【摘要】： 目的1)檢驗(yàn)前庭膜性迷路對(duì)腺病毒載體的通透性,從而探索在外淋巴中直接給腺病毒載體對(duì)于內(nèi)耳毛細(xì)胞再生研究的可行性。2)新生鼠的耳蝸上皮轉(zhuǎn)染math1產(chǎn)生異位新生的毛細(xì)胞,本實(shí)驗(yàn)為了解前庭上皮細(xì)胞是否存在同樣的具有分化潛能的感覺(jué)上皮。3)探索math1誘導(dǎo)的毛細(xì)胞樣的細(xì)胞的轉(zhuǎn)分化的形態(tài)學(xué)過(guò)程及分化條件、特點(diǎn)。 方法Ad-math1-EGFP載體的構(gòu)建及其轉(zhuǎn)染前庭上皮后表達(dá)的math1蛋白的檢測(cè)；本實(shí)驗(yàn)以鼠為研究對(duì)象,以中路窩途徑快速采集標(biāo)本,分別建立前庭膜迷路整體培養(yǎng)和前庭終器培養(yǎng)的模型,并檢驗(yàn)兩個(gè)不同模型轉(zhuǎn)染Ad-math1-EGFP載體后的效應(yīng)；采用前庭終器培養(yǎng)的模型,使正常培養(yǎng)的前庭上皮感染Ad-math1-EGFP,對(duì)轉(zhuǎn)染病毒后不同時(shí)間點(diǎn)的標(biāo)本固定,部分采取冰凍切片,運(yùn)用免疫組織化學(xué)進(jìn)行染色后觀察、采集實(shí)驗(yàn)數(shù)據(jù)；采用前庭終器培養(yǎng),并建立高、低增殖活性的前庭感覺(jué)上皮的模型,分別將它們轉(zhuǎn)染Ad-math1-EGFP,一定時(shí)間后固定、免疫組織化學(xué)染色后觀察、采集實(shí)驗(yàn)數(shù)據(jù)；本實(shí)驗(yàn)以萊卡熒光顯微鏡、SP2或者SP5的共聚焦顯微鏡對(duì)標(biāo)本進(jìn)行觀察并采集實(shí)驗(yàn)數(shù)據(jù)。 結(jié)果1)體外培養(yǎng),膜迷路整體模型和前庭終器培養(yǎng)模型的首次建立。前者,盡量保持前庭膜迷路的完整性并將它們貼壁培養(yǎng),培養(yǎng)液中給腺病毒載體,模擬在體時(shí)外淋巴給病毒；后者,去除前庭的膜性非感覺(jué)上皮并將剩余的感覺(jué)上皮貼壁培養(yǎng),培養(yǎng)液中給腺病毒載體,模擬在體時(shí)內(nèi)淋巴給病毒。2)膜迷路整體培養(yǎng)模式,培養(yǎng)液中給Ad-math1-EGFP載體,上皮細(xì)胞在非感覺(jué)上皮區(qū)出現(xiàn)極少量毛細(xì)胞樣的細(xì)胞；前庭終器培養(yǎng)模式,培養(yǎng)液中給Ad-math1-EGFP載體,非感覺(jué)上皮區(qū)有大量的方格狀細(xì)胞出現(xiàn),并在此處出現(xiàn)大量的毛細(xì)胞樣的細(xì)胞。3)異位新生的毛細(xì)胞樣的細(xì)胞具有纖毛,多成簇出現(xiàn),部分細(xì)胞共用一簇纖毛；細(xì)胞形態(tài)多樣,呈現(xiàn)為多邊形態(tài)、毛細(xì)胞樣的形態(tài)及兼有二者特點(diǎn)的柱狀。4)機(jī)械損傷的前庭終器,在損傷處的支持細(xì)胞增殖且在math1誘導(dǎo)下出現(xiàn)新生的毛細(xì)胞樣的細(xì)胞；正常培養(yǎng)的前庭終器感覺(jué)上皮區(qū)極少數(shù)增殖細(xì)胞且在math1誘導(dǎo)下未見(jiàn)新生的毛細(xì)胞樣的細(xì)胞；慶大霉素造模后前庭終器感覺(jué)上皮區(qū)幾乎沒(méi)有增殖細(xì)胞且在math1誘導(dǎo)下未見(jiàn)新生的毛細(xì)胞樣的細(xì)胞5)Math1誘導(dǎo)的新生毛細(xì)胞樣的細(xì)胞多來(lái)源于一些高增殖活性、方格狀的細(xì)胞,其分化的過(guò)程中多不經(jīng)歷細(xì)胞的增殖,但分裂中的細(xì)胞有可能被轉(zhuǎn)染并具備毛細(xì)胞的特點(diǎn)。 結(jié)論1)前庭膜性迷路是阻止腺病毒載體從外淋巴進(jìn)入內(nèi)淋巴的屏障。2)新生鼠的前庭上皮中存在一類(lèi)高增殖活性的細(xì)胞,貼壁培養(yǎng)時(shí),它們可以遷延、增殖,并在math1誘導(dǎo)下可轉(zhuǎn)分化為異位的毛細(xì)胞樣細(xì)胞。3)具有高增殖潛能的前體細(xì)胞轉(zhuǎn)染math1后易產(chǎn)生毛細(xì)胞樣的細(xì)胞。4)math1誘導(dǎo)的毛細(xì)胞樣的細(xì)胞的過(guò)程是一個(gè)不依賴(lài)于細(xì)胞增殖的轉(zhuǎn)分化的過(guò)程。5)毛細(xì)胞樣的細(xì)胞形態(tài)多樣,并具備其行使功能的基本結(jié)構(gòu)靜纖毛。
[Abstract]:Objective 1) to examine the permeability of vestibular membranous labyrinth to adenovirus vector, and to explore the feasibility of using the adenovirus vector directly to study the regeneration of inner ear hair cells in the external lymph nodes (.2). The cochlear epithelium of the newborn rats was transfected with Math1 to produce ectopic hair cells. This experiment is to understand whether the vestibular epithelial cells have the same differentiation. Potential epithelia.3) explore the morphological process, differentiation conditions and characteristics of Math1 induced hairy cell like cell transdifferentiation.
Methods the construction of the Ad-math1-EGFP vector and the detection of Math1 protein expressed after the vestibular epithelium were detected. In this experiment, the rat was used as the research object. The model of the vestibular membrane labyrinth culture and the vestibular terminal culture was established by the medium fossa pathway, and the effect of the two different model transfected Ad-math1-EGFP vectors was tested. Using the model of vestibule terminal culture, the normal cultured vestibule epithelium was infected with Ad-math1-EGFP, the specimens