【作者】 赵杰；

【导师】 蔡可丽；

【作者基本信息】 山东大学 ， 眼科学， 2005， 硕士

【摘要】 目的 雷帕霉素是一种新型的大环内酯类免疫抑制剂,近年来被广泛运用于器官移植、恶性肿瘤治疗、预防冠状动脉狭窄等,眼科已有研究应用于动物的角膜移植。后发性白内障是白内障术后最严重的并发症之一,其原因是由于白内障术后残留的晶体上皮细胞增殖、移行、转化,从而在后囊膜上形成纤维膜,严重影响术后视力的恢复。本研究在体外培养兔晶体上皮细胞的基础之上,旨在研究兔晶体细胞的体外生长特性,观察不同浓度雷帕霉素对晶体上皮细胞形态、增殖等生物学活性的影响,并对其可能的作用机制加以初步探讨,为临床阐明后囊混浊的发病机理和探讨防治措施提供实验依据。 方法 晶体上皮细胞来自健康成年兔(1.5kg-2.0kg)的晶体囊膜,将其放入DMEM培养基中培养,达汇合点后细胞传代,第2-3代细胞用于实验。 (1)用组织块培养法进行兔晶体上皮细胞的原代培养,光镜下观察细胞的形态。 (2)将2-3代兔晶体上皮细胞以5×10~4个/ml接种于96孔板,24小时后分别加入雷帕霉素(0ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml)。加药后于培养24小时后,光镜下观察细胞形态,以MTT法测细胞增殖活性。 (3)将2-3代兔晶体上皮细胞以5×10~4个/ml接种于铺有玻片的6孔板中,24小时后分别加入雷帕霉素(0ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml)。加药后于培养24小时后,光镜下观察细胞形态,取出细胞爬片,行HE染色,并以免疫组化SP法对PCNA进行检测。 (4)取雷帕霉素(0ng/ml,20ng/ml)处理过的兔晶体上皮细胞各10~6个,以流式细胞仪测细胞周期。 (5)取雷帕霉素(0ng/ml,5ng/ml,10ng/ml,20ng/ml,40ng/ml,80ng/ml)处理过的兔晶体上皮细胞各10~6个,以RT-PCR测P27kipl mRNA的表达。更多还原

【Abstract】 Objective Rapamycin was a kind of new macrolides immumosuppressive drug, which can inhibit the proliferation of many cells. In recent years, it was widely used in organ transplantation and tumor trerapy. It also served as smear layer of blood vessel cage to prevent the restenosis of coronal artery. In ophthalmology, the cornea transplantation of annimals had been studied. Posterior capsular opacification (PCO) was the most serious complications of cataract extract. The cause was the residual lens epithelial cells post-surgery proliferated, migrated, converted, and formatted into fibrous membrane. Then the recovery of vision was seriously influenced. This study aimed to observe the bioactivity of rabbit lens epithelial cells cultured in vitro and to evaluate the inhibitory reaction of different concentrations rapamycin on the proliferation of rabbit lens epithelial cells.Methods A randomized controlled study was performed.(l)Rabbit lens epithelial cells were isolated from healthy rabbits in either gender with a body mass from 1.5kg to 2.0kg and cultured in vitro. Cells morphological changes were observed with light microscope. (2)Rabbit lens epithelial cells were placed in 96-well flat bottomed tissue culture plates at an initial concentration of 5 X 104cells/ml. After incubated for 24 hours, multiple concentrations (0ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml) of RAPA were added to the wells. The cells were then incubated with drugs for 24 hours to measure proliferation by the MTT colorimetry.(3)Rabbit lens epithelial cells were placed on the carry sheet glass in 6-well flat bottomed tissue culture plates at an initial concentration of 5 X 104cells/ml. After incubated for 24 hours, multiple concentrations (Ong/ml, 5ng/ml, 10ng/ml, 20ng/ml, 40ng/ml, 80ng/ml) of RAPA were added to the wells. The cells were then incubated with drugs for 24 hours. On the basis of HE staining, the expression of proliferating cell nuclear antigen (PCNA) in lens epithelial cells were observed through immunocytochemistry.(4)Rabbit lens epithelial cells were placed in 25cm2 tissue culture flask at an initial concentration of 5X 104cells/ml. After incubated for 24 hours, multiple concentrations (Ong/ml, 2 Ong/ml) of RAPA were added to the flasks. The cells were then incubated with drugs for 24 hours. The change of cell cycles and apoptosis were observed by flow cytometer (FCM). (5)Rabbit lens epithelial cells were placed in 25cm2 tissue culture flask at an initial concentration of 5 X 104cells/ml. After incubated for 24 hours, multiple concentrations (Ong/ml, 5ng/ml, 1 Ong/ml, 20ng/ml, 40ng/ml, 80ng/ml) of RAPA were added to the flasks. The cells were then incubated with drugs for 24 hours. The expression levels of P27kipl mRNA were analyzed by reverse transcription-polymerase chain reaction (RT-PCR).Results: After the application of RAPA, RLECs showed different inhibition effect of proliferation. RAPA decreased rabbit lens epithelial cells proliferation significantly at a certain range of concentrations.(1) We observed that rabbit lens epithelial cells were irregular in morphology with light microscope.(2)Within a certain range of concentrations(5-80ng/ml), RAPA could significantly decrease rabbit lens epithelial cells proliferation(P<0.05). Within the concentrations range of 10-80ng/ml, cell proliferation effect decreased dramatically(P<0.01). However, within the range of concentration (20-80ng/ml), RAPA has no significantdifference to the proliferation of rabbit lens epithelial cells(P>0.05). (3)HE stainning showed that RLECs were large, polygon appearance and rich cutoplasm; nucleus were in middle, small, and blue apprearance. After affected by RAPA, the cells contracted and the nucleus get larger. Within a certain range of concentrations (5-80ng/ml), the expression of PCNA showed negative staining with the increasing concentrations of RAPA (PO.05).(4)FCM showed that the S-phase cells decreased, and that cell function became more inactive. There have no evident signs of apoptosis. (5)RT-PCR showed that the expression level of P27kipl mRNA increased with the increasing concentrations of RAPA (P<0.05).Conclusion These experiments confirmed that RAPA has powerful inhibition effects on RLECs proliferation. The effect was enhanced as concentration increased. The expression of PCNA decreased while the expression of P27kipl mRNA increased with the increase of the concentration of RAPA. The cell cycle was affected with RAPA. RAPA might had no influence on apoptosis of RLECs. The possible mechanism might be the expression of P27kipl prevents the cell cycle from Gl phrase to S phrase. This work provided an experimental basis for the mechanism and prevention of posterior capsular opacity after cataract surgery.更多还原