吸烟在甲状腺相关性眼病氧化应激中的作用及机制研究

发布时间：2019-05-17 09:02

【摘要】：研究目的:甲状腺相关性眼病是是眼眶病中较常见的一种,且发病率逐渐上升,可引起外观及视功能的损伤,临床表现多样,疾病后果严重,具体发病原因不明确,故近年来一直是困扰眼科医生的难题。而吸烟被证明是甲状腺相关性眼病(TAO)的重要危险因素,另外,有研究发现吸烟与氧化应激存在相关性,而我们已知氧化应激在TAO的疾病过程中发挥着重要作用。香烟的成分十分复杂,其中公认的毒性成分为尼古丁,目前国内外在其他疾病方面针对吸烟的研究多采用香烟烟雾提取物(CSE)及尼古丁两种物质为刺激因素,眼眶成纤维细胞作为TAO的最终效应细胞,是近年来TAO相关研究的经典研究模型。故本研究采用原代培养的眼外肌来源眼眶成纤维细胞,研究香烟烟雾提取物及尼古丁作用于TAO患者及正常人眼眶成纤维细胞后,各种氧化应激指标ROS、MDA、8-OHdG的变化,探索香烟烟雾提取物及尼古丁在TAO这一疾病中是否通过氧化应激发挥作用,其具体的剂量关系,并通过检测香烟烟雾提取物及尼古丁对于眼眶成纤维细胞NF-kB通路的作用来进一步探索其可能的作用机制。方法:1、取TAO患者及正常人的眼外肌组织,因标本量少故采取组织块法进行原代培养,获得稳定传代的眼眶成纤维细胞,免疫组化的方法鉴定。2、用MTT法检测香烟烟雾提取物及尼古丁作用于眼眶成纤维细胞24小时及72小时后细胞的活性。3、ELISA法检测香烟烟雾提取物及尼古丁作用于眼眶成纤维细胞24小时及72小时后8-OHdG指标的变化。4、分光光度计法检测香烟烟雾提取物及尼古丁作用于眼眶成纤维细胞24小时及72小时后MDA指标的变化。5、酶标仪法检测香烟烟雾提取物及尼古丁作用于眼眶成纤维细胞24小时后ROS指标的变化6、RT-PCR法检测香烟烟雾提取物及尼古丁作用于眼眶成纤维细胞24小时后NF-kB相关基因的表达情况。结果:1、本实验成功通过组织块法原代培养眼外肌来源的成纤维细胞,能够稳定传代,且经过鉴定为眼眶成纤维细胞,能够成功建立细胞系后作为甲状腺相关性眼病的细胞模型,为后续试验提供可能。2、MTT检测结果显示24小时刺激,TAO患者眼眶成纤维细胞在CSE刺激下细胞活力下降幅度更大,而在72小时刺激条件下,低浓度和高浓度的尼古丁呈现了相似的杀伤性。可见吸烟的损伤作用随刺激时间的增强呈一定的累积作用。3、在24小时刺激条件下,尼古丁10ug/ml可显著升高两种细胞体内8-OHdG的含量;在72小时刺激条件下,CSE单独刺激和低浓度尼古丁即可引起8-OHdG产生的增加,可见高剂量的尼古丁可在短时间内即可促进8-OHdG含量的突然上升,而低剂量的尼古丁导致8-OHdG的缓慢积累。4、在同时给予CSE和尼古丁10ug/ml刺激之后,MDA含量相比CSE单独刺激以及阴性对照组均有显著的增加。在72小时刺激之后,观察细胞体内MDA含量变化,所有刺激条件对比阴性对照组MDA表达均有大幅度的增加。5、吸烟可通过增加ROS含量发挥作用。高浓度尼古丁的作用效果最明显。6、在TAO患者眼眶成纤维细胞中,CSE刺激之后NF-κB RNA水平显著性上调。可见吸烟通过上调NF-κB基因的表达发挥作用。结论:吸烟可导致眼眶成纤维细胞氧化应激指标MDA、8-OHdG及ROS增高,且尼古丁作为香烟中的主要毒性成分发挥作用,即使低浓度的尼古丁,也可随时间累积而发挥较强的作用,说明吸烟可通过引起氧化应激而参与TAO病理过程。其中可能的分子通路为NF-κB,吸烟可上调NF-κB基因的表达。
[Abstract]:The purpose of the study is that the thyroid-related eye disease is one of the most common diseases in the orbit, and the incidence of the thyroid-related eye disease is increasing gradually, which can cause the appearance and the visual function damage, the clinical manifestations are various, the disease is serious, the specific cause of the disease is not clear, so it has been a difficult problem for the ophthalmologist in recent years. Smoking has been shown to be an important risk factor for thyroid-associated ophthalmopathy (TAO) and, in addition, studies have found that there is a correlation between smoking and oxidative stress, and we know that oxidative stress plays an important role in the disease of TAO. The composition of the cigarette is very complex, and the recognized toxic component is nicotine. At present, the cigarette smoke extract (CSE) and the nicotine are used as the stimulating factor for the research of smoking in other diseases at home and abroad, and the orbital fibroblasts are the final effector cells of the TAO. It is a classic study model of TAO-related research in recent years. The primary cultured extraocular muscle derived orbital fibroblasts were used to study the changes of ROS, MDA,8-OHdG of cigarette smoke extract and nicotine in patients with TAO and normal human orbital fibroblasts. To explore the role of cigarette smoke extract and nicotine in the treatment of TAO by oxidative stress, its specific dose relationship, and to further explore its possible mechanism by detecting the effect of cigarette smoke extract and nicotine on the NF-kB pathway of the orbital fibroblasts. Methods:1. The extraocular muscle tissues of the patients with TAO and the normal persons were