were fixed at different time points after the virus transfection, some frozen sections were taken, the experimental data were observed with immunohistochemical staining, the experimental data were collected, the vestibular terminal culture was used, and the high and low proliferative vestibular sensory epithelium was established. The models were transfected to Ad-math1-EGFP, fixed at a certain time and observed by immunohistochemical staining, and the experimental data were collected. The specimens were observed by Lycra fluorescence microscope, SP2 or SP5 confocal microscopy.
Results 1) in vitro culture, the whole model of the membranous labyrinth and the vestibular terminal culture model was first established. The former was to maintain the integrity of the vestibular membrane labyrinth and put them on the wall. The culture liquid was given to the adenovirus vector, and the virus was simulated in the body. The latter, the membranous non sensory epithelium of the vestibule was removed and the remaining sensory epithelium was adhered to the wall. Culture, incubate the adenovirus vector, simulate the whole culture mode of the membranous labyrinth of the endolymphatic virus.2 in vivo, the Ad-math1-EGFP carrier is given in the culture fluid, and the epithelial cells appear very small hair cell like cells in the non sensory epithelium; the vestibule terminal culture mode, the Ad-math1-EGFP carrier in the culture fluid, and the non sensory epithelium have a large number. There is a large number of hair cell like cells.3 here and there are ciliate cells with cilia, multiple clusters and a cluster of cilia in some cells; the cells are multiform, hairy cells like morphology and a column like.4 with two characteristics. The proliferation of the supporting cells in the injury and the emergence of new hair cell like cells under the induction of Math1; the normal cultured vestibule terminals felt a few proliferating cells in the epithelial area and no new hair cell like cells were found under the induction of Math1; the vestibular end organ of gentamicin was almost no proliferating cells in the sensory cortex and was induced by Math1. No new hair cell like cells 5.) Math1 induced cell like cells are mostly derived from a number of highly proliferative, square cells that do not undergo cell proliferation in the process of differentiation, but the cells in the split are likely to be transfected and possess the characteristics of hair cells.
Conclusion 1) vestibular membranous labyrinth is a kind of high proliferative cell in the vestibule epithelium of newborn rats, which prevents adenovirus vector from external lymph nodes into the endolymphatic lymph. In the wall culture, they can be extended, proliferate, and can be converted into heterotopic hair cell like cells.3 under the induction of Math1, and the precursor cells with high proliferation potential can be transferred to.2. The process of Math1 induced hair cell like cells is a process of.5 that does not depend on the proliferation of cell proliferation,.5) the hair cell like cells are diverse, and have the basic structure of the basic structure of its function of Math1.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R764

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