人工内耳淋巴液模拟环境对嗅球神经干细胞体外培养的实验性研究
发布时间：2018-08-28 13:28
【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。
[Abstract]:Objective:
Sensorineural hearing loss, as a common clinical disease, seriously affects the lives of people in modern society. Hearing AIDS and cochlear implantation can improve the hearing of patients to a certain extent, but it has certain limitations in the field of clinical application. Stem cell replacement therapy as a replacement of damaged hair cells. More and more attention has been paid to the potential means of neurons involved in the auditory pathway. At present, many laboratories are devoted to transplanting stem cells or precursor cells (NSC/NPCS) into the cochlea, auditory pathway and hope to promote hearing by replacing damaged hair cells, spiral neurons or other neurons in the auditory pathway with stem cell implantation. Functional recovery. Cell apoptosis (also known as programmed death) occurs when the graft enters the host, which is caused by immune rejection and microenvironment in the transplantation area. For example, embryonic stem cells, adult stem cells or composite stem cells enter the perilymphatic environment or endolymphatic environment respectively through a round window or middle-level, and in a relatively short period of time. A large number of stem cell necrosis and apoptosis occur in the inner ear, but only a small number of cells survive. Understanding the relationship between the endolymphatic and endolymphatic environments and transplanted stem cells provides an effective way to understand and detect the effects of the lymphatic environment of the inner ear as a transplantation environment on the graft, and can further influence the microenvironment and the graft through regulation. One-step medical exploration provides the basis, so the study of the endolymphatic and endolymphatic environment and stem cell mechanism is helpful to find an effective way of transplantation.
Method:
1. isolation, culture and identification of NSC/NPCS from the olfactory bulb of embryonic rats.
The olfactory bulb neural stem cells from E12.5-14.SD rat embryos were cultured in vitro and subcultured. The growth of the neural stem cells was observed under microscope. The cultured neural stem cells were identified by Nestin and other antibodies.
2. effects of artificial inner ear lymph environment on embryonic rat olfactory neural stem cells
MTT method was used to observe the cell viability after 12 h/24 h/48 h culture in the simulated environment of inner ear lymph; the apoptosis rate of NSCs/NPCs and the ratio of apoptotic cells in early and late stages were measured by flow cytometry at different time points; the activity of Caspase-3 enzyme was observed; and the ionic environment of inner ear lymph was induced by light microscope and transmission electron microscope. The morphological changes of NSCs/NPCs were analyzed.
3. change the mechanism of apoptosis of olfactory bulb stem cells by changing the concentration of ionic components in the lymph nodes.
Cells were cultured in endolymphatic medium containing 5 mm, 30 mM, 50 mM, 70 mM, and 150 mM potassium ions for 24 hours. Cell viability was observed by MTT assay after 24 hours of treatment with different potassium ion concentration similar to that of endolymphatic medium. The changes of Caspase-3 enzyme activity and apoptosis morphology were observed by Hoechst 3342-PI staining, and the morphological changes of NSCs/NPCs induced by ionic environment of inner ear lymph were analyzed by optical microscope and transmission electron microscope.
Result:
Cell viability, survival rate and the number of living cells in the medium containing perilymphatic fluid were better than those in the endolymphatic fluid group at all time points; the apoptosis rate in the perilymphatic fluid group increased significantly at 48 h, while the apoptosis rate in the endolymphatic fluid group increased significantly at 12 h; the morphological observation of the endolymphatic and endolymphatic fluid under light and electron microscopy at 24 h It was found that sudden cell death occurred in the endolymphatic group, mainly cell necrosis, and apoptotic cell bodies appeared slowly in the perilymphatic group, and most of the peripheral cells were normal.
The survival and apoptosis of olfactory bulb stem cells were different in different concentrations of potassium ions. The cell viability of 150mM was the lowest, the number of necrotic cells and late apoptotic cells was the highest and the ratio of living cells was the lowest. The cell viability and the number of living cells were the highest in 5mM and 30mM groups. Activation suggested that the potassium concentration in the cell culture medium was related to the activation of Caspase-3. Among them, the cell viability in the 30mM group was the best and the apoptosis rate was low. We considered that the high potassium concentration in extracellular fluid (25mM) reported in the literature could promote the differentiation and proliferation of stem cells and reduce apoptosis. The proliferation and differentiation of stem cells are largely determined by the concentration of potassium ions in the culture medium. The effect of different concentration of K + on the activity of olfactory bulb stem cells is different. The change of K + concentration is closely related to the apoptosis and necrosis of stem cells. Extracellular K + concentration of 30 mM can reduce the rate of apoptotic cells. Not only can it not increase cell viability and reduce apoptosis, but also become one of the related factors of apoptosis and necrosis.
Conclusion:
1. NSCs/NPCs with the ability of stem cell proliferation, differentiation and stable passage can be isolated and cultured from the olfactory bulb of 12.5-14.5 pregnant rat embryos.
2. The endolymphatic environment is more disadvantageous to the survival of stem cells than the peripheral lymphatic environment; stem cells die in large numbers in the inner ear, necrosis and most of them can not migrate to the location of inner ear hair cells may be related to the high potassium environment in the lymphatic environment of the inner ear.
