RSCs移植联合COP-1免疫治疗对青光眼模型鼠RGCs的保护作用

发布时间：2018-05-24 11:30

本文选题：青光眼 + 视网膜干细胞　；参考：《中南大学》2012年博士论文

【摘要】：第一章鼠青光眼模型的建立和视网膜干细胞的培养及鉴定目的：建立鼠慢性青光眼模型,观察各组鼠眼压的变化。培养视网膜干细胞(retinal stem cells,RSCs),鉴定并观察其分化特点。方法：取正常成年SD大鼠,采用532-二极管激光行270°角膜缘血管网及三条浅层巩膜静脉光凝,建立鼠慢性青光眼模型。取正常成年SD大鼠眼球,仔细分离出睫状体边缘区的视网膜组织块(包括色素组织),消化成单细胞悬液后置于含碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)、B27的无血清培养液中进行干细胞培养,免疫细胞化学染色鉴定细胞的表型及分化特征。结果：成功建立21只大鼠慢性青光眼模型,在激光光凝后14d,眼压达最高峰,其平均值为31.6±3.2mmHg,对侧对照眼为16.8±1.7mmHg(P0.001),21d后眼压基本趋于稳定状态；21d时各实验组眼压差异无显著性。培养的RSCs中干细胞标记物神经丝蛋白(Nestin)表达阳性,细胞增殖标志物5-溴脱氧尿核苷(BrdU)表达阳性。RSCs分化细胞中神经元标志物神经元特异性烯醇化酶(NSE)表达阳性,胶质细胞标志物胶质纤维酸性蛋白(GFAP)表达阳性。结论：采用532二极管激光角巩膜缘血管网及浅层巩膜静脉光凝能成功升高鼠眼内压,建立大鼠慢性青光眼模型；睫状体边缘区的视网膜组织块消化培养法能成功地培养出成年鼠RSCs,且RSCs能分化成神经元和胶质细胞。第二章RSCs移植和COP-1联合治疗后青光眼模型鼠IFN-γ的表达差异目的：探讨RSCs移植和多聚物-1(Copolymer-1,COP-1)联合治疗慢性青光眼模型鼠后眼内房水及血中IFN-y的表达差异,明确RSCs移植和COP-1联合治疗青光眼的机制。方法：采用532二极管激光光凝建立SD大鼠慢性青光眼模型,实验分为六组：①PBS/PBS治疗组：青光眼模型鼠后足皮下注射PBS,七天后玻璃体腔内注射PBS;②PBS/RSCs治疗组：青光眼模型鼠后足皮下注射PBS,七天后玻璃体腔内注射携带绿色荧光蛋白报告基因的慢病毒载体(Lentivirus-GFP+)转染的RSCs;③PBS/COP-1治疗组：青光眼模型鼠后足皮下注射0.2mg COP-1,七天后玻璃体腔内注射PBS;④RSCs/COP-1治疗组：青光眼模型鼠后足皮下注射0.2mg COP-1,七天后玻璃体腔内注射Lentivirus-GFP+转染的RSCs;⑤青光眼模型组：不做任何治疗；⑥正常对照组。采用ELISA检测各组眼内房水及血中IFN-y含量；并采用hoechst染色观察各组视网膜神经节细胞(retinal ganglion cells,RGCs)的凋亡情况。采用免疫组织化学及全视网膜铺片观察移植的RSCs的融合情况。结果：RSCs/COP-1治疗组较其它组房水及血中IFN-γ的含量明显减低(P0.05),分别为2371.9ng/L和710.9ng/L,RSCs/COP-1治疗组较其它组RGCs凋亡细胞数明显减少(P0.05)。移植的RSCs融合入宿主视网膜神经节细胞层和神经纤维层中。结论：RSCs多植和COP-1联合治疗可以通过减少慢性青光眼模型鼠眼内房水及血中IFN-γ含量,阻止RGCs的凋亡,其机制之一可能与COP-1介导的自身性免疫保护作用及干细胞的免疫调节作用有关。移植的RSCs融合到宿主视网膜中。第三章RSCs移植和COP-1联合治疗对青光眼模型鼠RGCs的保护作用目的：探讨RSCs移植和COP-1联合治疗对慢性青光眼模型鼠RGCs的保护作用。方法：采用532二极管激光光凝建立SD大鼠慢性青光眼模型,实验分为四组：①PBS/PBS治疗组：青光眼模型鼠后足皮下注射PBS,七天后玻璃体腔内注射PBS;②PBS/RSCs治疗组：青光眼模型鼠后足皮下注射PBS,七天后玻璃体腔内注射RSCs；③PBS/COP-1治疗组：青光眼模型鼠后足皮下注射0.2mg COP-1,七天后玻璃体腔内注射PBS；④RSCs/COP-1治疗组：青光眼模型鼠后足皮下注射0.2mgCOP-1,七天后玻璃体腔内注射RSCs;(?)用免疫组织化学、Real-time RT-PCR、Western Blot等方法研究各组视网膜中神经营养因子BDNF.IGF-Ⅰ的表达差异,通过视网膜切片及TUNEL染色检测各组RGCs的凋亡,并进行RGCs计数。结果:RSCs/COP-1治疗组较其它各组神经营养因子BDNF、IGF-Ⅰ蛋白及mRNA表达明显增高(P0.05),同时RGCs数目增多(P0.05),RGCs凋亡细胞数减少(P0.05)。结论：RSCs移植和COP-1联合治疗可以通过局部分泌神经营养因子BDNF、IGF-I减少慢性青光眼模型鼠RGCs凋亡,保护RGCs。
[Abstract]:Chapter 1 Establishment of rat glaucoma model and culture and identification of retinal stem cells
