硅油对晶状体蛋白体外积聚性影响研究

发布时间：2018-05-29 06:45

本文选题：晶状体蛋白αB(CRYAB) + 白内障　；参考：《中国科学院研究生院(上海应用物理研究所)》2017年硕士论文

【摘要】：硅油具有低密度和高表面张力的理化特性,可以作为一种重要的玻璃体眼内压填充材料,用于治疗复杂性视网膜脱落症。但在使用过程中,也带来了一系列的眼科并发症,包括角膜病、前房硅油乳化、青光眼和白内障等。在硅油填充手术六个月后,白内障的发病率高达百分之百,但具体的致病分子机制仍不明确,有报导认为可能是硅油眼内填充手术结合多种因素诱发了白内障等疾病。然而在这些分析中都没有谈及硅油分子本身是否会对晶状体蛋白有直接的影响。早期有文献报道称硅油能够诱导某些蛋白的积聚,形成较大的蛋白积聚体,具体表现为蛋白类生物医药存储于残留硅油的容器内会导致蛋白发生积聚,如胰岛素、抗体等。为探讨硅油导致白内障的分子机制,本论文研究了硅油对晶状体蛋白积聚特性的影响。形成白内障最重要的因素是晶状体蛋白积聚形成淀粉样纤维,改变了原本透明的晶状体,使得晶状体白化。晶状体蛋白有多种组分,在晶状体内高度有序地组织以维持晶状体的高度透明,其成分在生命周期内基本不会变化。但随着个体年龄的增长,晶状体蛋白会越发不稳定,更容易形成淀粉样纤维,故白内障的发病与个体年龄成正相关性。晶状体蛋白分为α、β、γ三个家族。β和γ家族蛋白在晶状体内功能尚不十分明确。在所有晶体蛋白中,α晶体蛋白占有50%,其组织中浓度可以达到几百毫克每毫升。α晶体蛋白分为αA-和αB-晶体蛋白,其功能是作为分子伴侣预防β和γ晶状体蛋白的积聚,维持晶状体的透明度,其中,αA和αB晶体蛋白在其它组织中均存在,αB更为存在更为普遍。因此,我们推测硅油填充手术后白内障的发病,可能是由于硅油直接与晶状体蛋白相互作用,使晶状体蛋白发生积聚导致的。我们以关键的晶状体蛋白αB(CRYAB)作为研究对象,在体外研究硅油对晶状体蛋白积聚性的影响。在本文中,我们使用了多种手段来研究CRYAB溶液在加入硅油后的积聚特性的变化。检测蛋白溶液在硅油存在条件下OD值的变化,发现硅油存在时蛋白溶液的OD值明显高于未含有硅油的对照组,且在一定的时间范围内,随着时间的延长,OD值也随之增加,说明硅油促进积聚的效果愈加明显。我们还使用纳米颗粒分析仪分析了蛋白积聚颗粒大小的变化,进一步证实了在一定时间范围内,硅油的存在能使蛋白积聚颗粒的粒度更大以及颗粒物浓度更高。对蛋白形成积聚物还使用了刚果红染色鉴定法,发现积聚物能被刚果红染成红色,且在偏振光显微镜下观察程苹果绿色,鉴定蛋白积聚物可能为淀粉样积聚。此外,使用硫磺素(ThT)测定蛋白质β片层结构含量随积聚过程的变化,结果发现随孵育时间延长ThT荧光值升高,也说明晶状体蛋白可能发生了淀粉样性质的积聚。使用圆二色谱仪测量晶状体蛋白的二级结构变化,发现α螺旋含量下降,β片层含量升高,说明α螺旋结构向β片层结构转变。此外,使用荧光分子ANS结合蛋白质分子的疏水区域,结果表明蛋白质表面的的疏水性增强,且加入硅油组的疏水性强于对照组,而且有随时间增长的趋势。使用了原子力显微镜和透射电镜对蛋白积聚物微观形貌进行表征,发现晶状体蛋白积聚后以寡聚颗粒、短纤维的形态存在。结合以上结果,我们推测硅油分子通过结合蛋白表面的疏水区域,使得内部疏水区域进一步暴露于水中,破坏蛋白结构的原有稳定性,使得晶状体蛋白分子加速向淀粉样纤维化积聚。这可能是硅油导致白内障的分子机制。
[Abstract]:Silicone oil has the physical and chemical properties of low density and high surface tension. It can be used as an important vitreous intraocular pressure filling material for the treatment of complicated retinal detachment. But in the process of use, it also brings a series of ophthalmic complications, including cornea disease, anterior chamber silicone emulsification, glaucoma and cataract, etc. in silicone oil filling operation six After a month, the incidence of cataracts was up to one hundred percent, but the specific molecular mechanism was not clear. It was reported that silicone oil eye fill surgery combined with multiple factors to induce cataracts. However, none of these analyses said that the silicone oil itself would have a direct impact on the crystallin. It is reported that silicone oil can induce the accumulation of some protein and form a large protein accumulation, which is manifested in the accumulation of protein in the container of residual silicone oil, such as insulin and antibody. In order to explore the molecular mechanism of cataract caused by silicone oil, this paper studies the crystalline protein product of silicone oil. The most important factor in cataract formation. The most important factor in the formation of cataracts is the accumulation of crystallin forming amyloid fibers, changing the original transparent lens and making the lens whitening. There are many components of the lens protein, which is highly organized in the crystalline body to maintain the high transparency of the lens, and the composition of the lens will not change in the life cycle. However, with the growth of individual age, the crystallin will become more unstable and more likely to form amyloid fibers, so the incidence of cataracts is positively related to the age of the individual. Crystallin are divided into three families: alpha, beta, and gamma. The function of beta and gamma family proteins in the crystalline body is not ten distinct. In all crystal proteins, alpha crystal protein occupies 5. 