干细胞与RPE细胞间的线粒体转运以及对其功能影响的初步研究

发布时间：2018-07-03 15:21

本文选题：干细胞 + 隧道纳米管　；参考：《第三军医大学》2017年硕士论文

【摘要】：研究背景和目的视网膜变性类疾病包括:视网膜色素变性疾病、黄斑变性疾病、Stargardt病、视锥视杆细胞发育不良等疾病。此类疾病的共同特点在于视网膜的关键细胞(包括感光细胞、视网膜色素上皮细胞)的变性、凋亡和坏死,从而导致患者的视功能不可逆的丧失。由于变性和凋亡的感光细胞或视网膜色素上皮(retinal pigment epithelial,RPE)细胞不能够进行自我再生,该类疾病目前尚无有效的治疗方法。RPE是单层的色素上皮细胞,位于视网膜神经上皮层与脉络膜之间,其主要功能包括:构成视网膜外屏障、吞噬感光细胞的外节、黑色素能吸收可见光起遮光暗房作用、分泌多种生长因子、细胞因子和神经营养因子以及抗氧化作用等。RPE细胞在发挥其正常生理功能的同时,随之产生了大量的活性氧类物质(reactive oxygen species,ROS)。线粒体是细胞内ROS清除的关键细胞器,也是细胞内最先最易受到氧化损伤的部位。有研究显示RPE细胞线粒体的功能障碍与AMD等视网膜变性类疾病、糖尿病视网膜病变等疾病的发生、发病机制等有着密切的联系。移植健康的视网膜细胞或者健康的视网膜组织,已成为目前最具有前景的治疗策略。干细胞移植治疗视网膜变性类疾病的主要治疗机制包括:细胞替代效应(cell replacement effect)、营养支持效应(by stander effect)、移植区微环境免疫调控(immune-regulatory effect)作用等。干细胞来源的RPE细胞移植是目前治疗湿性AMD等致盲性眼病颇具前景的治疗手段。Rustom等研究人员在体外培养大鼠嗜铬细胞瘤细胞(PC12细胞)时,发现两个相邻但未直接接触的细胞之间可以通过建立一个直的细长的管道进行连接,将其命名为隧道纳米管(tunneling nanotubes,TNT)。并且发现该结构可以作为细胞间物质相互交换、信息相互交流的通道。近来有研究显示间充质干细胞能够通过线粒体交换改善受者细胞的功能。本研究的主要目的是探索干细胞及人胚胎干细胞来源的视网膜色素上皮细胞(human embryonic stem cell-derived retinal pigment epithelium,h ESC-RPE)能否与原代分离的遗传性视网膜色素变性模型的皇家外科学院大鼠(Royal college of surgeons rat,RCS rat)的RPE细胞间形成TNT结构、可否进行线粒体转运、线粒体转运方向以及线粒体转运后对RCS大鼠RPE细胞的ROS水平、细胞增殖能力和细胞凋亡水平的影响。初步研究能否通过线粒体转运机制参与干细胞治疗视网膜变性疾病。方法:第一部分:小鼠神经干细胞与RCS大鼠RPE细胞的线粒体转运及其对RPE细胞功能影响的研究1.分别用含有线粒体特异标记物Mitotracker-red及Mitotracker-green的培养液标记RCS大鼠原代分离的RPE细胞(RCS-RPE)与小鼠神经干细胞(mouse neural stem cells,m NSC)后,将其按照1:1的比例等量直接共培养24 h后,于激光扫描共聚焦显微镜下观察mNSC与RCS-RPE细胞之间TNT的形成及线粒体转运。2.将RCS-RPE细胞与已转染绿色荧光蛋白(green fluorescent protein,GFP)病毒的mNSC 1:1等量直接共培养48h,然后行流式细胞分选,分选出GFP阴性的RCS-RPE细胞。3.收集分选出的RCS-RPE细胞,与单独培养的RCS-RPE细胞,采用流式细胞术检测细胞ROS水平、细胞周期及细胞凋亡水平。第二部分:正常与损伤的人RPE细胞系(ARPE-19细胞)间线粒体转运及其对损伤ARPE-19细胞功能影响的研究1.分别用含有线粒体特异性标记物Mitotracker-red及Mitotracker-green的培养液标记正常ARPE-19细胞和用碘酸钠(NaIO3)预先处理24h的ARPE-19细胞,1:1等量直接共培养24 h后,于激光扫描共聚焦显微镜下观察TNT的形成以及线粒体转运现象。2.将200ug/ml Na IO3预处理24h的ARPE-19细胞与转染GFP病毒的ARPE-19细胞等量直接共培养24h,然后进行流式细胞分选,分选出未带GFP的ARPE-19细胞。3.收集分选出的ARPE-19细胞、200ug/ml Na IO3预处理并单独培养24h的ARPE-19细胞、200ug/ml Na IO3预处理并Transwell小室与正常ARPE-19细胞间接共培养的ARPE-19细胞,采用流式细胞术检测细胞ROS水平、细胞周期及细胞凋亡水平。第三部分:hESC-RPE细胞与ARPE-19细胞及RCS-RPE细胞间TNT的形成及线粒体转运方向的初步研究1.分别用含有线粒体特异性标记物Mitotracker-red及Mitotracker-green的培养液标记hESC-RPE与正常ARPE-19细胞,将其按照1:1比例等量直接共培养24 h后,于激光扫描共聚焦显微镜下观察TNT的形成及线粒体转运情况。2.分别用含有线粒体特异标记物Mitotracker-red及Mitotracker-green的培养液标记hESC-RPE与200ug/ml Na IO3预处理24h的ARPE-19细胞,按照1:1比例等量直接共培养24 h后,于激光扫描共聚焦显微镜下观察TNT的形成及线粒体转运现象。3.分别用含有线粒体特异标记物Mitotracker-red及Mitotracker-green的培养液标记hESC-RPE与2000ug/mlNaIO3预处理的ARPE-19细胞,1:1等量直接共培养24 h后,于激光扫描共聚焦显微镜下观察TNT的形成及线粒体转运的现象。4.分别用含有线粒体特异标记物Mitotracker-red及Mitotracker-green的培养液标记h ESC-RPE与RCS-RPE,1:1等量直接共培养24 h后,于激光扫描共聚焦显微镜下观察TNT的形成及线粒体转运的情况。结果:第一部分:小鼠神经干细胞与RCS-RPE细胞的线粒体转运及其对RPE细胞功能影响的研究1.通过激光扫描共聚焦显微镜观察,mNSC与RCS-RPE细胞之间可见TNT的形成,并且可观察到mNSC将自身线粒体转运到RCS-RPE细胞中。2.体外m NSC与RCS-RPE细胞直接共培养体系下,直接共培养组相比单独培养组RPE细胞的ROS水平有所降低、细胞增殖能力提高、凋亡水平降低,差异均具有统计学意义。说明干细胞能够通过线粒体转运的机制,改善RCS-RPE细胞的增殖、凋亡功能。第二部分:正常与损伤的人RPE细胞系(ARPE-19细胞)间线粒体转运及其对损伤ARPE-19细胞功能影响的研究1.正常ARPE-19细胞与损伤的ARPE-19细胞之间,可以借助激光扫描共聚焦显微镜观察到其间TNT的形成,线粒体从正常ARPE-19细胞向损伤的ARPE-19细胞转运。2.损伤的ARPE-19细胞与正常ARPE-19细胞直接共培养后,ROS水平较单独培养组及Transwell小室间接共培养组均明显降低;增殖能力较单独培养组有所增加,胞凋亡水平较单独培养组有所降低,差异具有统计学意义。表明正常的RPE细胞可以向损伤的RPE细胞转运线粒体,以挽救损伤RPE细胞的功能。第三部分:hESC-RPE细胞与人RPE细胞系(ARPE-19细胞)及RCS大鼠RPE细胞间TNT的形成及线粒体转运方向的初步研究1.通过激光扫描共聚焦显微镜观察到正常ARPE-19细胞与hESC-RPE细胞间可以形成TNT,并且线粒体从正常ARPE-19细胞向h ESC-RPE细胞方向转运。2.借助激光扫描共聚焦显微镜观察到200ug/ml Na IO3预处理的ARPE-19细胞与h ESC-RPE细胞间可以观察到TNT形成,线粒体从200ug/ml Na IO3预处理的ARPE-19细胞向hESC-RPE细胞方向转运。3.借助激光扫描共聚焦显微镜观察到2000ug/ml NaIO3预处理的ARPE-19细胞与h ESC-RPE细胞间可以观察到TNT形成,线粒体从2000ug/mlNaIO3预处理的ARPE-19细胞向hESC-RPE细胞方向转运。4.通过激光扫描共聚焦显微镜可以观察到h ESC-RPE细胞与RCS大鼠RPE细胞间TNT形成,线粒体从RCS大鼠RPE细胞向hESC-RPE细胞方向转运。结论:1.神经干细胞能够通过线粒体转运的机制,改善视网膜变性大鼠RPE细胞的增殖、凋亡功能。2.正常ARPE-19细胞可通过向受损ARPE-19细胞转运线粒体的机制,挽救氧化损伤的RPE细胞的ROS水平、细胞增殖、凋亡功能。3.hESC-RPE细胞可与正常及损伤的ARPE-19细胞间形成TNT结构。h ESC-RPE细胞可与视网膜变性大鼠RPE细胞之间形成TNT结构。线粒体从正常的ARPE-19细胞、损伤的ARPE-19细胞、视网膜变性大鼠RCS-RPE细胞向h ESC-RPE细胞方向转运。我们推测可能是由于线粒体从成熟程度较高的向线粒体成熟程度相对较低的细胞方向转运有关。
