本文選題：DMSO + DAPT�。� 參考：《福建醫(yī)科大學(xué)》2012年碩士論文

【摘要】：感音神經(jīng)性聾是導(dǎo)致言語交流障礙的常見致殘疾病，嚴(yán)重影響人類的生活質(zhì)量，其實(shí)質(zhì)多在于耳蝸毛細(xì)胞的變性、壞死，目前尚無根治辦法。近年來人們?cè)诜肿由飳W(xué)、分子遺傳學(xué)、基因工程技術(shù)等現(xiàn)代學(xué)科取得的發(fā)展，使我們能借助新的工具和手段對(duì)毛細(xì)胞再生進(jìn)行進(jìn)一步探索。本實(shí)驗(yàn)的目的主要是研究小分子物質(zhì)在耳蝸毛細(xì)胞再生中的作用，涉及的小分子物質(zhì)主要包括：DMSO、DAPT和miRNA。 第一部分二甲基亞砜對(duì)在體耳蝸毛細(xì)胞的毒性作用研究 目的：研究二甲基亞砜（DMSO）對(duì)在體耳蝸毛細(xì)胞的毒性作用。 方法：正常SD大鼠40只,隨機(jī)分成4組，人工外淋巴液對(duì)照組8只；0.1%DMSO溶液組8只；1%DMSO溶液組8只；5%DMSO溶液組16只。每個(gè)實(shí)驗(yàn)耳注入不同濃度DMSO溶液。術(shù)前1天和術(shù)后7天分別進(jìn)行ABR（click和tone burst）檢測(cè)。術(shù)后7天取耳蝸進(jìn)行免疫組化和HE染色觀察毛細(xì)胞變化。5%DMSO組導(dǎo)入術(shù)后2h、4h、6h、12h各取2耳行TUNEL染色。 結(jié)果：人工外淋巴液和0.1%DMSO導(dǎo)入后于32k處引起30dBSPL的閾移，其余各頻率ABR未見明顯提高，形態(tài)學(xué)未見明顯異常；1%DMSO即可引起明顯的HC損傷，以O(shè)HC丟失為主；5%DMSO所造成的損傷較1%組重，并導(dǎo)致明顯的IHC缺失，后兩種濃度均使ABR各頻率閾值明顯提高。冰凍切片示DMSO亦造成基底膜SC的損傷。5%組術(shù)后2h行TUNEL染色見陽性染色。 結(jié)論：DMSO對(duì)毛細(xì)胞的損傷存在劑量依賴性，損傷程度自耳蝸底回向頂回逐漸減輕。DMSO不僅損傷HC，也對(duì)SC造成破壞。DMSO對(duì)耳蝸內(nèi)細(xì)胞的毒性可能與誘發(fā)細(xì)胞凋亡有關(guān)。 第二部分DAPT對(duì)在體耳蝸毛細(xì)胞再生的作用 目的：探討DAPT對(duì)耳蝸毛細(xì)胞再生的作用。 方法：健康SD乳鼠大鼠（n=15）隨機(jī)分成3組，，滲透泵植入組5只，導(dǎo)入術(shù)后14天實(shí)驗(yàn)耳激光共聚焦顯微鏡下觀察；鼓階打孔連續(xù)給藥和間斷給藥組各5只，術(shù)后7天實(shí)驗(yàn)耳激光共聚焦顯微鏡下觀察。 結(jié)果：滲透泵植入組實(shí)驗(yàn)耳基底膜可見異位myosinVIIa和phalloidin雙陽性染色細(xì)胞，但發(fā)生率低；此外，部分實(shí)驗(yàn)耳頂回纖毛開口朝向發(fā)生明顯改變，多數(shù)靜纖毛束結(jié)構(gòu)消失，僅見散在phalloidin陽性染色點(diǎn)。鼓階打孔單次持續(xù)或間歇給藥組因DMSO濃度較高造成HC、SC損傷嚴(yán)重，未見明顯異位myosinVIIa或phalloidin陽性染色細(xì)胞。 結(jié)論：DAPT可能對(duì)乳鼠毛細(xì)胞的靜纖毛產(chǎn)生影響。滲透泵植入給藥法由于能長(zhǎng)期、定量地將藥物穩(wěn)定導(dǎo)入內(nèi)耳，因此更適于研究DAPT在耳蝸毛細(xì)胞再生的作用。 第三部分MiRNA在內(nèi)耳毛細(xì)胞再生中作用的初步探討 目的：研究新生大鼠和成年大鼠耳蝸基底膜的miRNA表達(dá)變化，以期發(fā)現(xiàn)miRNA在耳蝸毛細(xì)胞再生中可能具有的作用。 方法：取P0期及P30SD大鼠各9只，利用microarry對(duì)兩種大鼠耳蝸基底膜的miRNA表達(dá)模式進(jìn)行分析，并通過基因本體論分析和相關(guān)信號(hào)通路分析進(jìn)行進(jìn)一步探討。 結(jié)果：microarray分析發(fā)現(xiàn)有16種miRNA在成年大鼠中表達(dá)高于新生大鼠,2種miRNA在成年大鼠中表達(dá)略有下調(diào)。基因本體論分析發(fā)現(xiàn)差異性表達(dá)的miRNA功能涉及多個(gè)細(xì)胞生命活動(dòng)過程，其中具有最大富集度的功能與調(diào)節(jié)TGF-β信號(hào)有關(guān)。相關(guān)信號(hào)通路分析發(fā)現(xiàn)有19個(gè)信號(hào)轉(zhuǎn)導(dǎo)通路出現(xiàn)上調(diào)，而14個(gè)信號(hào)轉(zhuǎn)導(dǎo)通路則出現(xiàn)了下調(diào)。 結(jié)論：新生大鼠和成年大鼠聽覺上皮miRNA表達(dá)存在差異，基因本體論分析和相應(yīng)信號(hào)通路分析發(fā)現(xiàn)該變化可能與Corti器的發(fā)育成熟、細(xì)胞日常功能維持和內(nèi)耳毛細(xì)胞增殖能力的喪失緊密相關(guān)，并且TGFβ信號(hào)在毛細(xì)胞再生中可能具有重要作用。
[Abstract]:Sensorineural deafness is a common deformity causing speech communication disorder, which seriously affects the quality of life of human beings. In fact, the quality of the human life is mainly due to the degeneration and necrosis of the cochlear hair cells. At present, there is no radical cure. In recent years, the development of modern disciplines, such as molecular biology, molecular genetics and genetic engineering techniques, has made us able to help with the new The purpose of this experiment is to study the role of small molecules in the regeneration of the cochlear hair cells. The small molecules involved mainly include: DMSO, DAPT and miRNA..
Part 1 toxicity of two methyl sulfoxide on cochlear hair cells in vivo
Objective: To study the toxic effect of two methyl sulfoxide (DMSO) on cochlear hair cells in vivo.
Methods: 40 normal SD rats were randomly divided into 4 groups, 8 of the artificial peripheral lymph fluid control group, 8 in the 0.1%DMSO solution group, 8 in the 1%DMSO solution group and 16 in the 5%DMSO solution group. Each experimental ear was injected with different concentrations of DMSO solution. The ABR (click and tone burst) were detected for 1 days before and 7 days after the operation. The cochlea was immunohistochemical and HE on the 7 day after the operation. The changes of hair cells were observed by staining. After.5%DMSO, 2h, 4h, 6h and 12h were taken in each group, and 2 ears were stained with TUNEL.
