骨髓间充质干细胞对光损伤视网膜的保护作用与机制

发布时间：2018-03-02 12:45

  本文选题：骨髓间充质干细胞　切入点：碱性成纤维细胞生长因子　出处：《福建医科大学》2013年博士论文　论文类型：学位论文

【摘要】：随着对间充质干细胞（mesenchymal stem cells, MSCs）认识的深入，MSCs的定义已经超出了作为一种具有多向分化潜能成体干细胞的概念。MSCs被认为是多源的细胞群体，广泛参与了组织修复。MSCs可能通过细胞因子分泌和调节机体微环境促进损伤修复过程，然而其确切的机制尚不清楚。本研究采用体外模型探讨视网膜光损伤对MSCs细胞因子表达变化的影响，并进一步阐述细胞因子表达变化在MSCs参与视网膜变性过程的作用。通过体内移植研究MSCs在视网膜光损伤中的作用及移植介导的宿主细胞因子表达变化。 第一部分视网膜光损伤动物模型研究 目的：建立视网膜光损伤动物模型，探讨光损伤对视网膜组织结构的影响及光损伤诱导的内源性细胞因子表达变化。 方法：6~8W SD大鼠经散瞳后，进行12h光照（5klux），12h避光，，每日反复。并于强光暴露后1d、2d、5d、7d、14d摘除眼球。采用透射电镜观察光照早期视网膜超微结构的改变。HE染色观察强光暴露对视网膜组织结构的影响。末端脱氧核苷酸标记法（TUNEL）检测光照后视网膜细胞凋亡情况。采用墨汁灌注及视网膜铺片法评估光照后视网膜微循环通透性改变。免疫荧光检测视网膜细胞因子bFGF、BDNF、CNTF的表达变化。 结果：强光暴露24h后，视网膜超微结构出现改变，光感受器细胞外节膜盘大量脱落，线粒体膜肿胀，核电子密度增高。视网膜组织学结构随光照时间的延长而持续破坏，视网膜外核层进行性变薄。视网膜细胞凋亡于光照后持续存在，主要分布于外核层及视网膜色素上皮层。强光暴露视网膜微血管通透性增强，墨汁灌注出现点状渗出，光照14d后出现片状渗漏，位于深层。强光暴露引起视网膜bFGF、BDNF、CNTF及GFAP表达上调。其中CNTF及GFAP表达上调随光照时间延长而增强。 结论：强光暴露可以引起视网膜组织结构破坏并出现反应性细胞因子表达上调，视网膜光损伤伴随胶质细胞反应增强。 第二部分视网膜光损伤对骨髓间充质干细胞神经保护功能的影响 目的：探讨光损伤视网膜匀浆上清液对MSCs细胞因子表达的影响及细胞因子表达变化在MSCs参与视网膜变性过程的作用。 方法：全骨髓贴壁法培养MSCs，并进行流式细胞术、成骨及成脂分化鉴定。制备正常及光损伤视网膜匀浆上清液，分别诱导第3～5代MSCs2d及5d。采用RT-qPCR及Western-blotting检测bFGF、BDNF、CNTF表达情况并与未诱导组MSCs比较。收集未诱导及诱导后MSCs培养上清液，制备条件培养液，培养视网膜片24h。收集视网膜片培养液，通过LDH释放水平评估不同条件培养液对视网膜细胞膜完整性的影响。RT-qPCR检测不同条件培养液对视网膜bcl-2及bax表达的影响。构建含bFGF干扰序列的慢病毒载体，转染MSCs并进一步诱导转染后的MSCs，观察bFGF表达下调对MSCs发挥视网膜神经保护作用的影响。 结果：MSCs可以表达bFGF、BDNF、CNTF。经光损伤视网膜匀浆上清液诱导后，MSCs表达bFGF及CNTF显著增强（P＜0.05）。MSCs条件培养液上调视网膜片bcl-2表达，抑制bax表达及LDH释放。光损伤视网膜匀浆上清液诱导后的MSCs条件培养液较其它条件培养液显著提高其上调视网膜片bcl-2表达，抑制bax表达及LDH释放的能力（均P＜0.05）。bFGF表达下调后的MSCs条件培养液仍可上调视网膜片bcl-2表达，抑制LDH释放。但各条件培养液组间比较无显著性差异（P＞0.05）。 结论：损伤刺激可以促进MSCs细胞因子表达，增强MSCs的视网膜神经保护作用，损伤诱导的bFGF表达上调在MSCs应对视网膜变性中具有重要作用。 第三部分经静脉移植骨髓间充质干细胞对光损伤视网膜的影响 目的：探讨MSCs静脉移植对光损伤视网膜细胞凋亡的影响及移植后细胞迁移和移植介导的宿主细胞因子表达变化。 方法：建立视网膜光损伤动物模型，MSCs注射组于强光暴露48h后，经静脉移植MSCs，剂量5×106个/ml，DMEM注射组接受等体积的DMEM注射。采用TUNEL比较光照14d后单纯光照组、DMEM注射组及MSCs注射组视网膜细胞凋亡情况，HE染色比较各组视网膜外核层厚度变化情况，Western-blotting检测光照1d、2d、5d、7d、14d各组视网膜CNTF及GFAP的表达情况。免疫荧光双重标记检测CNTF与GFAP的定位情况。采用GFP标记的MSCs静脉移植观察移植后细胞在视网膜的横向及纵向分布情况。 结果：经静脉移植的MSCs可以移行并迁移出视网膜血管，移植细胞主要分布于视网膜外核层、脉络膜等。MSCs移植显著抑制视网膜外核层厚度变薄（P＜0.05）及强光暴露引起的视网膜细胞凋亡（P＜0.05）。Western-blotting显示MSCs注射组大鼠视网膜表达CNTF及GFAP显著增强（P＜0.05）。免疫荧光双重标记显示二者具有共同定位。 结论：静脉移植MSCs具有对光损伤视网膜的神经保护作用，其机制可能与移植介导的胶质细胞反应增强有关。