[Abstract]:Background and objective retina degeneration diseases include diseases such as retinitis pigmentosa, macular degeneration, Stargardt disease, cone optic dysplasia, and other diseases. The common characteristics of such diseases are the degeneration, apoptosis and necrosis of the key cells of the retina (including photoreceptors, retinoid epithelial cells), resulting in the disease. An irreversible loss of visual function. Due to degeneration and apoptosis of photoreceptor cells or retinal pigment epithelium (retinal pigment epithelial, RPE) cells are not capable of self regeneration. There is no effective treatment for this kind of disease at present,.RPE is a single layer of pigment epithelial cells, located between the retinal neuroepithelial layer and the choroid membrane, which is mainly located between the retina and the choroid. The functions include: the outer barrier of the retina, which engulf the outer segments of the photoreceptor cells, and the melanin can absorb visible light from the dark room, secrete a variety of growth factors, and.RPE cells, such as cytokine and neurotrophic factor, as well as the antioxidant activity, produce a large number of active oxygen substances (reactive). Oxygen species, ROS). Mitochondria are the key organelles of intracellular ROS scavenging and the first site of the most vulnerable oxidative damage in cells. Studies have shown that the dysfunction of mitochondria in RPE cells is closely related to the pathogenesis of retinal degeneration diseases such as AMD and other diseases such as diabetic retinopathy, and the pathogenesis of the disease. Retinal cells or healthy retinal tissues have become the most promising therapeutic strategies. The main mechanisms of treatment for retinal degeneration diseases by stem cell transplantation include cell replacement effect (cell replacement effect), nutritional support effect (by stander effect), and microenvironmental immunoregulation in transplant areas (immune-regulatory Effect) function and so on. Stem cell derived RPE cell transplantation is a promising treatment for blind eye diseases such as wet AMD,.Rustom and other researchers in the culture of pheochromocytoma cells (PC12 cells) in vitro, and found that two adjacent but not direct contact cells can be established by establishing a straight and slender pipe. It is called tunneling nanotubes (TNT). It is found that the structure can be used as a channel for intercellular exchange and exchange of information. Recently, studies have shown that mesenchymal stem cells can improve the power of the recipient cells through mitochondrial exchange. The main purpose of this study is to explore stem cells and human beings. Can the embryonic stem cell derived retinal pigment epithelial cells (human embryonic stem cell-derived retinal pigment epithelium, H ESC-RPE) form a structure between the Royal Academy of surgery (Royal College of) cells of the hereditary retinal pigment degeneration model isolated from the original generation Body transport, mitochondrial transport direction and the effect of mitochondrial transport on the level of ROS, cell proliferation and apoptosis of RPE cells in RCS rats. Preliminary study