Results: the threshold shift of 30dBSPL was caused by the introduction of artificial lymph and 0.1%DMSO at 32K, the other frequency ABR was not obviously improved, and the morphology was not obviously abnormal. 1%DMSO could cause obvious HC damage and OHC loss. The damage caused by 5%DMSO was more serious than that of 1% groups, and the loss of IHC was obviously guided, and the latter two concentrations were all frequency of ABR. The rate of threshold increased significantly. Frozen sections showed that DMSO also caused SC damage in the basement membrane..5% group showed positive staining after 2H TUNEL staining.
Conclusion: the damage of DMSO to hair cells is dose-dependent, and the degree of damage is gradually reduced from the back of the cochlear back to the apex of the cochlea. The damage of.DMSO not only damage HC, but also the toxicity of.DMSO to the inner cells of the cochlea may be related to the apoptosis of the cells.
The second part is the effect of DAPT on the regeneration of cochlear hair cells in vivo.
Objective: To investigate the effect of DAPT on the regeneration of cochlear hair cells.
Methods: healthy SD rat rats (n=15) were randomly divided into 3 groups, 5 were implanted in the osmotic pump group, and the experimental ears were observed under confocal microscope for 14 days after the operation, 5 in the drums and in the discontinuous administration group, and the experimental ear laser confocal microscope was observed for 7 days after the operation.
Results: ectopic myosinVIIa and phalloidin double positive staining cells were found in the basilar membrane of the osmotic pump group, but the occurrence rate was low. In addition, the cilium opening orientation of the top of the ear was obviously changed, most of the static cilium structure disappeared and only scattered in the phalloidin positive staining points. The high concentration of DMSO caused severe damage to HC and SC, and no obvious ectopic myosinVIIa or phalloidin positive staining cells were found.
Conclusion: DAPT may affect the static cilia of the mouse hair cells. The osmotic pump implantation is more suitable for the study of the role of DAPT in the cochlear hair cell regeneration because of the long-term and quantitative introduction of the drug into the inner ear.
The third part is the preliminary study of the role of MiRNA in hair cell regeneration of inner ear.
Objective: To study the expression of miRNA in the cochlea basement membrane of neonatal rats and adult rats, in order to find out the possible role of miRNA in the regeneration of cochlear hair cells.
Methods: 9 rats of P0 and P30SD rats were used to analyze the miRNA expression pattern of the basilar membrane of the cochlea of two rats by microarry, and further explored through the analysis of gene ontology and the analysis of the related signal pathways.
Results: the expression of 16 kinds of miRNA in adult rats was higher than that of the newborn rats, and the expression of 2 miRNA in adult rats was slightly down. The gene ontology analysis found that the function of differential expression of miRNA involved multiple cell life activities, and the function of the maximum enrichment was related to the regulation of TGF- beta signal. Signal pathway analysis showed that 19 signal transduction pathways were upregulated, while 14 signal transduction pathways were down regulated.
Conclusion: the expression of miRNA in the auditory epithelium of the newborn rats and adult rats is different. The gene ontology analysis and the corresponding signal pathway analysis found that the changes may be related to the maturation of the Corti apparatus, the maintenance of the daily function of the cells and the loss of the proliferation ability of the inner ear hair cells, and the TGF beta signal may be heavy in the regeneration of the hair cells. It's going to work.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764.35

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李華偉;雛雞基底乳頭慶大霉素?fù)p傷后TGFβ_1、TGFβ_2、TGFβ_3的表達(dá)[J];臨床耳鼻咽喉科雜志;1998年10期

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