Objective: to establish a rat model of chronic glaucoma and observe the changes in intraocular pressure (IOP) of each group. Retinal stem cells (RSCs) was cultured and the differentiation characteristics were observed and observed.
Methods: Taking the normal adult SD rats, using the 532- diode laser in 270 degree corneal limbus network and three superficial scleral vein photocoagulation, the rat model of chronic glaucoma was established. The retina tissue block (including pigment tissue, including the pigment tissue) in the rim of the ciliary body was carefully separated from the normal adult SD rat. Fibroblast growth factor (bFGF), epidermal growth factor (EGF), and serum-free culture of B27 were cultured for stem cells. Immunocytochemical staining was used to identify the phenotype and differentiation of the cells.
Results: 21 rat chronic glaucoma models were successfully established. In 14d after laser photocoagulation, the peak pressure reached the peak, the average value was 31.6 + 3.2mmHg, the contralateral control eyes were 16.8 + 1.7mmHg (P0.001). The intraocular pressure after 21d tended to be stable, and the difference in intraocular pressure of the experimental groups at 21d was not obvious. The cultured RSCs was the stem cell marker neurofilament protein (Nes). Tin) expressed positive, the cell proliferation marker 5- bromodeoxyuridine (BrdU) expression positive.RSCs differentiated cells, neuron specific enolase (NSE) expression positive, glial cell marker glial fibrillary acidic protein (GFAP) expression positive.
Conclusion: the 532 diode laser cornea sclera vascular network and superficial scleral vein photocoagulation can successfully raise the rat intraocular pressure and establish the rat model of chronic glaucoma. The retinal tissue block digestion and culture method of the ciliary body edge region can successfully develop the adult rat RSCs, and RSCs can differentiate into neurons and glia cells.
The second chapter is the difference of IFN- gamma expression in glaucoma model rats after RSCs transplantation and COP-1 combined treatment.
Objective: To investigate the difference in the expression of IFN-y in aqueous humor and blood in the treatment of chronic glaucoma model rats combined with RSCs transplantation and -1 (Copolymer-1, COP-1), and to clarify the mechanism of combination of RSCs transplantation and COP-1 in the treatment of glaucoma.
