小分子物质在耳蜗毛细胞再生中的作用

发布时间：2018-06-13 14:48

本文选题：DMSO + DAPT　；参考：《福建医科大学》2012年硕士论文

【摘要】：感音神经性聋是导致言语交流障碍的常见致残疾病，严重影响人类的生活质量，其实质多在于耳蜗毛细胞的变性、坏死，目前尚无根治办法。近年来人们在分子生物学、分子遗传学、基因工程技术等现代学科取得的发展，使我们能借助新的工具和手段对毛细胞再生进行进一步探索。本实验的目的主要是研究小分子物质在耳蜗毛细胞再生中的作用，涉及的小分子物质主要包括：DMSO、DAPT和miRNA。第一部分二甲基亚砜对在体耳蜗毛细胞的毒性作用研究目的：研究二甲基亚砜（DMSO）对在体耳蜗毛细胞的毒性作用。方法：正常SD大鼠40只,随机分成4组，人工外淋巴液对照组8只；0.1%DMSO溶液组8只；1%DMSO溶液组8只；5%DMSO溶液组16只。每个实验耳注入不同浓度DMSO溶液。术前1天和术后7天分别进行ABR（click和tone burst）检测。术后7天取耳蜗进行免疫组化和HE染色观察毛细胞变化。5%DMSO组导入术后2h、4h、6h、12h各取2耳行TUNEL染色。结果：人工外淋巴液和0.1%DMSO导入后于32k处引起30dBSPL的阈移，其余各频率ABR未见明显提高，形态学未见明显异常；1%DMSO即可引起明显的HC损伤，以OHC丢失为主；5%DMSO所造成的损伤较1%组重，并导致明显的IHC缺失，后两种浓度均使ABR各频率阈值明显提高。冰冻切片示DMSO亦造成基底膜SC的损伤。5%组术后2h行TUNEL染色见阳性染色。结论：DMSO对毛细胞的损伤存在剂量依赖性，损伤程度自耳蜗底回向顶回逐渐减轻。DMSO不仅损伤HC，也对SC造成破坏。DMSO对耳蜗内细胞的毒性可能与诱发细胞凋亡有关。第二部分DAPT对在体耳蜗毛细胞再生的作用目的：探讨DAPT对耳蜗毛细胞再生的作用。方法：健康SD乳鼠大鼠（n=15）随机分成3组，，渗透泵植入组5只，导入术后14天实验耳激光共聚焦显微镜下观察；鼓阶打孔连续给药和间断给药组各5只，术后7天实验耳激光共聚焦显微镜下观察。结果：渗透泵植入组实验耳基底膜可见异位myosinVIIa和phalloidin双阳性染色细胞，但发生率低；此外，部分实验耳顶回纤毛开口朝向发生明显改变，多数静纤毛束结构消失，仅见散在phalloidin阳性染色点。鼓阶打孔单次持续或间歇给药组因DMSO浓度较高造成HC、SC损伤严重，未见明显异位myosinVIIa或phalloidin阳性染色细胞。结论：DAPT可能对乳鼠毛细胞的静纤毛产生影响。渗透泵植入给药法由于能长期、定量地将药物稳定导入内耳，因此更适于研究DAPT在耳蜗毛细胞再生的作用。第三部分MiRNA在内耳毛细胞再生中作用的初步探讨目的：研究新生大鼠和成年大鼠耳蜗基底膜的miRNA表达变化，以期发现miRNA在耳蜗毛细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对两种大鼠耳蜗基底膜的miRNA表达模式进行分析，并通过基因本体论分析和相关信号通路分析进行进一步探讨。结果：microarray分析发现有16种miRNA在成年大鼠中表达高于新生大鼠,2种miRNA在成年大鼠中表达略有下调。基因本体论分析发现差异性表达的miRNA功能涉及多个细胞生命活动过程，其中具有最大富集度的功能与调节TGF-β信号有关。相关信号通路分析发现有19个信号转导通路出现上调，而14个信号转导通路则出现了下调。结论：新生大鼠和成年大鼠听觉上皮miRNA表达存在差异，基因本体论分析和相应信号通路分析发现该变化可能与Corti器的发育成熟、细胞日常功能维持和内耳毛细胞增殖能力的丧失紧密相关，并且TGFβ信号在毛细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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764.35

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