collected, and the primary culture was performed by the tissue-block method, and the orbital fibroblast and the immunohistochemical method of the stable passage were obtained. The activity of the cigarette smoke extract and the nicotine on the cell after 24 hours and 72 hours of the orbital fibroblasts was detected by the MTT method.3. The activity of the cigarette smoke extract and the nicotine on the orbital fibroblasts for 24 hours and 72 hours after 72 hours was detected by the ELISA method. The changes of the activity of the cigarette smoke extract and the nicotine on the content of MDA in 24 hours and 72 hours of the orbital fibroblasts were detected by the spectrophotometer. The expression of NF-kB related gene after 24 hours of orbital fibroblasts was detected by RT-PCR. Results:1. In this experiment, the fibroblasts of the extraocular muscle derived from the primary culture of the extraocular muscle were successfully cultured by the tissue-block method, and the cell line can be successfully established as the cell model of the thyroid-related ophthalmopathy after the cell line was successfully established. The results of the MTT assay showed that the activity of the orbital fibroblasts in the patients with TAO was greater than that of the CSE, while the low concentration and high concentration of nicotine exhibited similar anti-personnel effects at 72-hour stimulation. It can be seen that the damage effect of cigarette smoking has a certain accumulation function with the enhancement of the stimulation time.3. Under the condition of 24-hour stimulation, the content of 8-OHdG in the two cells can be significantly increased under the condition of 24-hour stimulation, and the increase of 8-OHdG can be caused by the single stimulation of CSE and low concentration of nicotine under the condition of 72-hour stimulation. A high dose of nicotine could promote a sudden rise in the content of 8-OHdG in a short period of time, while low-dose nicotine results in a slow accumulation of 8-OHdG.4. After both CSE and nicotine 10 ug/ ml stimulation, the MDA content was significantly increased as compared with the CSE alone and in the negative control group. After 72 hours of stimulation, the content of MDA in the cells was observed, and the expression of MDA in the negative control group was significantly increased in all the stimulation conditions.5. Smoking could play a role in increasing the ROS content. The effect of high concentration of nicotine was most obvious.6. In the orbital fibroblasts of patients with TAO, the level of NF-EMAB RNA was up-regulated after CSE stimulation. It can be seen that smoking plays a role by up-regulation of the expression of the NF-B-B gene. Conclusion: Smoking can lead to the increase of MDA,8-OHdG and ROS in the oxidative stress of the orbital fibroblasts, and the nicotine functions as the main toxic component in the cigarette. It is indicated that smoking can be involved in the TAO pathological process by causing oxidative stress. Among them, the possible molecular pathway is NF-B-B, and smoking can increase the expression of NF-B-B gene.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R581;R771.3

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