玻璃体切割术后并发性白内障发病机制的实验研究

发布时间：2019-06-05 00:43

【摘要】：目的：通过检测玻璃体切割术后前房氧浓度和炎性因子的变化、晶状体纤维细胞凋亡情况以、caspase-8和DLAD基因表达的变化以及硅油填充术后晶状体基因表达谱的变化,探讨玻璃体切割术后并发性白内障的可能发病机制。方法：分五部分进行研究,第一部分：建立兔眼玻璃体切割术后并发性白内障的动物模型,术后裂隙灯观察晶状体混浊程度并照相。第二部分：于术后1天、3天、7天、1月、3月抽取前房房水检测氧浓度和IL-2、TNF-a的变化；第三部分：于术后1天、3天、7天、1月、3月取兔眼晶状体,病理学观察晶状体纤维细胞形态、原位末端核苷标记(TUNEL)法检测其凋亡并计算凋亡指数(AI)、免疫组化法测定Caspase-8的表达。第四部分：采用半定量RT-PCR技术测定术后1天、3天、7天、1月、3月晶状体上DLAD基因的表达情况。第五部分：应用DNA芯片技术对硅油填充术后晶状体的基因表达谱进行差异表达谱分析,并采用实时定量PCR进行验证。结果：成功建立了玻璃体切割术后并发性白内障的动物模型；玻璃体切割术后前房氧含量和炎性因子IL-2、TNF-a有不同程度的增加；玻璃体切割术后晶状体纤维细胞凋亡增加,细胞内Caspase-8明显表达；玻璃体切割术后晶状体的DLAD基因随时间延长表达逐渐减弱；芯片结果表明硅油填充术后晶状体变化的基因主要有氧化应激、炎症反应、细胞凋亡、信号传导、细胞分化粘附等相关基因。实时定量PCR与芯片检测结果具有相同的方向性,且数据非常接近。结论：玻璃体切割术后晶状体周围微环境的变化导致了某些调控机制的破坏,使晶状体纤维细胞凋亡增加,而细胞器的降解减少,这种动态平衡的破坏,从而导致了白内障的发生。硅油填充术后并发性白内障的发生发展涉及多基因改变；实时定量PCR进一步验证了基因芯片的可靠性。
[Abstract]:Objective: to detect the changes of anterior chamber oxygen concentration and inflammatory factors, apoptosis of lens fiber cells, the expression of caspase-8 and DLAD genes and the changes of lens gene expression profile after silicone oil filling. To investigate the possible pathogenesis of complicated cataract after vitrectomy. Methods: the study was carried out in five parts. In the first part, the animal model of complicated cataract after vitrectomy was established, and the degree of lens opacification was observed and photographed by slit lamp after operation. The second part: the changes of oxygen concentration and IL-2,TNF-a were detected by extracting aqueous humor from anterior chamber 1 day, 3 days, 7 days, 1 month and 3 months after operation. The third part: the lens of rabbit eyes was taken 1 day, 3 days, 7 days, 1 month and 3 months after operation. The morphology of lens fiber cells was observed pathologically. Apoptosis was detected by in situ terminal nucleosides labeling (TUNEL) method and the apoptosis index (AI), was calculated. The expression of Caspase-8 was detected by immunohistochemistry. Part IV: the expression of DLAD gene in lens was measured by semi-quantitative RT-PCR at 1 day, 3 days, 7 days, 1 month and 3 months after operation. In the fifth part, the differential expression profiles of lens after silicone oil filling were analyzed by DNA chip technique, and verified by real-time quantitative PCR. Results: the animal model of complicated cataract after vitrectomy was successfully established, and the oxygen content and inflammatory factor IL-2,TNF-a in the anterior chamber were increased in varying degrees. After vitrification, the apoptosis of lens fiber cells increased and the expression of Caspase-8 was obvious, and the expression of DLAD gene in lens decreased gradually with the prolongation of time. The results of microarray showed that the main genes of lens changes after silicone oil filling were oxidative stress, inflammatory reaction, apoptosis, signal transduction, cell differentiation and adhesion and so on. The real-time quantitative PCR has the same directivity as the chip detection results, and the data are very close. Conclusion: the changes of microenvironment around lens after vitrectomy lead to the destruction of some regulatory mechanisms, which increase the apoptosis of lens fiber cells and decrease the degradation of organelle, which destroys the dynamic balance. This leads to the occurrence of cataracts. The occurrence and development of complicated cataract after silicone oil filling involves polygene changes, and the reliability of gene chip is further verified by real-time quantitative PCR.
【学位授予单位】：山东中医药大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R779.6

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