【作者】 王梦航；

【导师】 邱建华；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉头颈外科， 2010， 博士

【摘要】 目的:感音神经性耳聋作为临床上的一种常见疾病,严重影响着现代社会人们的生活,临床上应用助听器及人工耳蜗植入技术从一定程度上改善了患者的听力。但在临床应用领域具有一定的局限性。干细胞替代治疗作为一种取代受损毛细胞及相关听觉通路神经元的潜在手段,越来越受到人们的关注。目前,多个实验室致力于将干细胞或前体细胞(NSC/NPCS)移植进入耳蜗、听觉通路并且希望通过干细胞的植入取代受损的毛细胞、螺旋神经元或听觉通路其他神经元而促进听觉功能恢复。移植物进入宿主体内大多发生细胞凋亡(又称程序性的死亡),是由于免疫排斥因素、移植区微环境等原因造成。例如胚胎干细胞、成体干细胞或复合型干细胞通过圆窗或中阶分别进入外淋巴液环境或内淋巴液环境,在较短时间内出现大量干细胞坏死和凋亡,仅有少量细胞生存。了解内外淋巴液环境与移植干细胞的关系能够提供有效途径去了解和探测内耳淋巴液环境作为移植环境对于移植物的影响,并可以进一步通过调控对微环境和移植物造成影响,为进一步医学探索提供依据,因此研究内外淋巴液环境与干细胞机理有助于找到有效的移植途径。方法:1.胚胎大鼠嗅球NSC/NPCS的分离、培养、鉴定。取E12.5-14.SD大鼠胚胎的嗅球组织,体外培养嗅球神经干细胞并传代,显微镜下观察嗅球神经干细胞的生长状况,并且用Nestin等抗体对培养的嗅球神经干细胞进行鉴定。2.人工内耳淋巴液模拟环境对胚胎大鼠嗅神经干细胞影响的研究运用MTT法在内耳淋巴液模拟环境体外培养12h/24h/48h后对细胞活力进行观察;观察不同时间点流式细胞仪测定NSCs/NPCs的凋亡率、早晚期凋亡细胞比率变化;观察Caspase-3酶的活性变化;并运用光学显微镜、透射电镜对内耳淋巴液离子环境引起的NSCs/NPCs形态学的变化进行分析。3.改变内外淋巴液离子组分浓度对嗅球干细胞凋亡机制的研究用含有5mM,30mM,50mM,70mM,150mM钾离子浓度的类似内淋巴液培养基,观察干细胞在以上溶液处理24h后的变化。运用MTT法对不同钾离子浓度类似内淋巴液培养基处理24h的细胞活力进行观察;观察流式细胞仪测定的细胞凋亡率、早晚期凋亡比率变化;观察Caspase-3酶的活性变化;Hoechst3342-PI染色观察凋亡形态学变化;并运用光学显微镜、透射电镜对内耳淋巴液离子环境引起的NSCs/NPCs形态学的变化进行分析。结果:含有外淋巴液的培养基在所有时间段的细胞活力、生存率、活细胞数均好于内淋巴液组;外淋巴液组在48h时细胞凋亡率才出现明显的上升趋势,而内淋巴液组在12h时就出现凋亡率的大幅度上升;对24h内外淋巴液光镜和电镜的形态学观察发现:内淋巴液组出现的是局部细胞突发性死亡,以细胞坏死为主而外淋巴液组则是缓慢出现的凋亡细胞小体,并且周围细胞大多正常。不同浓度的钾离子造成嗅球干细胞生存凋亡状况的不同,其中150mM的细胞活力最低,坏死细胞和晚期凋亡细胞数最多且活细胞比率最少,而5mM和30mM组细胞的活力以及活细胞数是最多的。并且K+>50mM后Caspase-3随着钾离子浓度的升高而升高激活,提示细胞培养基中的钾离子浓度与Caspase-3的激活相关。其中30mM组细胞活力在各组中最好,且凋亡率低,我们考虑与文献上所报道的细胞外液中的高钾离子浓度(25mM)可以促进干细胞分化增殖,并且减少凋亡具有相关性。本实验发现干细胞的增殖和分化很大程度上被培养基中钾离子的浓度所决定,K+浓度不同对嗅球干细胞的活性影响就不同。K+浓度的变化与干细胞凋亡、坏死的发生密切相关,30mM的细胞外K+浓度可以减少凋亡细胞发生的比率,而>50mM浓度时,高钾离子不但不能增加细胞活力,减少凋亡发生,而且成为细胞凋亡坏死的相关因素之一。结论:1.从孕12.5-14.5大鼠胚胎嗅球组织中可以分离并且培养出具有干细胞增殖、分化能力且能够稳定传代的NSCs/NPCs。2.内淋巴液环境与外淋巴液环境相比对于干细胞的生存更为不利;干细胞在内耳中大量死亡、坏死并且大多数不能迁移到内耳毛细胞所在的位置可能与内耳的淋巴液环境中高钾的环境有关。3.钾离子浓度的变化可以引发干细胞凋亡和坏死,并且细胞外液中过高的钾离子浓度,不再是凋亡的抑制因素,可以成为细胞剧烈性坏死的促发因素之一。更多还原