whether or not through mitochondrial transport mechanism to participate in stem cell treatment of retinal degeneration disease. Methods: the first part: mouse neural stem cells and RCS rat RPE cells The study of body transport and its effect on the function of RPE cells 1., respectively, to mark the primary isolated RPE cells (RCS-RPE) of RCS rats and the mouse neural stem cells (mouse neural stem cells, m NSC) with the culture solution containing the mitochondrial specific markers Mitotracker-red and Mitotracker-green, respectively, and then directly co culture them according to the proportion of the mouse neural stem cells and m NSC. The formation of TNT and mitochondrial transport between mNSC and RCS-RPE cells were observed by laser scanning confocal microscopy..2. was directly co cultured with RCS-RPE cells and mNSC 1:1 equal to green fluorescent protein, GFP virus, and then separated by flow cytometry. RCS-RPE cells and isolated RCS-RPE cells, using flow cytometry to detect cell ROS level, cell cycle and cell apoptosis level. Second part: mitochondrial transport between normal and injured human RPE cell line (ARPE-19 cell) and Study on the function response to damage of ARPE-19 cells, respectively, contain mitochondrial specific marker M Itotracker-red and Mitotracker-green culture medium labeled normal ARPE-19 cells and 24h ARPE-19 cells treated with sodium iodate (NaIO3). 1:1 equal quantities were directly co cultured for 24 h, and the formation of TNT and mitochondrial transport were observed under the laser scanning confocal microscope..2. 200ug/ml Na pretreated and transfected. The ARPE-19 cells of the virus were directly co cultured with 24h, then the flow cytometry was carried out to separate the ARPE-19 cells from the ARPE-19 cells without GFP, and the 200ug/ml Na IO3 pretreated and individually cultured for ARPE-19 cells of 24h. Cell, cell ROS, cell cycle and apoptosis level were detected by flow cytometry. Third part: the formation of TNT between hESC-RPE cells and ARPE-19 cells and RCS-RPE cells and the preliminary study on the direction of mitochondrial transport, 1. respectively labeled hESC-RPE with the culture solution containing mitochondrial specific markers, Mitotracker-red and Mitotracker-green, respectively The normal ARPE-19 cells were directly co cultured for 24 h according to the proportion of 1:1, and the formation of TNT and the mitochondrial transport were observed under the laser scanning confocal microscope..2. was used to mark hESC-RPE and 200ug/ml Na IO3 pretreatment 24h, respectively, with the medium containing mitochondrial specific markers Mitotracker-red and Mitotracker-green. After a direct co culture of 24 h according to the proportion of 1:1, the formation of TNT and the mitochondrial transport were observed under the laser scanning confocal microscope..3. was labeled with hESC-RPE and 2000ug/mlNaIO3 with the culture solution containing the mitochondrial specific markers Mitotracker-red and Mitotracker-green respectively. The 1:1 equal amount was directly co cultured with 24. After h, the formation of TNT and mitochondrial transport were observed under the laser scanning confocal microscope..4. was marked with H ESC-RPE and RCS-RPE with mitochondria specific markers Mitotracker-red and Mitotracker-green respectively. The 1:1 equivalent was directly co cultured with 24 h, and the formation and mitochondria of TNT were observed under the laser scanning confocal microscope. Results: Part 1: the first part: mitochondrial transport of mouse neural stem cells and RCS-RPE cells and the effect of RCS-RPE cells on the function of RPE cells. 