光刺激对光基因化干细胞增殖和自我更新影响的研究

发布时间：2018-05-30 00:23

  本文选题：光遗传学 + 胚胎干细胞　； 参考：《第三军医大学》2014年博士论文

【摘要】：视网膜变性疾病是一类严重的致盲性眼病,我国每年近200万患者因该类疾病导致失明。以干细胞移植为代表的细胞替代疗法有望恢复变性视网膜的感光能力，具有良好的临床应用前景，目前已经进入临床试验阶段。干细胞分化来源的神经元必须通过与宿主视网膜存留的神经元建立有功能的突触连接，才能恢复视觉信息的传递。目前多种研究提供了移植入视网膜的干细胞分化来源的细胞与宿主神经元细胞建立突触连接的形态学证据，但由于技术瓶颈的限制，未能检测突触连接的功能。光遗传学技术的发展为探讨这一问题提供了平台，通过基因转染，使干细胞稳定表达光敏感阳离子通道蛋白和绿色荧光蛋白(channelrhodopsin2, ChR2; green fluorescent protein, GFP)，移植入视网膜后，GFP用于示踪干细胞的定位，470nm波长的蓝光可激活细胞ChR2引起兴奋，然后通过膜片钳技术记录周围宿主神经元的突触后电流，研究移植干细胞来源的神经元与宿主视网膜神经元的功能整合。而进行该研究的前提是构建表达ChR2的光基因化干细胞，并对光基因化干细胞生物学特征是否发生改变进行评估。但目前光刺激对光基因化神经干细胞和胚胎干细胞的增殖和自我更新是否产生影响尚缺乏明确的数据支持。 以往研究提示，干细胞的增殖及自我更新受到胞内和胞外多种因素的调控。如胚胎干细胞(mouse embryonic stem cells, mESCs)的Oct4/Sox2/Nanog核心转录因子形成调控环路能够保持mESCs的干性稳定；神经干细胞(neural stem cells, NSCs)中的p21/p27对于调控NSCs的适度增殖及保持干细胞库的稳定具有重要意义。干细胞的干性状态维持不仅仅依赖内源性调控因子的活动，多种外界因素比如受体激动剂如白介素抑制因子(leukemia inhibitory factor, LIF)、机械力及离子环境均可影响干细胞的状态。尤其是分布在干细胞膜表面的离子通道活动改变与干细胞的增殖和自我更新等过程密切相关。以往由于缺乏特异快速的刺激手段，离子通道活动改变对于干细胞干性状态的调控还需要深入的研究。光遗传学技术具有时间和空间精确性的优势，特别适用于研究离子通道功能改变对干细胞生物学特性的影响。 因此，本课题通过携带ChR2-GFP的慢病毒感染mESCs和NSCs，FACS筛选构建稳定表达ChR2-GFP的mESCs和NSCs，并进一步评价光刺激对ChR2-GFP-V6.5mESCs和ChR2-GFP-C17.2NSCs增殖和自我更新的影响，并初步探讨与之相关的分子机制。 方法： 1.携带ChR2-GFP慢病毒感染V6.5mESCs和C17.2NSCs，并通过荧光活化细胞分选系统(fluorescence activated cell sorting, FACS)筛选ChR2-GFP阳性的细胞，进行扩增； 2.通过免疫荧光染色及Real-time PCR检测ChR2-GFP-V6.5mESCs和ChR2-GFP-C17.2NSCs干细胞标记物的表达水平；通过光刺激膜片钳检测470nm波长的蓝光是否能诱导ChR2-GFP-V6.5mESCs和ChR2-GFP-C17.2NSCs产生内向的光电流； 3.通过细胞形态学观察、细胞计数实验及干细胞标记物检测确定表达ChR2-GFP对mESCs和NSCs形态及增殖的影响； 4.通过光刺激后细胞计数、流式分析细胞周期及Ki67染色半定量分析确定光刺激对细胞增殖及周期进展的影响；并通过台盼蓝染色及流式分析细胞凋亡，确定光刺激是否影响细胞活性； 5.通过Real-time PCR检测光刺激对ChR2-GFP-V6.5mESCs核心转录因子的影响，，通过AP染色确定自我更新能力的改变；并检测光刺激后不同胚层标记物表达水平的改变。 6.通过Western-blot检测光刺激后ChR2-GFP-V6.5mESCs的ERK活性改变及ChR2-GFP-C17.2NSCs中p21和p27表达水平的改变。 结果： 1．携带ChR2-GFP的慢病毒感染C17.2NSCs和V6.5mESCs，观察到ChR2-GFP的表达。通过FACS筛选出稳定表达ChR2-GFP的单细胞来源的C17.2NSCs和V6.5mESCs。 2．ChR2-GFP-C17.2NSCs和ChR2-GFP-V6.5mESCs形态及增殖无显著变化；且均表达相应干细胞的标记物，具备干细胞的特征。 3. ChR2-GFP-C17.2NSCs和ChR2-GFP-V6.5mESCs对470nm波长的蓝光刺激均有反应，ChR2激活产生内向的光电流，且电流幅值具有光强度依赖效应。 4.光刺激明显抑制ChR2-GFP-C17.2NSCs的增殖速度及阻碍细胞周期中G1/S期转换；光刺激明显抑制ChR2-GFP-V6.5mESCs的增殖速度及减少S期比例，同时显著降低维持自我更新的核心转录因子Oct4、Sox2、Nanog、Esrrb及Klf4的表达水平，减少单细胞形成AP阳性克隆的数量；光刺激不影响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细胞膜去极化，上调p21和p27的表达水平，参与抑制细胞增殖及周期进展。光刺激后引起ChR2-GFP-V6.5mESCs的胚层特异性相关基因的表达上调，尤其是外胚层相关基因Cdx2、Fgf5及Nestin的表达水平显著升高；光刺激后ERK分子的磷酸化水平显著升高。光刺激后下调ChR2-GFP-V6.5mESCs克隆内细胞之间缝隙连接蛋白Cx43表达水平。 结论： 1.成功构建光基因化的小鼠神经干细胞和胚胎干细胞，可用于干细胞移植入变性视网膜后功能整合的研究。 2.光刺激抑制光基因化的小鼠神经干细胞和胚胎干细胞的增殖、细胞周期进展及自我更新，还可引发胚胎干细胞分化的启动，提示在处理细胞过程中要做好避光处理，避免影响干细胞生物学特性。 3.光刺激改变光基因化干细胞的细胞膜电生理特性，通过影响多种胞内信号通路的活动参与调控细胞的增殖及自我更新，进一步证实干细胞的电生理特性的改变可以影响干细胞增殖及自我更新过程。
[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.
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