视网膜祖细胞移植治疗视网膜变性性疾病的相关研究
发布时间：2018-08-29 12:44
【摘要】：一系列累及视网膜的疾病都会造成视网膜感光细胞和其他视网膜神经元的丢失,这些疾病包括遗传性疾病,比如视网膜色素变性；还包括许多发病率更高的疾病,比如黄斑变性、视网膜脱离、青光眼、糖尿病视网膜病变等等。由于视网膜同颅内的脑神经组织一样,缺乏有效的自我修复机能,所以以上这些疾病通常都会造成患者视力不可逆性的丧失。临床上对这些疾病造成的视力损伤多是采用支持保护性治疗,真正能挽救和恢复的视力有限。这些疾病造成视力损伤的根本原因是视网膜功能细胞的死亡、减少,所以对这类细胞的替换或再生治疗是有希望治愈疾病并恢复视力的有效手段。 近年的研究热点之一是,通过手术注射的方式将视网膜祖细胞或前体细胞移植入病变的视网膜下。有对动物疾病模型的研究表明,注入受体视网膜下的细胞可以向受体视网膜移行,并能够表达某些成熟视网膜细胞的蛋白标志,且能够检测到视网膜功能的改善。所以细胞移植治疗视网膜变性性疾病是一种可行的、有希望的治疗手段。如何通过体外培养的方式,扩增得到大量的可供于研究及移植治疗的细胞,是研究者需要解决的一个问题。而作为细胞移植受体的疾病动物模型,对于我们深入理解这类疾病有着重要意义；观察并了解动物模型疾病的发生、发展过程及其中形态和功能的变化,是十分必要的。这样,我们不仅对疾病过程中各方面的改变建立了一个基线标准,同时也使得对后续的干预治疗的效果进行观察和评估成为可能。 本研究将通过三部分,来介绍人类视网膜祖细胞(human retinal progenitor cell, hRPC)移植入视网膜变性小鼠的相关研究。第一部分：人类视网膜祖细胞的体外培养；第二部分：对于rhodopsin-/-小鼠视网膜变性过程的动态连续观察；第三部分：rhodopsin-/-小鼠视网膜下hRPC移植后的视网膜形态和功能变化的动态观察。 第一部分：人类视网膜祖细胞的低氧培养 目的：比较人类视网膜祖细胞(hRPC)在常氧浓度培养(20%氧浓度)和低氧浓度培养(3%氧浓度)下各方面性状的差异,如细胞的自我更新、增殖及细胞向视网膜细胞分化的能力等。初步研究相关的细胞因子及信号通路,了解相关现象发生的机制。 方法：从人类胎儿视网膜中分离得到视网膜祖细胞,进行体外培养。培养条件分为常氧培养(20%氧浓度)和低氧培养(3%氧浓度),观察细胞在两种培养条件下生物学特性的差异,并在特定时间点收集各组细胞进行相关细胞因子的蛋白表达水平检测(蛋白免疫印迹实验、免疫细胞化学检测)和基因mRNA水平检测(聚合酶链式反应),通过这些检测的结果推断各细胞因子(低氧诱导因子)在低氧培养中发挥的作用。 结果：hRPC在低氧培养(3%氧浓度)条件下细胞增殖曲线及MTT实验均提示较好的细胞增殖水平,实时荧光定量PCR和细胞免疫化学染色也分别从相关基因mRNA水平及蛋白表达水平方而说明了细胞增殖水平的增强,如Ki67、CyclinD1。而且低氧培养条件下的hRPC维持了向视网膜细胞分化的能力,Klf4、c-Myc在基因和蛋白水平表达均高于常氧培养条件。低氧诱导因子la(Hypoxia inducible factor-1a, HIF-1a)在低氧培养的细胞中表达高于常氧培养,且表达水平随时间而变化。 结论：本研究证明低氧浓度培养环境有利于人类视网膜祖细胞的体外扩增,并能够维持细胞向视网膜细胞分化的能力。低氧诱导因子1a可能在其中发挥关键作用。低氧培养模式可以为视网膜变性性疾病的研究和今后可能的细胞移植治疗提供大量未分化状态的人类视网膜祖细胞。 第二部分：Rhodopsin-/-鼠视网膜变性过程的在体动态观察 目的：视网膜色素变性(RP)和老年性黄斑变性(AMD)是两类主要的造成不可逆性失明的视网膜变性性疾病。啮齿类动物模型是我们理解这类疾病的重要工具。之前的研究已经表明视网膜紫质是视网膜光传导过程中必不可少的要素,同时也是视杆细胞外节重要的结构蛋白。Rhodopsin-/-鼠不表达视网膜紫质,不形成视杆细胞外节,在出生后短短的几个月内视杆细胞便会消失,继之是视锥细胞功能和数量的丧失。本部分研究采用频域光学相干断层扫描(Spectral Domain Optical Coherence Tomography, SD-OCT)动态连续性观察rhodopsin-/鼠视网膜形态方面的变化。 方法：选择出生后第3、6、9和12周的rhodopsin-/鼠(C57B16rhodopsin基因敲除)和野生型C57B16鼠用于本研究。SD-OCT采用放射状体积扫描模式(以视盘为中心,直径1.3mm的扫描范围)。每个体积扫描包含100个B扫描(每个B扫描包含1000个A扫描)。选取特定的扫描进行视网膜外核层(outer nuclear layer,ONL)厚度的测量。