0%, the concentration in the tissue can reach several hundred milligrams per milliliter. Alpha crystal protein is divided into alpha A- and alpha B- crystal protein. Its function is to prevent the accumulation of beta and gamma lens protein as a molecular chaperone and maintain the transparency of the lens. Among them, alpha A and alpha B crystal protein exist in other tissues, and the alpha B is more common. Therefore, we speculate that the protein is more common. The incidence of cataract after silicone oil filling may be due to the accumulation of silica oil directly with the lens protein, resulting in the accumulation of lens protein. We studied the effect of silicone oil on the accumulation of lens protein in vitro. In this article, we used a variety of methods in this paper. The change in the accumulation characteristics of CRYAB solution after adding silicone oil was studied. The change in the OD value of the protein solution under the presence of silicone oil was detected. It was found that the OD value of the protein solution in the presence of silicone oil was obviously higher than that of the control group without the silicone oil, and the OD value increased with the time extension, indicating that the accumulation of silicon oil promoted accumulation. We also used the nano particle analyzer to analyze the changes in the size of the protein accumulation particles, and further confirmed that the existence of silicon oil could make the particle size of the protein particles larger and the concentration of particles higher in a certain time range. The accumulation of protein in the accumulation of protein was also used in Congo red staining identification method, and the accumulation of protein was found to be accumulated. The substance can be dyed red by Congo red, and the apple green is observed under the polarized light microscope. The identification protein accumulation may be amyloid accumulation. In addition, the changes in the structure of protein beta layer with the change of the accumulation process with sulphur (ThT) have been detected. The results show that the fluorescence value of ThT increases with the prolonged incubation time, which also indicates the possible hair of the lens protein. The accumulation of amyloid properties was produced. Using a circular two chromatograph to measure the two structure changes of the crystallin, it was found that the content of alpha helix decreased and the content of the beta lamella increased, indicating that the alpha spiral structure changed into the structure of the beta lamellar structure. In addition, the hydrophobic region of the protein surface was increased by using the fluorescent molecule ANS to combine the hydrophobic region of the protein molecule. The hydrophobicity of the silicone oil group was stronger than that of the control group, and there was a tendency to increase with time. The micromorphology of the protein accumulation was characterized by atomic force microscopy and transmission electron microscopy. It was found that after the accumulation of crystallin, oligomeric particles and short fibers were found. The hydrophobic region of the white surface makes the internal hydrophobic region further exposed to the water and destroys the original stability of the protein structure, making the molecules of the lens protein accelerating to amyloidosis accumulation. This may be the molecular mechanism of the cataract caused by silicone oil.
【学位授予单位】：中国科学院研究生院(上海应用物理研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R776.1

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