本文選題：光遺傳學(xué) + 胚胎干細(xì)胞　； 參考：《第三軍醫(yī)大學(xué)》2014年博士論文

【摘要】：視網(wǎng)膜變性疾病是一類嚴(yán)重的致盲性眼病,我國每年近200萬患者因該類疾病導(dǎo)致失明。以干細(xì)胞移植為代表的細(xì)胞替代療法有望恢復(fù)變性視網(wǎng)膜的感光能力，具有良好的臨床應(yīng)用前景，目前已經(jīng)進(jìn)入臨床試驗(yàn)階段。干細(xì)胞分化來源的神經(jīng)元必須通過與宿主視網(wǎng)膜存留的神經(jīng)元建立有功能的突觸連接，才能恢復(fù)視覺信息的傳遞。目前多種研究提供了移植入視網(wǎng)膜的干細(xì)胞分化來源的細(xì)胞與宿主神經(jīng)元細(xì)胞建立突觸連接的形態(tài)學(xué)證據(jù)，但由于技術(shù)瓶頸的限制，未能檢測突觸連接的功能。光遺傳學(xué)技術(shù)的發(fā)展為探討這一問題提供了平臺(tái)，通過基因轉(zhuǎn)染，使干細(xì)胞穩(wěn)定表達(dá)光敏感陽離子通道蛋白和綠色熒光蛋白(channelrhodopsin2, ChR2; green fluorescent protein, GFP)，移植入視網(wǎng)膜后，GFP用于示蹤干細(xì)胞的定位，470nm波長的藍(lán)光可激活細(xì)胞ChR2引起興奮，然后通過膜片鉗技術(shù)記錄周圍宿主神經(jīng)元的突觸后電流，研究移植干細(xì)胞來源的神經(jīng)元與宿主視網(wǎng)膜神經(jīng)元的功能整合。而進(jìn)行該研究的前提是構(gòu)建表達(dá)ChR2的光基因化干細(xì)胞，并對光基因化干細(xì)胞生物學(xué)特征是否發(fā)生改變進(jìn)行評估。但目前光刺激對光基因化神經(jīng)干細(xì)胞和胚胎干細(xì)胞的增殖和自我更新是否產(chǎn)生影響尚缺乏明確的數(shù)據(jù)支持。 以往研究提示，干細(xì)胞的增殖及自我更新受到胞內(nèi)和胞外多種因素的調(diào)控。如胚胎干細(xì)胞(mouse embryonic stem cells, mESCs)的Oct4/Sox2/Nanog核心轉(zhuǎn)錄因子形成調(diào)控環(huán)路能夠保持mESCs的干性穩(wěn)定；神經(jīng)干細(xì)胞(neural stem cells, NSCs)中的p21/p27對于調(diào)控NSCs的適度增殖及保持干細(xì)胞庫的穩(wěn)定具有重要意義。干細(xì)胞的干性狀態(tài)維持不僅僅依賴內(nèi)源性調(diào)控因子的活動(dòng)，多種外界因素比如受體激動(dòng)劑如白介素抑制因子(leukemia inhibitory factor, LIF)、機(jī)械力及離子環(huán)境均可影響干細(xì)胞的狀態(tài)。尤其是分布在干細(xì)胞膜表面的離子通道活動(dòng)改變與干細(xì)胞的增殖和自我更新等過程密切相關(guān)。以往由于缺乏特異快速的刺激手段，離子通道活動(dòng)改變對于干細(xì)胞干性狀態(tài)的調(diào)控還需要深入的研究。光遺傳學(xué)技術(shù)具有時(shí)間和空間精確性的優(yōu)勢，特別適用于研究離子通道功能改變對干細(xì)胞生物學(xué)特性的影響。 因此，本課題通過攜帶ChR2-GFP的慢病毒感染mESCs和NSCs，F(xiàn)ACS篩選構(gòu)建穩(wěn)定表達(dá)ChR2-GFP的mESCs和NSCs，并進(jìn)一步評價(jià)光刺激對ChR2-GFP-V6.5mESCs和ChR2-GFP-C17.2NSCs增殖和自我更新的影響，并初步探討與之相關(guān)的分子機(jī)制。 方法： 1.攜帶ChR2-GFP慢病毒感染V6.5mESCs和C17.2NSCs，并通過熒光活化細(xì)胞分選系統(tǒng)(fluorescence activated cell sorting, FACS)篩選ChR2-GFP陽性的細(xì)胞，進(jìn)行擴(kuò)增； 2.通過免疫熒光染色及Real-time PCR檢測ChR2-GFP-V6.5mESCs和ChR2-GFP-C17.2NSCs干細(xì)胞標(biāo)記物的表達(dá)水平；通過光刺激膜片鉗檢測470nm波長的藍(lán)光是否能誘導(dǎo)ChR2-GFP-V6.5mESCs和ChR2-GFP-C17.2NSCs產(chǎn)生內(nèi)向的光電流； 3.通過細(xì)胞形態(tài)學(xué)觀察、細(xì)胞計(jì)數(shù)實(shí)驗(yàn)及干細(xì)胞標(biāo)記物檢測確定表達(dá)ChR2-GFP對mESCs和NSCs形態(tài)及增殖的影響； 4.通過光刺激后細(xì)胞計(jì)數(shù)、流式分析細(xì)胞周期及Ki67染色半定量分析確定光刺激對細(xì)胞增殖及周期進(jìn)展的影響；并通過臺(tái)盼藍(lán)染色及流式分析細(xì)胞凋亡，確定光刺激是否影響細(xì)胞活性； 5.通過Real-time PCR檢測光刺激對ChR2-GFP-V6.5mESCs核心轉(zhuǎn)錄因子的影響，，通過AP染色確定自我更新能力的改變；并檢測光刺激后不同胚層標(biāo)記物表達(dá)水平的改變。 6.