人工內(nèi)耳淋巴液模擬環(huán)境對嗅球神經(jīng)干細胞體外培養(yǎng)的實驗性研究
發(fā)布時間：2018-08-28 13:28
【摘要】：目的: 感音神經(jīng)性耳聾作為臨床上的一種常見疾病,嚴重影響著現(xiàn)代社會人們的生活,臨床上應用助聽器及人工耳蝸植入技術(shù)從一定程度上改善了患者的聽力。但在臨床應用領(lǐng)域具有一定的局限性。干細胞替代治療作為一種取代受損毛細胞及相關(guān)聽覺通路神經(jīng)元的潛在手段,越來越受到人們的關(guān)注。目前,多個實驗室致力于將干細胞或前體細胞(NSC/NPCS)移植進入耳蝸、聽覺通路并且希望通過干細胞的植入取代受損的毛細胞、螺旋神經(jīng)元或聽覺通路其他神經(jīng)元而促進聽覺功能恢復。移植物進入宿主體內(nèi)大多發(fā)生細胞凋亡(又稱程序性的死亡),是由于免疫排斥因素、移植區(qū)微環(huán)境等原因造成。例如胚胎干細胞、成體干細胞或復合型干細胞通過圓窗或中階分別進入外淋巴液環(huán)境或內(nèi)淋巴液環(huán)境,在較短時間內(nèi)出現(xiàn)大量干細胞壞死和凋亡,僅有少量細胞生存。了解內(nèi)外淋巴液環(huán)境與移植干細胞的關(guān)系能夠提供有效途徑去了解和探測內(nèi)耳淋巴液環(huán)境作為移植環(huán)境對于移植物的影響,并可以進一步通過調(diào)控對微環(huán)境和移植物造成影響,為進一步醫(yī)學探索提供依據(jù),因此研究內(nèi)外淋巴液環(huán)境與干細胞機理有助于找到有效的移植途徑。 方法: 1.胚胎大鼠嗅球NSC/NPCS的分離、培養(yǎng)、鑒定。 取E12.5-14.SD大鼠胚胎的嗅球組織,體外培養(yǎng)嗅球神經(jīng)干細胞并傳代,顯微鏡下觀察嗅球神經(jīng)干細胞的生長狀況,并且用Nestin等抗體對培養(yǎng)的嗅球神經(jīng)干細胞進行鑒定。 2.人工內(nèi)耳淋巴液模擬環(huán)境對胚胎大鼠嗅神經(jīng)干細胞影響的研究 運用MTT法在內(nèi)耳淋巴液模擬環(huán)境體外培養(yǎng)12h/24h/48h后對細胞活力進行觀察;觀察不同時間點流式細胞儀測定NSCs/NPCs的凋亡率、早晚期凋亡細胞比率變化;觀察Caspase-3酶的活性變化;并運用光學顯微鏡、透射電鏡對內(nèi)耳淋巴液離子環(huán)境引起的NSCs/NPCs形態(tài)學的變化進行分析。 3.改變內(nèi)外淋巴液離子組分濃度對嗅球干細胞凋亡機制的研究 用含有5mM,30mM,50mM,70mM,150mM鉀離子濃度的類似內(nèi)淋巴液培養(yǎng)基,觀察干細胞在以上溶液處理24h后的變化。運用MTT法對不同鉀離子濃度類似內(nèi)淋巴液培養(yǎng)基處理24h的細胞活力進行觀察;觀察流式細胞儀測定的細胞凋亡率、早晚期凋亡比率變化;觀察Caspase-3酶的活性變化;Hoechst3342-PI染色觀察凋亡形態(tài)學變化;并運用光學顯微鏡、透射電鏡對內(nèi)耳淋巴液離子環(huán)境引起的NSCs/NPCs形態(tài)學的變化進行分析。 結(jié)果: 含有外淋巴液的培養(yǎng)基在所有時間段的細胞活力、生存率、活細胞數(shù)均好于內(nèi)淋巴液組;外淋巴液組在48h時細胞凋亡率才出現(xiàn)明顯的上升趨勢,而內(nèi)淋巴液組在12h時就出現(xiàn)凋亡率的大幅度上升;對24h內(nèi)外淋巴液光鏡和電鏡的形態(tài)學觀察發(fā)現(xiàn):內(nèi)淋巴液組出現(xiàn)的是局部細胞突發(fā)性死亡,以細胞壞死為主而外淋巴液組則是緩慢出現(xiàn)的凋亡細胞小體,并且周圍細胞大多正常。 不同濃度的鉀離子造成嗅球干細胞生存凋亡狀況的不同,其中150mM的細胞活力最低,壞死細胞和晚期凋亡細胞數(shù)最多且活細胞比率最少,而5mM和30mM組細胞的活力以及活細胞數(shù)是最多的。并且K+50mM后Caspase-3隨著鉀離子濃度的升高而升高激活,提示細胞培養(yǎng)基中的鉀離子濃度與Caspase-3的激活相關(guān)。其中30mM組細胞活力在各組中最好,且凋亡率低,我們考慮與文獻上所報道的細胞外液中的高鉀離子濃度(25mM)可以促進干細胞分化增殖,并且減少凋亡具有相關(guān)性。本實驗發(fā)現(xiàn)干細胞的增殖和分化很大程度上被培養(yǎng)基中鉀離子的濃度所決定,K+濃度不同對嗅球干細胞的活性影響就不同。K+濃度的變化與干細胞凋亡、壞死的發(fā)生密切相關(guān),30mM的細胞外K+濃度可以減少凋亡細胞發(fā)生的比率,而50mM濃度時,高鉀離子不但不能增加細胞活力,減少凋亡發(fā)生,而且成為細胞凋亡壞死的相關(guān)因素之一。 結(jié)論: 1.從孕12.5-14.5大鼠胚胎嗅球組織中可以分離并且培養(yǎng)出具有干細胞增殖、分化能力且能夠穩(wěn)定傳代的NSCs/NPCs。 2.內(nèi)淋巴液環(huán)境與外淋巴液環(huán)境相比對于干細胞的生存更為不利;干細胞在內(nèi)耳中大量死亡、壞死并且大多數(shù)不能遷移到內(nèi)耳毛細胞所在的位置可能與內(nèi)耳的淋巴液環(huán)境中高鉀的環(huán)境有關(guān)。 3.鉀離子濃度的變化可以引發(fā)干細胞凋亡和壞死,并且細胞外液中過高的鉀離子濃度,不再是凋亡的抑制因素,可以成為細胞劇烈性壞死的促發(fā)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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R764
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R764
【參考文獻】
相關(guān)期刊論文 前3條
1 陳福權(quán);邱建華;王錦玲;劉順利;米文娟;;大鼠嗅上皮神經(jīng)干細胞的分離及培養(yǎng)[J];臨床耳鼻咽喉科雜志;2006年14期
2 陳福權(quán),王錦玲,邱建華,劉順利,米文娟;胚胎大鼠嗅神經(jīng)干細胞的培養(yǎng)及分化特性[J];中華神經(jīng)外科疾病研究雜志;2004年04期
3 陳陽;陳福權(quán);邱建華;劉順利;米文娟;;毒性損傷大鼠耳蝸核移植嗅球神經(jīng)前體細胞的初步觀察[J];中華神經(jīng)外科疾病研究雜志;2007年01期
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