[Abstract]:As to the mesenchymal stem cells (mesenchymal stem cells, MSCs) the deeper understanding of the definition of MSCs has exceeded a kind of pluripotent adult stem cells are considered the concept of.MSCs multi cell population, widely involved in tissue repair.MSCs may promote injury repair process by cytokine secretion and regulate the body the micro environment, but its exact mechanism remains unclear. This study used an in vitro model of light induced retinal damage influence on the expression of MSCs cytokines, and further elaborates the changes in MSCs expression of cytokines involved in retinal degeneration process. The expression changes of host cell factor by in vivo transplantation of MSCs in retinal light injury and transplantation mediated.
The first part of the animal model of retinal light injury
Objective: to establish an animal model of retinal light injury and to explore the effect of light damage on the structure of retina and the changes of endogenous cytokine expression induced by light damage.
Methods: 6~8W SD rats after mydriasis, 12h light (5klux), 12h light, and light for repeated daily. After exposure to 1D, 2D, 5D, 7d, 14d of enucleation by transmission electron microscopy to observe the light early retinal ultrastructural changes were observed by.HE staining light exposure effect on retinal structure the terminal deoxynucleotidyl labeling (TUNEL) assay after irradiation, the apoptosis of retinal cells. By ink perfusion and retinal flatmount was evaluated after illumination change. Detection of bFGF retinal microvascular permeability, retinal cell factor immunofluorescence BDNF, the expression of CNTF.
Results: after exposure of 24h light, the retinal ultrastructure changed, photoreceptor cell outer membrane disc abscission, mitochondria swelling, nucleus electron density increased. The retinal tissue structure with the prolonging of UV irradiation time and sustained damage, the outer nuclear layer of retina thinning. Persistent apoptosis of retinal cells after being exposed to light. Mainly distributed in the outer nuclear layer and retinal pigment epithelium. Light exposure of retinal microvascular permeability, ink perfusion punctate exudate, after 14d light leakage in the deep lamellar, light exposure. The retina caused bFGF, BDNF, CNTF and GFAP. The expression of CNTF and GFAP expression increased with the irradiation time increased.