3. The change of potassium concentration can induce the apoptosis and necrosis of stem cells, and the excessive potassium concentration in extracellular fluid is no longer an inhibitor of apoptosis, can become one of the provocative factors of severe cell necrosis.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764
本文编号：2209525
[Abstract]:Objective:
Sensorineural hearing loss, as a common clinical disease, seriously affects the lives of people in modern society. Hearing AIDS and cochlear implantation can improve the hearing of patients to a certain extent, but it has certain limitations in the field of clinical application. Stem cell replacement therapy as a replacement of damaged hair cells. More and more attention has been paid to the potential means of neurons involved in the auditory pathway. At present, many laboratories are devoted to transplanting stem cells or precursor cells (NSC/NPCS) into the cochlea, auditory pathway and hope to promote hearing by replacing damaged hair cells, spiral neurons or other neurons in the auditory pathway with stem cell implantation. Functional recovery. Cell apoptosis (also known as programmed death) occurs when the graft enters the host, which is caused by immune rejection and microenvironment in the transplantation area. For example, embryonic stem cells, adult stem cells or composite stem cells enter the perilymphatic environment or endolymphatic environment respectively through a round window or middle-level, and in a relatively short period of time. A large number of stem cell necrosis and apoptosis occur in the inner ear, but only a small number of cells survive. Understanding the relationship between the endolymphatic and endolymphatic environments and transplanted stem cells provides an effective way to understand and detect the effects of the lymphatic environment of the inner ear as a transplantation environment on the graft, and can further influence the microenvironment and the graft through regulation. One-step medical exploration provides the basis, so the study of the endolymphatic and endolymphatic environment and stem cell mechanism is helpful to find an effective way of transplantation.
Method:
1. isolation, culture and identification of NSC/NPCS from the olfactory bulb of embryonic rats.
The olfactory bulb neural stem cells from E12.5-14.SD rat embryos were cultured in vitro and subcultured. The growth of the neural stem cells was observed under microscope. The cultured neural stem cells were identified by Nestin and other antibodies.
2. effects of artificial inner ear lymph environment on embryonic rat olfactory neural stem cells
MTT method was used to observe the cell viability after 12 h/24 h/48 h culture in the simulated environment of inner ear lymph; the apoptosis rate of NSCs/NPCs and the ratio of apoptotic cells in early and late stages were measured by flow cytometry at different time points; the activity of Caspase-3 enzyme was observed; and the ionic environment of inner ear lymph was induced by light microscope and transmission electron microscope. The morphological changes of NSCs/NPCs were analyzed.
3. change the mechanism of apoptosis of olfactory bulb stem cells by changing the concentration of ionic components in the lymph nodes.
Cells were cultured in endolymphatic medium containing 5 mm, 30 mM, 50 mM, 70 mM, and 150 mM potassium ions for 24 hours. Cell viability was observed by MTT assay after 24 hours of treatment with different potassium ion concentration similar to that of endolymphatic medium. The changes of Caspase-3 enzyme activity and apoptosis morphology were observed by Hoechst 3342-PI staining, and the morphological changes of NSCs/NPCs induced by ionic environment of inner ear lymph were analyzed by optical microscope and transmission electron microscope.
Result:
Cell viability, survival rate and the number of living cells in the medium containing perilymphatic fluid were better than those in the endolymphatic fluid group at all time points; the apoptosis rate in the perilymphatic fluid group increased significantly at 48 h, while the apoptosis rate in the endolymphatic fluid group increased significantly at 12 h; the morphological observation of the endolymphatic and endolymphatic fluid under light and electron microscopy at 24 h It was found that sudden cell death occurred in the endolymphatic group, mainly cell necrosis, and apoptotic cell bodies appeared slowly in the perilymphatic group, and most of the peripheral cells were normal.
The survival and apoptosis of olfactory bulb stem cells were different in different concentrations of potassium ions. The cell viability of 150mM was the lowest, the number of necrotic cells and late apoptotic cells was the highest and the ratio of living cells was the lowest. The cell viability and the number of living cells were the highest in 5mM and 30mM groups. Activation suggested that the potassium concentration in the cell culture medium was related to the activation of Caspase-3. Among them, the cell viability in the 30mM group was the best and the apoptosis rate was low. We considered that the high potassium concentration in extracellular fluid (25mM) reported in the literature could promote the differentiation and proliferation of stem cells and reduce apoptosis. The proliferation and differentiation of stem cells are largely determined by the concentration of potassium ions in the culture medium. The effect of different concentration of K + on the activity of olfactory bulb stem cells is different. The change of K + concentration is closely related to the apoptosis and necrosis of stem cells. Extracellular K + concentration of 30 mM can reduce the rate of apoptotic cells. Not only can it not increase cell viability and reduce apoptosis, but also become one of the related factors of apoptosis and necrosis.
Conclusion:
1. NSCs/NPCs with the ability of stem cell proliferation, differentiation and stable passage can be isolated and cultured from the olfactory bulb of 12.5-14.5 pregnant rat embryos.
2. The endolymphatic environment is more disadvantageous to the survival of stem cells than the peripheral lymphatic environment; stem cells die in large numbers in the inner ear, necrosis and most of them can not migrate to the location of inner ear hair cells may be related to the high potassium environment in the lymphatic environment of the inner ear.
3. The change of potassium concentration can induce the apoptosis and necrosis of stem cells, and the excessive potassium concentration in extracellular fluid is no longer an inhibitor of apoptosis, can become one of the provocative factors of severe cell necrosis.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R764
【参考文献】
相关期刊论文 前3条
1 陈福权;邱建华;王锦玲;刘顺利;米文娟;;大鼠嗅上皮神经干细胞的分离及培养[J];临床耳鼻咽喉科杂志;2006年14期
2 陈福权,王锦玲,邱建华,刘顺利,米文娟;胚胎大鼠嗅神经干细胞的培养及分化特性[J];中华神经外科疾病研究杂志;2004年04期
3 陈阳;陈福权;邱建华;刘顺利;米文娟;;毒性损伤大鼠耳蜗核移植嗅球神经前体细胞的初步观察[J];中华神经外科疾病研究杂志;2007年01期
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