在相应的时间点进行视网膜组织学检查,并将得到的数据和SD-OCT所获得的数据进行比较。记录暗适应和明适应条件下的视网膜电图(Electroretinograms, ERG),对视网膜功能学变化和形态学变化进行相关性分析。 结果：SD-OCT测量数据显示rhodopsin-/-鼠视网膜外核层厚度在其出生后第3周至第12周逐渐变薄。第3、6、9和12周的厚度值分别为40.6±1.61μm,27.9±1.65μm,14.5±0.7μm和6.0±0.78μm。在rhodopsin-/鼠视网膜无法观察到视杆细胞外节。视网膜组织学检测数据显示出同OCT检测相同的趋势。在第3周时,rhodopsin-/鼠视网膜外核层有9-10层细胞核,而C57B16鼠有11-12层细胞核；到第12周时,rhodopsin-/-鼠外核层细胞核层数已经减低到1-2层,而野生型小鼠细胞核层数稳定在11-12层。Rhodopsin-/鼠的明适应和暗适应条件下的ERG均不能见到明显a波,而b波波幅也随年龄增长而降低,同形态学观察到的结果一致。而野生型C57B16鼠在各时间点形态学和功能学的测量值基本稳定。 结论：通过SD-OCT测量得到的数据确认了rhodopsin-/鼠外核层厚度在其出生后逐渐变薄,而在野生型小鼠中则没有发现变化。并且视网膜组织学检测和ERG功能学的定量检测都证实了OCT所得到的结果。因此,SD-OCT可以作为一种无创性的、有效并且可靠的研究工具,用于动态观察视网膜变性性疾病动物模型的疾病变化过程。 第三部分：Rhodopsin-/-鼠视网膜下视网膜祖细胞的移植 目的：将体外培养扩增的人视网膜祖细胞通过手术方式注入药物(环孢素)免疫抑制的rhodopsin-/-鼠视网膜下,观察细胞移植后的存活、移行、和受体视网膜结合及分化等情况,评估移植后视网膜变性小鼠的视网膜形态(组织学检测和SD-OCT检测)和功能(ERG)的相关变化。 方法：移植细胞为体外低氧浓度(3%)培养的hRPC(细胞代数为第7至第9代)。移植受体鼠rhodopsin-/-鼠经过环孢素免疫抑制。通过手术方式,将hRPC(对照组注入PBS)注射入小鼠视网膜下。于术后第3天和第3周对术眼进行OCT观察,并于第3周OCT观察结束后进行ERG检测,随后处死小鼠进行视网膜组织学检测。 结果：在移植之后的第3天和第3周分别进行OCT观察,均可在移植成功的小鼠视网膜下见到移植细胞存在,并能够通过随后的组织学检查证实。对组织学切片进行相关免疫染色后可以观察到rhodopsin-/鼠视网膜下存活的移植细胞,但是在移植后第3周也几乎观察不到hRPC向受体鼠视网膜的移行和整合。在对移植细胞小鼠和对照小鼠(仅在视网膜下注入PBS)的视网膜功能学检查(ERG)中未见明显差异。 结论：通过环孢素对细胞移植受体鼠进行免疫抑制,可以提高移植细胞的存活率。移植后细胞并未向受体视网膜移行和整合,视网膜功能检测未见改善。 一、主要研究结果 1.人类视网膜祖细胞在低氧浓度培养条件下表现出较好的增殖趋势,增殖相关因子Ki67、Cyclin D1在基因和蛋白水平的表达也高于常氧培养；而低氧条件下干细胞特性相关因子c-Myc、Klf4也同样有较高的基因和蛋白表达水平。低氧诱导因子1a在低氧培养的细胞中有表达,而在常氧培养细胞中仅有低量表达。 2. SD-OCT测量数据显示rhodopsin-/-鼠视网膜外核层厚度在其出生后第3周至第12周逐渐变薄。视网膜组织学检测也显示出相同的趋势。Rhodopsin-/-鼠的ERG不能见到明显a波,而b波波幅也随年龄增长而降低,同形态学观察到的结果一致。而野生型C57B16鼠在各时间点形态学和功能学的测量值基本稳定。 3. SD-OCT可在移植成功的小鼠视网膜下见到移植细胞存在,并能够通过随后的组织学检查证实。组织学切片可以观察到rhodopsin-/鼠视网膜下存活的移植细胞,但几乎观察不到细胞向受体鼠视网膜的移行和整合。在对移植细胞小鼠和对照小鼠(视网膜下注入PBS)的视网膜功能学检查(ERG)中未见明显差异。 二、研究结论 1.低氧浓度培养条件有利于人类视网膜祖细胞的体外扩增,并能够维持细胞的分化能力。低氧培养条件下,细胞所表现出来的性状可能受到低氧诱导因子1a的调控。低氧浓度培养可作为人类视网膜祖细胞常规的培养方式。 2.通过SD-OCT测量得到的数据确认了rhodopsin-/鼠外核层厚度在其出生后逐渐变薄。并且视网膜组织学检测和ERG功能学的定量检测都证实了OCT所得到的结果。SD-OCT可以作为一种无创性的、有效并且可靠的研究工具,用于动态观察视网膜变性性疾病动物模型的疾病变化过程。 3.通过环孢素对rhodopsin-/鼠进行免疫抑制,可以提高移植细胞的存活,但移植后细胞在观察时间内并未向受体视网膜移行和整合。细胞移植后未见视网膜功能的改善。
