡對(duì)老年性聾的保護(hù)作用 實(shí)驗(yàn)一：聽(tīng)皮層X(jué)IAP高表達(dá)對(duì)老年性聾高頻聽(tīng)力的保護(hù)作用 1、立體定向注射Ad-XIAP構(gòu)建初級(jí)聽(tīng)皮層X(jué)IAP高表達(dá)的老年性聾動(dòng)物模型； 2、觀察Ad-XIAP感染初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞效率，Wb方法檢測(cè)初級(jí)聽(tīng)皮層X(jué)IAP蛋白表達(dá)水平； 3、Ad-XIAP感染初級(jí)聽(tīng)皮層不同時(shí)相點(diǎn)對(duì)老年性聾模型小鼠高頻聽(tīng)力的保護(hù)作用，篩選最有效的Ad-XIAP感染時(shí)間； 實(shí)驗(yàn)二：XIAP高表達(dá)抑制初級(jí)聽(tīng)皮層GABA能神經(jīng)元凋亡及機(jī)制研究 1、免疫熒光化學(xué)染色方法檢測(cè)老年性聾動(dòng)物模型初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞XIAP高表 達(dá)對(duì)caspase-3表達(dá)的抑制作用和對(duì)GABA能神經(jīng)元的保護(hù)作用； 2、TUNEL實(shí)驗(yàn)明確老年組小鼠初級(jí)聽(tīng)皮層存在GABA能神經(jīng)元凋亡現(xiàn)象； 3、TUNEL法和免疫熒光化學(xué)方法檢測(cè)老年性聾動(dòng)物模型初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞XIAP高表達(dá)對(duì)GABA能神經(jīng)元凋亡的抑制作用。 結(jié)果： 1、無(wú)血清培養(yǎng)法體外培養(yǎng)皮層神經(jīng)細(xì)胞，經(jīng)β-Tubulin Ⅲ免疫熒光化學(xué)染色方法鑒定細(xì)胞純度80%。 2、Ad-XIAP感染體外培養(yǎng)皮層神經(jīng)細(xì)胞后細(xì)胞內(nèi)XIAP基因相對(duì)表達(dá)量升高； 3、Ad-XIAP感染體外培養(yǎng)皮層神經(jīng)細(xì)胞72小時(shí)細(xì)胞內(nèi)XIAP基因相對(duì)表達(dá)量達(dá)到峰值(p0.05)，此時(shí)細(xì)胞活性高于正常組細(xì)胞(p0.05)。 4、成功構(gòu)建了初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞內(nèi)XIAP高表達(dá)的老年性聾動(dòng)物模型。 5、Ad-XIAP感染初級(jí)聽(tīng)皮層組織2周時(shí)組織中XIAP蛋白表達(dá)量最高(p0.05)，老年性聾模型小鼠高頻聽(tīng)閾值改善最明顯(p0.05)。 6、生理狀態(tài)下，老年組小鼠初級(jí)聽(tīng)皮層GABA能神經(jīng)元數(shù)目少于青年組，GABA能神經(jīng)元凋亡數(shù)多于青年組。 7、Ad-XIAP感染初級(jí)聽(tīng)皮層組織2周時(shí)，局部組織中capase-3表達(dá)、GABA神經(jīng)元凋亡低于對(duì)照組(p0.05)。 結(jié)論: 1、重組腺病毒載體能有效感染皮層神經(jīng)細(xì)胞，介導(dǎo)細(xì)胞內(nèi)XIAP高表達(dá)并維持細(xì)胞活性。 2、XIAP在初級(jí)聽(tīng)皮層神經(jīng)細(xì)胞中高表達(dá)能有效緩解老年性聾高頻聽(tīng)力下降； 3、老年性聾初級(jí)聽(tīng)皮層存在GABA能神經(jīng)元凋亡現(xiàn)象； 4、XIAP能夠抑制老年性聾初級(jí)聽(tīng)皮層組織中caspase-3表達(dá)和GABA能神經(jīng)元凋亡。 5、上述研究提示XIAP通過(guò)caspase-3途徑抑制初級(jí)聽(tīng)皮層GABA能神經(jīng)元凋亡可能作為老年性聾疾病新的治療靶點(diǎn)。
[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錛

本文編號(hào)：1807405