通過Western-blot檢測光刺激后ChR2-GFP-V6.5mESCs的ERK活性改變及ChR2-GFP-C17.2NSCs中p21和p27表達(dá)水平的改變。 結(jié)果： 1．?dāng)y帶ChR2-GFP的慢病毒感染C17.2NSCs和V6.5mESCs，觀察到ChR2-GFP的表達(dá)。通過FACS篩選出穩(wěn)定表達(dá)ChR2-GFP的單細(xì)胞來源的C17.2NSCs和V6.5mESCs。 2．ChR2-GFP-C17.2NSCs和ChR2-GFP-V6.5mESCs形態(tài)及增殖無顯著變化；且均表達(dá)相應(yīng)干細(xì)胞的標(biāo)記物，具備干細(xì)胞的特征。 3. ChR2-GFP-C17.2NSCs和ChR2-GFP-V6.5mESCs對470nm波長的藍(lán)光刺激均有反應(yīng)，ChR2激活產(chǎn)生內(nèi)向的光電流，且電流幅值具有光強(qiáng)度依賴效應(yīng)。 4.光刺激明顯抑制ChR2-GFP-C17.2NSCs的增殖速度及阻礙細(xì)胞周期中G1/S期轉(zhuǎn)換；光刺激明顯抑制ChR2-GFP-V6.5mESCs的增殖速度及減少S期比例，同時(shí)顯著降低維持自我更新的核心轉(zhuǎn)錄因子Oct4、Sox2、Nanog、Esrrb及Klf4的表達(dá)水平，減少單細(xì)胞形成AP陽性克隆的數(shù)量；光刺激不影響ChR2-GFP-C17.2NSCs和ChR2-GFP-V6.5mESCs的活性，也不引起凋亡。 5.光刺激后引起ChR2-GFP-C17.2NSCs細(xì)胞膜去極化，上調(diào)p21和p27的表達(dá)水平，參與抑制細(xì)胞增殖及周期進(jìn)展。光刺激后引起ChR2-GFP-V6.5mESCs的胚層特異性相關(guān)基因的表達(dá)上調(diào)，尤其是外胚層相關(guān)基因Cdx2、Fgf5及Nestin的表達(dá)水平顯著升高；光刺激后ERK分子的磷酸化水平顯著升高。光刺激后下調(diào)ChR2-GFP-V6.5mESCs克隆內(nèi)細(xì)胞之間縫隙連接蛋白Cx43表達(dá)水平。 結(jié)論： 1.成功構(gòu)建光基因化的小鼠神經(jīng)干細(xì)胞和胚胎干細(xì)胞，可用于干細(xì)胞移植入變性視網(wǎng)膜后功能整合的研究。 2.光刺激抑制光基因化的小鼠神經(jīng)干細(xì)胞和胚胎干細(xì)胞的增殖、細(xì)胞周期進(jìn)展及自我更新，還可引發(fā)胚胎干細(xì)胞分化的啟動(dòng)，提示在處理細(xì)胞過程中要做好避光處理，避免影響干細(xì)胞生物學(xué)特性。 3.光刺激改變光基因化干細(xì)胞的細(xì)胞膜電生理特性，通過影響多種胞內(nèi)信號(hào)通路的活動(dòng)參與調(diào)控細(xì)胞的增殖及自我更新，進(jìn)一步證實(shí)干細(xì)胞的電生理特性的改變可以影響干細(xì)胞增殖及自我更新過程。
[Abstract]:Retinal degeneration is a serious type of blinding disease. Nearly 2 million of the patients in China are blinded by this disease every year. Cell replacement therapy, represented by stem cell transplantation, is expected to restore the photoreceptor ability of the denatured retina. It has a good clinical application prospect. It is now in the clinical trial stage. The source of stem cell differentiation Through the establishment of functional synaptic connections with the neurons of the host retina, the transmission of visual information can be restored. Many studies have provided morphological evidence for the establishment of synaptic connections between the cells of the stem cell differentiation of the transplanted retina and the cells of the host neurons. The development of optical genetics technology provides a platform for exploring this problem. Through gene transfection, the stem cells are stabilized to express photosensitive cation channel protein and green fluorescent protein (channelrhodopsin2, ChR2; green fluorescent protein, GFP). After transplantation into the retina, GFP is used to trace the location of stem cells, 47 The 0nm wavelength blue light activates the excitation of the cell ChR2, and then the postsynaptic current of the peripheral host neurons is recorded by the patch clamp technique, and the function integration of the cells derived from the transplanted stem cells and the host retina neurons is studied. The premise of this study is to construct the ChR2 - based photo gene stem cells and to gene the photo gene. However, there is no clear data support for the effect of light stimulation on the proliferation and self renewal of neurogene neural stem cells and embryonic stem cells.
Previous studies have suggested that the proliferation and self renewal of stem cells are regulated by a variety of factors within and outside the cell. For example, the regulatory loop of the Oct4/Sox2/Nanog core transcription factor formation of the mouse embryonic stem cells (mESCs) can maintain the dry stability of the mESCs; the p21/p27 in the neural stem cells (neural stem cells, NSCs) is modulated. It is of great significance to control the moderate proliferation of NSCs and to maintain the stability of the stem cell bank. The dry state of stem cells is not only dependent on the activity of endogenous regulators, but a variety of external factors such as receptor agonists such as leukemia inhibitory factor (LIF), mechanical force and ionic environment can affect the state of stem cells. Especially, the changes of ion channel activity on the surface of stem cells are closely related to the process of stem cell proliferation and self renewal. In the past, the regulation of the dry state of stem cells is still necessary for the regulation of the dry state of stem cells due to the lack of specific and rapid stimulus. The advantage of sex is particularly suitable for studying the effects of ion channel function changes on the biological characteristics of stem cells.
Therefore, we screened the mESCs and NSCs for stable expression of ChR2-GFP by mESCs and NSCs with ChR2-GFP and FACS, and further evaluated the effect of light stimulation on the proliferation and self renewal of ChR2-GFP-V6.5mESCs and ChR2-GFP-C17.2NSCs, and preliminarily discussed the related molecular mechanisms.
Method錛

本文編號(hào)：1953091