Conclusion: strong light exposure can cause the destruction of retinal tissue structure and the expression of reactive cytokines.
The effect of retinal light damage on the neuroprotective function of bone marrow mesenchymal stem cells in the second part
Objective: To investigate the effect of light injured retinal homogenate supernatant on the expression of MSCs cytokines and the expression of cytokines in the process of MSCs's involvement in retinal degeneration.
Methods: whole bone marrow adherent culture method and MSCs, flow cytometry, osteogenic and adipogenic differentiation. Preparation of normal and light damage to the retina homogenate, respectively by third ~ 5 generations of MSCs2d and 5d. by RT-qPCR and Western-blotting to detect bFGF expression of CNTF and BDNF, and non induced group MSCs. Collect without induction and after MSCs culture supernatant, preparation of conditioned medium, cultured retinal slices of 24h. retinal slice culture fluid collection, evaluation of different effects of conditioned medium on retinal cell membrane integrity in different culture conditions to detect the influence of.RT-qPCR solution on the expression of retinal Bcl-2 and Bax through the release levels of LDH lentiviral vectors containing bFGF. The transfection of MSCs interference sequence, and further induce MSCs after transfection, to observe the effect of down-regulation of bFGF expression may play a protective role in retinal nerve on MSCs.
Results: MSCs expression of bFGF, BDNF, CNTF. after light damage to the retina homogenate after induction of MSCs expression of bFGF and CNTF increased significantly (P < 0.05).MSCs expression in liquid culture conditions by retinal Bcl-2, inhibit the expression of Bax and LDH. The release of light damage of retinal homogenate induced after MSCs conditioned medium than other conditions medium significantly enhanced the expression of Bcl-2 by retinal slices, inhibition of Bax expression and LDH release (P < 0.05) after downregulation of.BFGF MSCs conditioned medium can increase retinal bcl-2 expression, inhibiting the release of LDH. But the culture conditions showed no significant difference between the liquid group (P > 0.05).
Conclusion: injury stimulation can promote the expression of MSCs cytokines, enhance the retinal neuroprotective effect of MSCs, and increase the expression of bFGF induced by injury. MSCs plays an important role in the treatment of retinal degeneration.
The effect of bone marrow mesenchymal stem cells transplanted into the third part of the vein on the light damage of the retina
Objective: To investigate the effect of MSCs vein transplantation on retinal cell apoptosis induced by light injury and the changes of cell migration and transplantation mediated host cell factor expression after transplantation.
Methods: to establish the animal model of retinal light damage, MSCs injection group in light exposed 48h after intravenous transplantation of MSCs, dose of 5 * 106 /ml, DMEM injection group received equal volume of DMEM injection. With TUNEL after 14d light irradiation group, DMEM injection group and MSCs injection group of retinal cell apoptosis. Comparison of the thickness of the outer nuclear layer changes each retina HE staining, detection of Western-blotting light 1D, 2D, 5D, 7d, CNTF and 14d groups the expression of retinal GFAP. The localization of the double labeled immunofluorescence detection of CNTF and GFAP. Using GFP labeled MSCs vein graft after transplantation were observed in the horizontal and vertical distribution the retina.
Results: after intravenous transplantation of MSCs can migrate and migrate out of retinal vessels, transplanted cells were mainly distributed in the outer nuclear layer of retina, choroid transplantation.MSCs significantly inhibit the retinal outer nuclear layer thickness (P < 0.05) and exposure to light induced retinal cell apoptosis (P < 0.05).Western-blotting display MSCs retinal injection group rats the expression of CNTF and GFAP increased significantly (P < 0.05). Double immunofluorescent labeling showed the two have a common location.
Conclusion: venous transplantation of MSCs has a neuroprotective effect on light damaged retina, and its mechanism may be related to the enhanced reaction of glia mediated by transplantation.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1


本文编号：1556579