本文選題：青光眼 + 視網(wǎng)膜干細胞　； 參考：《中南大學》2012年博士論文

【摘要】：第一章鼠青光眼模型的建立和視網(wǎng)膜干細胞的培養(yǎng)及鑒定 目的：建立鼠慢性青光眼模型,觀察各組鼠眼壓的變化。培養(yǎng)視網(wǎng)膜干細胞(retinal stem cells,RSCs),鑒定并觀察其分化特點。 方法：取正常成年SD大鼠,采用532-二極管激光行270°角膜緣血管網(wǎng)及三條淺層鞏膜靜脈光凝,建立鼠慢性青光眼模型。取正常成年SD大鼠眼球,仔細分離出睫狀體邊緣區(qū)的視網(wǎng)膜組織塊(包括色素組織),消化成單細胞懸液后置于含堿性成纖維細胞生長因子(bFGF)、表皮生長因子(EGF)、B27的無血清培養(yǎng)液中進行干細胞培養(yǎng),免疫細胞化學染色鑒定細胞的表型及分化特征。 結(jié)果：成功建立21只大鼠慢性青光眼模型,在激光光凝后14d,眼壓達最高峰,其平均值為31.6±3.2mmHg,對側(cè)對照眼為16.8±1.7mmHg(P0.001),21d后眼壓基本趨于穩(wěn)定狀態(tài)；21d時各實驗組眼壓差異無顯著性。培養(yǎng)的RSCs中干細胞標記物神經(jīng)絲蛋白(Nestin)表達陽性,細胞增殖標志物5-溴脫氧尿核苷(BrdU)表達陽性。RSCs分化細胞中神經(jīng)元標志物神經(jīng)元特異性烯醇化酶(NSE)表達陽性,膠質(zhì)細胞標志物膠質(zhì)纖維酸性蛋白(GFAP)表達陽性。 結(jié)論：采用532二極管激光角鞏膜緣血管網(wǎng)及淺層鞏膜靜脈光凝能成功升高鼠眼內(nèi)壓,建立大鼠慢性青光眼模型；睫狀體邊緣區(qū)的視網(wǎng)膜組織塊消化培養(yǎng)法能成功地培養(yǎng)出成年鼠RSCs,且RSCs能分化成神經(jīng)元和膠質(zhì)細胞。 第二章RSCs移植和COP-1聯(lián)合治療后青光眼模型鼠IFN-γ的表達差異 目的：探討RSCs移植和多聚物-1(Copolymer-1,COP-1)聯(lián)合治療慢性青光眼模型鼠后眼內(nèi)房水及血中IFN-y的表達差異,明確RSCs移植和COP-1聯(lián)合治療青光眼的機制。 方法：采用532二極管激光光凝建立SD大鼠慢性青光眼模型,實驗分為六組：①PBS/PBS治療組：青光眼模型鼠后足皮下注射PBS,七天后玻璃體腔內(nèi)注射PBS;②PBS/RSCs治療組：青光眼模型鼠后足皮下注射PBS,七天后玻璃體腔內(nèi)注射攜帶綠色熒光蛋白報告基因的慢病毒載體(Lentivirus-GFP+)轉(zhuǎn)染的RSCs;③PBS/COP-1治療組：青光眼模型鼠后足皮下注射0.2mg COP-1,七天后玻璃體腔內(nèi)注射PBS;④RSCs/COP-1治療組：青光眼模型鼠后足皮下注射0.2mg COP-1,七天后玻璃體腔內(nèi)注射Lentivirus-GFP+轉(zhuǎn)染的RSCs;⑤青光眼模型組：不做任何治療；⑥正常對照組。采用ELISA檢測各組眼內(nèi)房水及血中IFN-y含量；并采用hoechst染色觀察各組視網(wǎng)膜神經(jīng)節(jié)細胞(retinal ganglion cells,RGCs)的凋亡情況。采用免疫組織化學及全視網(wǎng)膜鋪片觀察移植的RSCs的融合情況。 結(jié)果：RSCs/COP-1治療組較其它組房水及血中IFN-γ的含量明顯減低(P0.05),分別為2371.9ng/L和710.9ng/L,RSCs/COP-1治療組較其它組RGCs凋亡細胞數(shù)明顯減少(P0.05)。移植的RSCs融合入宿主視網(wǎng)膜神經(jīng)節(jié)細胞層和神經(jīng)纖維層中。 結(jié)論：RSCs多植和COP-1聯(lián)合治療可以通過減少慢性青光眼模型鼠眼內(nèi)房水及血中IFN-γ含量,阻止RGCs的凋亡,其機制之一可能與COP-1介導的自身性免疫保護作用及干細胞的免疫調(diào)節(jié)作用有關。移植的RSCs融合到宿主視網(wǎng)膜中。 第三章RSCs移植和COP-1聯(lián)合治療對青光眼模型鼠RGCs的保護作用 目的：探討RSCs移植和COP-1聯(lián)合治療對慢性青光眼模型鼠RGCs的保護作用。 方法：采用532二極管激光光凝建立SD大鼠慢性青光眼模型,實驗分為四組：①PBS/PBS治療組：青光眼模型鼠后足皮下注射PBS,七天后玻璃體腔內(nèi)注射PBS;②PBS/RSCs治療組：青光眼模型鼠后足皮下注射PBS,七天后玻璃體腔內(nèi)注射RSCs；③PBS/COP-1治療組：青光眼模型鼠后足皮下注射0.2mg COP-1,七天后玻璃體腔內(nèi)注射PBS；④RSCs/COP-1治療組：青光眼模型鼠后足皮下注射0.2mgCOP-1,七天后玻璃體腔內(nèi)注射RSCs;(?)用免疫組織化學、Real-time RT-PCR、Western Blot等方法研究各組視網(wǎng)膜中神經(jīng)營養(yǎng)因子BDNF.IGF-Ⅰ的表達差異,通過視網(wǎng)膜切片及TUNEL染色檢測各組RGCs的凋亡,并進行RGCs計數(shù)。 結(jié)果:RSCs/COP-1治療組較其它各組神經(jīng)營養(yǎng)因子BDNF、IGF-Ⅰ蛋白及mRNA表達明顯增高(P0.05),同時RGCs數(shù)目增多(P0.05),RGCs凋亡細胞數(shù)減少(P0.05)。 結(jié)論：RSCs移植和COP-1聯(lián)合治療可以通過局部分泌神經(jīng)營養(yǎng)因子BDNF、IGF-I減少慢性青光眼模型鼠RGCs凋亡,保護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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R775;R-332

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