Math1诱导的前庭毛细胞再生的转分化过程与细胞增殖

发布时间：2018-07-18 07:17

【摘要】： 目的1)检验前庭膜性迷路对腺病毒载体的通透性,从而探索在外淋巴中直接给腺病毒载体对于内耳毛细胞再生研究的可行性。2)新生鼠的耳蜗上皮转染math1产生异位新生的毛细胞,本实验为了解前庭上皮细胞是否存在同样的具有分化潜能的感觉上皮。3)探索math1诱导的毛细胞样的细胞的转分化的形态学过程及分化条件、特点。方法Ad-math1-EGFP载体的构建及其转染前庭上皮后表达的math1蛋白的检测；本实验以鼠为研究对象,以中路窝途径快速采集标本,分别建立前庭膜迷路整体培养和前庭终器培养的模型,并检验两个不同模型转染Ad-math1-EGFP载体后的效应；采用前庭终器培养的模型,使正常培养的前庭上皮感染Ad-math1-EGFP,对转染病毒后不同时间点的标本固定,部分采取冰冻切片,运用免疫组织化学进行染色后观察、采集实验数据；采用前庭终器培养,并建立高、低增殖活性的前庭感觉上皮的模型,分别将它们转染Ad-math1-EGFP,一定时间后固定、免疫组织化学染色后观察、采集实验数据；本实验以莱卡荧光显微镜、SP2或者SP5的共聚焦显微镜对标本进行观察并采集实验数据。结果1)体外培养,膜迷路整体模型和前庭终器培养模型的首次建立。前者,尽量保持前庭膜迷路的完整性并将它们贴壁培养,培养液中给腺病毒载体,模拟在体时外淋巴给病毒；后者,去除前庭的膜性非感觉上皮并将剩余的感觉上皮贴壁培养,培养液中给腺病毒载体,模拟在体时内淋巴给病毒。2)膜迷路整体培养模式,培养液中给Ad-math1-EGFP载体,上皮细胞在非感觉上皮区出现极少量毛细胞样的细胞；前庭终器培养模式,培养液中给Ad-math1-EGFP载体,非感觉上皮区有大量的方格状细胞出现,并在此处出现大量的毛细胞样的细胞。3)异位新生的毛细胞样的细胞具有纤毛,多成簇出现,部分细胞共用一簇纤毛；细胞形态多样,呈现为多边形态、毛细胞样的形态及兼有二者特点的柱状。4)机械损伤的前庭终器,在损伤处的支持细胞增殖且在math1诱导下出现新生的毛细胞样的细胞；正常培养的前庭终器感觉上皮区极少数增殖细胞且在math1诱导下未见新生的毛细胞样的细胞；庆大霉素造模后前庭终器感觉上皮区几乎没有增殖细胞且在math1诱导下未见新生的毛细胞样的细胞5)Math1诱导的新生毛细胞样的细胞多来源于一些高增殖活性、方格状的细胞,其分化的过程中多不经历细胞的增殖,但分裂中的细胞有可能被转染并具备毛细胞的特点。结论1)前庭膜性迷路是阻止腺病毒载体从外淋巴进入内淋巴的屏障。2)新生鼠的前庭上皮中存在一类高增殖活性的细胞,贴壁培养时,它们可以迁延、增殖,并在math1诱导下可转分化为异位的毛细胞样细胞。3)具有高增殖潜能的前体细胞转染math1后易产生毛细胞样的细胞。4)math1诱导的毛细胞样的细胞的过程是一个不依赖于细胞增殖的转分化的过程。5)毛细胞样的细胞形态多样,并具备其行使功能的基本结构静纤毛。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764

【共引文献】