[Abstract]:A series of diseases involving the retina can cause the loss of photoreceptors and other retinal neurons. These diseases include hereditary diseases, such as retinitis pigmentosa, and many more common diseases, such as macular degeneration, retinal detachment, glaucoma, diabetic retinopathy and so on. These diseases usually result in irreversible loss of vision. Clinically, most of the visual impairment caused by these diseases is caused by supportive protective treatment, which can really save and restore limited vision. This is due to the death and decrease of retinal functional cells, so replacement or regeneration of these cells is an effective means of hopefully curing the disease and restoring vision.
In recent years, one of the research hotspots is the transplantation of retinal progenitor cells or precursor cells into the diseased retina by surgical injection. Studies on animal disease models have shown that cells injected into the recipient retina can migrate to the recipient retina, and can express some protein markers of mature retinal cells, and can be detected. So cell transplantation is a feasible and promising treatment for retinal degenerative diseases. How to amplify a large number of cells for research and transplantation through in vitro culture is a problem that researchers need to solve. Animal models are of great importance to our understanding of these diseases; it is necessary to observe and understand the occurrence, development and morphological and functional changes of animal models. Thus, we not only establish a baseline standard for changes in various aspects of the disease process, but also make follow-up interventions and treatments possible. It is possible to observe and evaluate the effect.
In this study, three parts will be introduced to study the transplantation of human retinal progenitor cell (hRPC) into mice with retinal degeneration. The dynamic changes of retinal morphology and function after subretinal hRPC transplantation in rhodopsin-/- mice.