Methods: the chronic glaucoma model of SD rats was established by 532 diode laser photocoagulation. The experiment was divided into six groups: (1) the PBS/PBS treatment group: the glaucoma model rat was injected PBS subcutaneously and injected PBS in the vitreous cavity seven days later; (2) the PBS/RSCs treatment group: the glaucoma model rat was injected PBS in the posterior foot skin, and the vitreous cavity was injected green in the vitreous cavity after seven days. RSCs transfected by the lentivirus carrier (Lentivirus-GFP+) of the reporter gene of the fluorescent protein; (3) PBS/COP-1 treatment group: 0.2mg COP-1 was injected subcutaneously in the posterior foot of glaucoma model rats and PBS in the vitreous cavity seven days later; (4) RSCs/COP-1 treatment group: the glaucoma model rat was injected with 0.2mg COP-1 subcutaneously, and Lentivirus-GFP+ in the vitreous cavity after seven days. Transfected RSCs; (5) glaucoma model group: without any treatment; 6. Normal control group. ELISA was used to detect the content of IFN-y in aqueous humor and blood in each group; and Hoechst staining was used to observe the apoptosis of retinal ganglion cells (retinal ganglion cells, RGCs) in each group. Immunohistochemistry and whole retina sheet were used to observe the transplantation. The fusion of RSCs.
Results: the content of IFN- gamma in the aqueous and blood of the RSCs/COP-1 group was significantly lower than that in the other groups (P0.05), 2371.9ng/L and 710.9ng/L respectively. The number of RGCs apoptotic cells in the RSCs/COP-1 treatment group decreased significantly (P0.05). The transplant RSCs fused into the retinal ganglion layer and the nerve fiber layer of the host retina.
Conclusion: the combined treatment of RSCs and COP-1 can prevent the apoptosis of RGCs by reducing the content of IFN- gamma in the ocular aqueous humor and blood of the chronic glaucoma model rats. One of the mechanisms may be related to the autoimmune protective action mediated by COP-1 and the immunoregulation of the stem cells. The transplanted RSCs is fused into the host retina.
The third chapter is the protective effect of RSCs transplantation combined with COP-1 on RGCs in glaucoma model rats.
Objective: To investigate the protective effect of RSCs transplantation combined with COP-1 on RGCs in chronic glaucoma model rats.
Methods: the chronic glaucoma model of SD rats was established by 532 diode laser photocoagulation. The experiment was divided into four groups: (1) the PBS/PBS treatment group: the glaucoma model rats were injected PBS subcutaneously and injected PBS in the vitreous cavity seven days later; (2) the PBS/RSCs treatment group: the glaucoma model rats were injected PBS in the posterior foot skin, and RSCs in the vitreous cavity after seven days; (3) PBS/ COP-1 treatment group: glaucoma model rats were injected with 0.2mg COP-1 subcutaneously and injected PBS in the vitreous cavity seven days later; (4) RSCs/COP-1 treatment group: glaucoma model rats were injected subcutaneously with 0.2mgCOP-1, and RSCs was injected into the vitreous cavity seven days later; (?) using immunohistochemistry, Real-time RT-PCR, Western Blot and other methods to study the retina God in each group. The difference of expression of BDNF.IGF- I was observed. The apoptosis of RGCs in each group was detected by retina slices and TUNEL staining, and RGCs was counted.
Results: the expression of neurotrophic factor BDNF, IGF- I protein and mRNA in the RSCs/COP-1 group was significantly higher than that in other groups (P0.05), and the number of RGCs increased (P0.05), and the number of apoptotic cells in RGCs decreased (P0.05).
Conclusion: the combination of RSCs transplantation and COP-1 can reduce the apoptosis of RGCs in chronic glaucoma model rats and protect RGCs. through local secretion of neurotrophic factor BDNF and IGF-I.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R775;R-332

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