1. the formation of TNT was observed between mNSC and RCS-RPE cells by laser scanning confocal microscopy, and mNSC could be observed to transport its own mitochondria to.2. in vitro m NSC in RCS-RPE cells. Compared with the direct co culture system of RCS-RPE cells, the ROS level of RPE cells in the direct co culture group was lower than that in the single culture group. The cell proliferation ability and the apoptosis level were reduced. The difference was statistically significant. The second part: the stem cells could improve the proliferation and apoptosis function of RCS-RPE cells through the mitochondrial transport mechanism. Mitochondrial transport between the RPE cell line (ARPE-19 cell) and the damaged human RPE cell line (ARPE-19 cell) and its effect on the function of damaged ARPE-19 cells; between normal ARPE-19 cells and injured ARPE-19 cells, the formation of TNT can be observed by laser scanning confocal microscope, and mitochondria transfer.2 from normal ARPE-19 cells to damaged ARPE-19 cells. After direct co culture of damaged ARPE-19 cells and normal ARPE-19 cells, the level of ROS was significantly lower than that in the single culture group and the indirect co culture group of the Transwell compartment, and the proliferation ability was higher than that in the single culture group, and the apoptosis level was lower than that in the single culture group. The difference had the significance of the overall planning. It showed that the normal RPE cells could be damaged to the injury. RPE cells transport mitochondria to save the function of damaged RPE cells. Third part: a preliminary study on the formation of TNT and the direction of mitochondrial transport between hESC-RPE cells and human RPE cell lines (ARPE-19 cells) and RPE cells in RCS rats. 1. by laser scanning confocal microscopy, a TNT ARPE-19 cell and hESC-RPE cells can be observed to form TNT. And mitochondria transfer from normal ARPE-19 cells to h ESC-RPE cell direction and.2. by laser scanning confocal microscope to observe that TNT formation can be observed between ARPE-19 cells pretreated by 200ug/ml Na IO3 and H ESC-RPE cells. Scanning confocal microscopy observed that TNT formation was observed between ARPE-19 cells pretreated with 2000ug/ml NaIO3 and H ESC-RPE cells. Mitochondria from ARPE-19 cells pretreated by 2000ug/mlNaIO3 were transported to hESC-RPE cells in the direction of.4. through laser scanning confocal microscopy. Mitochondria transfer from RCS rat RPE cells to hESC-RPE cells. Conclusion: 1. neural stem cells can improve the proliferation of RPE cells in retinal degeneration rats through the mechanism of mitochondrial transport. The apoptosis function.2. normal ARPE-19 cells can save the ROS of RPE cells damaged by oxidative damage by the mechanism of transporting the mitochondria to the damaged ARPE-19 cells. The level, cell proliferation and apoptosis function.3.hESC-RPE cells can form the TNT structure with the normal and damaged ARPE-19 cells. The.H ESC-RPE cells can form the TNT structure with the RPE cells of the retina denatured rats. Mitochondria from normal ARPE-19 cells, damaged ARPE-19 cells, retinal degeneration rat RCS-RPE cells turn to h ESC-RPE cells. We speculate that mitochondria may be related to the transport of mitochondria from relatively high maturity to relatively low degree of mitochondria maturation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774

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