Part one: hypoxic culture of human retinal progenitor cells
AIM: To compare the characteristics of human retinal progenitor cells (hRPC) cultured in normoxia (20% oxygen concentration) and hypoxia (3% oxygen concentration), such as cell self-renewal, proliferation and differentiation to retinal cells. System.
METHODS: Retinal progenitor cells were isolated from human fetal retina and cultured in vitro. The culture conditions were divided into normoxic culture (20% oxygen concentration) and hypoxic culture (3% oxygen concentration). The differences of biological characteristics between the two cultures were observed and the expression of related cytokines in the cells was collected at a specific time point. Level detection (protein immunoblotting, immunocytochemical detection) and gene mRNA level detection (polymerase chain reaction) were used to infer the role of cytokines (hypoxia inducible factors) in hypoxia culture.
Results: The proliferation curve and MTT assay of hRPC in hypoxic culture (3% oxygen concentration) showed a good level of cell proliferation. Real-time fluorescence quantitative PCR and immunocytochemical staining also showed the enhancement of cell proliferation from the mRNA level and protein expression level of related genes, such as Ki67 and Cyclin D1. Hypoxia inducible factor-1a (HIF-1a) expression was higher in hypoxia-cultured cells than in normoxia-cultured cells, and the expression level of Klf4 and c-Myc changed with time.
CONCLUSION: This study demonstrates that hypoxia culture environment is conducive to the expansion of human retinal progenitor cells in vitro and can maintain the ability of cells to differentiate into retinal cells. Treatment provides a large number of undifferentiated human retinal progenitor cells.
The second part: in vivo dynamic observation of retinal degeneration in Rhodopsin-/- rats.
OBJECTIVE: Retinal pigment degeneration (RP) and age-related macular degeneration (AMD) are two major types of irreversible blindness-causing retinal degenerative diseases. Rodent animal models are an important tool for understanding these diseases. Previous studies have shown that retinal purple is an essential element in retinal light transmission, and at the same time Rhodopsin - / - mice do not express retinal purple, do not form rod extracellular segments, rod cells disappear within a few months after birth, followed by loss of cone cell function and number. Ence Tomography (SD-OCT) was used to observe the morphological changes of retina in rhodopsin-/ rats.
METHODS: Rhodopsin - / mouse (C57B16 rhodopsin knockout) and wild-type C57B16 mice at 3, 6, 9 and 12 weeks after birth were selected for this study. SD-OCT was performed in a radiovolume scan mode (with an optic disc as the center and a scan range of 1.3 mm in diameter). Each volume scan consisted of 100 B scans (each B scan consisted of 1,000 A scans). The thickness of the outer nuclear layer (ONL) was measured by scanning. Retinal histology was performed at the corresponding time points, and the data were compared with those obtained by SD-OCT. Electroretinograms (ERG) were recorded under dark and light adaptation conditions, and the changes of retinal function and shape were observed. Correlation analysis of state changes.
Results: SD-OCT measurements showed that the thickness of rhodopsin-/-rat retinal outer nuclear layer gradually thinned from the 3rd week to the 12th week after birth. The thickness values at the 3rd, 6th, 9th and 12th weeks were 40.6 (+ 1.61), 27.9 (+ 1.65), 14.5 (+ 0.7) and 6.0 (+ 0.78) microns, respectively. At week 3, there were 9-10 layers of nuclei in the rhodopsin-/ mouse extraretinal nucleus layer, and 11-12 layers in the C57B16 mouse. By week 12, the number of nuclei in the rhodopsin-/-mouse outer nucleus layer had been reduced to 1-2 layers, while the number of nuclei in the wild type mice was stable at 11-12 layers. The ERG under dark adaptation could not see a wave, but the amplitude of B wave decreased with age, which was consistent with the morphological observation. The morphological and functional measurements of wild type C57B16 mice were basically stable at all time points.
CONCLUSIONS: Data from SD-OCT measurements confirm that rhodopsin-/mouse outer nuclear layer thickness gradually thinned after birth, but no change was found in wild-type mice. The results of OCT were confirmed by histological examination and quantitative detection of ERG function. Therefore, SD-OCT can be used as a noninvasive and effective method. And a reliable research tool can be used to dynamically observe the changes of retinal degenerative diseases in animal models.