【Abstract】 ObjectiveSensorineural hearing loss (SNHL) is one of the most common disabilities. Hearing depends largely on hair cells (HCs) and their associated spiral ganglion neurons (SGNs), and defects in these cells produce permanent deafness. In mammalian, the cochlear HCs and SGNs do not regenerate or repair naturally following severe injury. Recently, several mammalian stem cell lines have suc-cessfully via the scala tympani, scala media and the scala vestibuli compartments transplanted into the perilymph (extracellular-like fluid, high Na+ low K+) and endolymph (intracellular-like fluid, high K+ low Na+), arrived at different posi-tions of inner ear, survived and differentiated into a variety of cell types. How-ever, It was reported that the survival rate of the cell grafts was comparatively low, the majority of transplanted stem cells may appotosis or die after trans-planted into new enviroment, it is hard to find the engrafted cells integrate into the cochlea epithelium and differentiate into new hair cell via inner ear fluids, indicating that inner fluids microenviroment especially the endolymph might be an challenging factor of transplanted cells survival. Moreover, until recently, the effect of inner ear fluids especially endolymph on the engrafted cells is still un-clear. In our experiment, we observated the inner ear lymphatic simulation envi-roment how affects the fates of NSCs in vitro culture experimental study and ex-plore high K+ how affect the survival and apoptosis feature of NSCs.Methods:1.Isolation and identification of neural stem cells derived from the olfactory bulb.We obtained the neural stem cells from the olfactory bulb of E12.5-14.5 SD rats embryos, observed the survival and passage ability, and then identified of neural stem cells derived from the olfactory bulb2. This study was designed to firstly investigate the effect of artificial endo-lymph and perilymph on neural precursor cells(NPCs) from the aspect of survival and apoptosis features.We had obtained and culture NSCs from olfactory bulb at the embryonic day 12.5–14.5 and prepare artifical PL, artifical EL. Then NSCs were exposed to artifical PL and artifical EL medium 24h and observed the changes.3. This study was designed to investigate the effect of artificial endolymph with altering potassium concentration medium on neural stem cells(NSCs) from the aspect of survival and apoptosis features.5mM, 30 mM, 50 mM, 70 mM, 150 mM potassium concentration of artifi-cial endolymph like solution cultured NSCs 24h consecutively. Cell viability, apoptosis rate and morphology changes were observed.Results:1.NSCs can obtained from olfactory bulb neural stem cells of SD rats em-bryos, the cell spheres in vitro culture, survived well and were stained nestin positive. 2. The cell viability and survival rate of NSCs of artificial EL medium was lower than artificial PL solutions medium. Also, most of cells were late apoptotic and necrotic cells in artificial EL medium .3. The results indicated that under the condition of 150mM and 70 mM po-tassium concentration solutions medium, the cell viability and survival rate of NPCs was lower than 5 mM and 30 mM potassium concentration solutions. Also, most of cells were late apoptotic and necrotic cells.Conclusion:1. These indicated that those cells were stained with neural stem cells marker. They hold the self-renewal ability and could generate daughter cells through asymmetric cell divisions.2. The endolymph is a challengeing environment for engraft stem cells.3. The high potassium concentration of endolymph might play an unex-pected role in the aspect of survival and apoptosis of transplanted stem cells and neural progenitor cells into inner ear.更多还原