The third part: transplantation of retinal progenitor cells from Rhodopsin-/- mice.
OBJECTIVE: To observe the survival, migration, and retinal binding and differentiation of human retinal progenitor cells (RPCs) after transplantation into rhodopsin - / - mice subretina, which is immunosuppressed by cyclosporine, and to evaluate the retinal morphology (histological examination and SD-OCT) of retinal degeneration mice after transplantation. Correlation between detection and function (ERG).
METHODS: The transplanted cells were cultured in vitro with hypoxia (3%) for 7 to 9 generations. The recipient rhodopsin - / - mice were subjected to cyclosporine immunosuppression. The hRPC (control group injected PBS) was injected into the retina of the mice by operation. OCT was observed on the 3rd day and 3rd week after operation, and OCT was observed on the 3rd week after operation. After ERG, the mice were sacrificed for histological examination of the retina.
RESULTS: OCT observation on the 3rd day and the 3rd week after transplantation showed that transplanted cells were present in the subretinal region of the transplanted mice and could be confirmed by subsequent histological examination. The migration and integration of hRPC into the retina of recipient mice were almost not observed at the 3rd week after transplantation. There was no significant difference in the retinal function test (ERG) between transplanted cell mice and control mice (only PBS was injected into the retina).
CONCLUSION: Cyclosporin can improve the survival rate of transplanted cells by immunosuppression in recipient mice. The transplanted cells did not migrate and integrate to the recipient retina, and the retinal function was not improved.
I. main findings
1. Human retinal progenitor cells showed a good proliferation trend under hypoxic conditions, and the expression of proliferation-related factors Ki67 and Cyclin D1 were also higher than those in normoxic conditions. The expression levels of stem cell characteristic-related factors c-Myc and Klf4 were also higher under hypoxic conditions. A was expressed in hypoxic culture cells but only in low oxygen cultured cells.
2. SD-OCT measurements showed that the thickness of the outer retinal nucleus of rhodopsin-/-mice gradually thinned from the 3rd week to the 12th week after birth. Retinal histological examination also showed the same trend. ERG of Rhodopsin-/-mice could not see obvious a wave, but the amplitude of B wave decreased with age, which was consistent with morphological observation. The morphological and functional measurements of 57B16 mice were stable at various time points.
3. SD-OCT can see the existence of transplanted cells under the retina of transplanted mice, and can be confirmed by subsequent histological examination. Histological section can observe the survival of transplanted cells under the retina of rhodopsin - / mice, but hardly observe the migration and integration of cells into the retina of recipient mice. There was no significant difference in retinal functional examination (ERG) between mice (subretinal injection of PBS).
Two, research conclusion
1. Hypoxic culture conditions are conducive to the expansion of human retinal progenitor cells in vitro, and can maintain the ability of cell differentiation.
2. Data from SD-OCT measurements confirmed that rhodopsin-/mouse outer nuclear layer thickness gradually thinned after birth. The results of OCT were confirmed by both histological and quantitative detection of ERG function. SD-OCT can be used as a noninvasive, effective and reliable research tool for dynamic observation of retinal degeneration. The changing course of disease in animal models of sexual diseases.
3. Immunosuppression of rhodopsin-/mouse by cyclosporine can improve the survival of transplanted cells, but the transplanted cells did not migrate and integrate to the recipient retina during the observation period. There was no improvement in retinal function after transplantation.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1
本文编号：2211246
[Abstract]:A series of diseases involving the retina can cause the loss of photoreceptors and other retinal neurons. These diseases include hereditary diseases, such as retinitis pigmentosa, and many more common diseases, such as macular degeneration, retinal detachment, glaucoma, diabetic retinopathy and so on. These diseases usually result in irreversible loss of vision. Clinically, most of the visual impairment caused by these diseases is caused by supportive protective treatment, which can really save and restore limited vision. This is due to the death and decrease of retinal functional cells, so replacement or regeneration of these cells is an effective means of hopefully curing the disease and restoring vision.
In recent years, one of the research hotspots is the transplantation of retinal progenitor cells or precursor cells into the diseased retina by surgical injection. Studies on animal disease models have shown that cells injected into the recipient retina can migrate to the recipient retina, and can express some protein markers of mature retinal cells, and can be detected. So cell transplantation is a feasible and promising treatment for retinal degenerative diseases. How to amplify a large number of cells for research and transplantation through in vitro culture is a problem that researchers need to solve. Animal models are of great importance to our understanding of these diseases; it is necessary to observe and understand the occurrence, development and morphological and functional changes of animal models. Thus, we not only establish a baseline standard for changes in various aspects of the disease process, but also make follow-up interventions and treatments possible. It is possible to observe and evaluate the effect.
In this study, three parts will be introduced to study the transplantation of human retinal progenitor cell (hRPC) into mice with retinal degeneration. The dynamic changes of retinal morphology and function after subretinal hRPC transplantation in rhodopsin-/- mice.
Part one: hypoxic culture of human retinal progenitor cells
AIM: To compare the characteristics of human retinal progenitor cells (hRPC) cultured in normoxia (20% oxygen concentration) and hypoxia (3% oxygen concentration), such as cell self-renewal, proliferation and differentiation to retinal cells. System.
METHODS: Retinal progenitor cells were isolated from human fetal retina and cultured in vitro. The culture conditions were divided into normoxic culture (20% oxygen concentration) and hypoxic culture (3% oxygen concentration). The differences of biological characteristics between the two cultures were observed and the expression of related cytokines in the cells was collected at a specific time point. Level detection (protein immunoblotting, immunocytochemical detection) and gene mRNA level detection (polymerase chain reaction) were used to infer the role of cytokines (hypoxia inducible factors) in hypoxia culture.
Results: The proliferation curve and MTT assay of hRPC in hypoxic culture (3% oxygen concentration) showed a good level of cell proliferation. Real-time fluorescence quantitative PCR and immunocytochemical staining also showed the enhancement of cell proliferation from the mRNA level and protein expression level of related genes, such as Ki67 and Cyclin D1. Hypoxia inducible factor-1a (HIF-1a) expression was higher in hypoxia-cultured cells than in normoxia-cultured cells, and the expression level of Klf4 and c-Myc changed with time.
CONCLUSION: This study demonstrates that hypoxia culture environment is conducive to the expansion of human retinal progenitor cells in vitro and can maintain the ability of cells to differentiate into retinal cells. Treatment provides a large number of undifferentiated human retinal progenitor cells.
The second part: in vivo dynamic observation of retinal degeneration in Rhodopsin-/- rats.
OBJECTIVE: Retinal pigment degeneration (RP) and age-related macular degeneration (AMD) are two major types of irreversible blindness-causing retinal degenerative diseases. Rodent animal models are an important tool for understanding these diseases. Previous studies have shown that retinal purple is an essential element in retinal light transmission, and at the same time Rhodopsin - / - mice do not express retinal purple, do not form rod extracellular segments, rod cells disappear within a few months after birth, followed by loss of cone cell function and number. Ence Tomography (SD-OCT) was used to observe the morphological changes of retina in rhodopsin-/ rats.
METHODS: Rhodopsin - / mouse (C57B16 rhodopsin knockout) and wild-type C57B16 mice at 3, 6, 9 and 12 weeks after birth were selected for this study. SD-OCT was performed in a radiovolume scan mode (with an optic disc as the center and a scan range of 1.3 mm in diameter). Each volume scan consisted of 100 B scans (each B scan consisted of 1,000 A scans). The thickness of the outer nuclear layer (ONL) was measured by scanning. Retinal histology was performed at the corresponding time points, and the data were compared with those obtained by SD-OCT. Electroretinograms (ERG) were recorded under dark and light adaptation conditions, and the changes of retinal function and shape were observed. Correlation analysis of state changes.
Results: SD-OCT measurements showed that the thickness of rhodopsin-/-rat retinal outer nuclear layer gradually thinned from the 3rd week to the 12th week after birth. The thickness values at the 3rd, 6th, 9th and 12th weeks were 40.6 (+ 1.61), 27.9 (+ 1.65), 14.5 (+ 0.7) and 6.0 (+ 0.78) microns, respectively. At week 3, there were 9-10 layers of nuclei in the rhodopsin-/ mouse extraretinal nucleus layer, and 11-12 layers in the C57B16 mouse. By week 12, the number of nuclei in the rhodopsin-/-mouse outer nucleus layer had been reduced to 1-2 layers, while the number of nuclei in the wild type mice was stable at 11-12 layers. The ERG under dark adaptation could not see a wave, but the amplitude of B wave decreased with age, which was consistent with the morphological observation. The morphological and functional measurements of wild type C57B16 mice were basically stable at all time points.
CONCLUSIONS: Data from SD-OCT measurements confirm that rhodopsin-/mouse outer nuclear layer thickness gradually thinned after birth, but no change was found in wild-type mice. The results of OCT were confirmed by histological examination and quantitative detection of ERG function. Therefore, SD-OCT can be used as a noninvasive and effective method. And a reliable research tool can be used to dynamically observe the changes of retinal degenerative diseases in animal models.
The third part: transplantation of retinal progenitor cells from Rhodopsin-/- mice.
OBJECTIVE: To observe the survival, migration, and retinal binding and differentiation of human retinal progenitor cells (RPCs) after transplantation into rhodopsin - / - mice subretina, which is immunosuppressed by cyclosporine, and to evaluate the retinal morphology (histological examination and SD-OCT) of retinal degeneration mice after transplantation. Correlation between detection and function (ERG).
METHODS: The transplanted cells were cultured in vitro with hypoxia (3%) for 7 to 9 generations. The recipient rhodopsin - / - mice were subjected to cyclosporine immunosuppression. The hRPC (control group injected PBS) was injected into the retina of the mice by operation. OCT was observed on the 3rd day and 3rd week after operation, and OCT was observed on the 3rd week after operation. After ERG, the mice were sacrificed for histological examination of the retina.
RESULTS: OCT observation on the 3rd day and the 3rd week after transplantation showed that transplanted cells were present in the subretinal region of the transplanted mice and could be confirmed by subsequent histological examination. The migration and integration of hRPC into the retina of recipient mice were almost not observed at the 3rd week after transplantation. There was no significant difference in the retinal function test (ERG) between transplanted cell mice and control mice (only PBS was injected into the retina).
CONCLUSION: Cyclosporin can improve the survival rate of transplanted cells by immunosuppression in recipient mice. The transplanted cells did not migrate and integrate to the recipient retina, and the retinal function was not improved.
I. main findings
1. Human retinal progenitor cells showed a good proliferation trend under hypoxic conditions, and the expression of proliferation-related factors Ki67 and Cyclin D1 were also higher than those in normoxic conditions. The expression levels of stem cell characteristic-related factors c-Myc and Klf4 were also higher under hypoxic conditions. A was expressed in hypoxic culture cells but only in low oxygen cultured cells.
2. SD-OCT measurements showed that the thickness of the outer retinal nucleus of rhodopsin-/-mice gradually thinned from the 3rd week to the 12th week after birth. Retinal histological examination also showed the same trend. ERG of Rhodopsin-/-mice could not see obvious a wave, but the amplitude of B wave decreased with age, which was consistent with morphological observation. The morphological and functional measurements of 57B16 mice were stable at various time points.
3. SD-OCT can see the existence of transplanted cells under the retina of transplanted mice, and can be confirmed by subsequent histological examination. Histological section can observe the survival of transplanted cells under the retina of rhodopsin - / mice, but hardly observe the migration and integration of cells into the retina of recipient mice. There was no significant difference in retinal functional examination (ERG) between mice (subretinal injection of PBS).
Two, research conclusion
1. Hypoxic culture conditions are conducive to the expansion of human retinal progenitor cells in vitro, and can maintain the ability of cell differentiation.
2. Data from SD-OCT measurements confirmed that rhodopsin-/mouse outer nuclear layer thickness gradually thinned after birth. The results of OCT were confirmed by both histological and quantitative detection of ERG function. SD-OCT can be used as a noninvasive, effective and reliable research tool for dynamic observation of retinal degeneration. The changing course of disease in animal models of sexual diseases.
3. Immunosuppression of rhodopsin-/mouse by cyclosporine can improve the survival of transplanted cells, but the transplanted cells did not migrate and integrate to the recipient retina during the observation period. There was no improvement in retinal function after transplantation.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1
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