1.8GHz微波辐射对人晶状体上皮细胞蛋白质表达影响的研究

发布时间：2018-04-02 00:34

本文选题：微波辐射　切入点：鸟枪法蛋白质组学　出处：《浙江大学》2012年博士论文

【摘要】：背景随着移动通讯设备的大面积普及引用,大众越来越关心生活环境中手机微波辐射可能产生的有害健康的效应。到目前为止,手机微波辐射对晶状体造成损伤的机制以及引发白内障病理过程是眼科学研究讨论的重点之一,也是争论的焦点。近几年手机微波辐射所致蛋白组学改变引起人们的关注。以鸟枪法为代表的新兴蛋白质组学技术在生命科学研究中的广泛应用,使得人们从整体水平研究蛋白质组成和调控活动规律成为可能。如果能从人晶状体上皮细胞的蛋白质组谱中筛选出与微波辐照密切相关的标志性蛋白,将有助于深入研究手机微波辐射所致晶状体损伤的机制。目的探讨1.8GHz微波辐射对体外培养的人晶状体上皮细胞蛋白表达的影响。方法体外培养人晶状体上皮细胞(hLECs)置于sXc-1800细胞辐照系统(发射217Hz脉冲调制的1.8GHz微波)内连续辐照2小时,辐照组辐照强度(specific absorption rate, SAR)为4W/kg,假辐照为0 W/kg。辐照后立即提取全蛋白裂解液,经过溶液内酶解所得样本直接进入Ettan多维液相色谱系统进行分离,而后通过LTQ-Orbitrap质谱仪进行质谱分析。运用化学计量法对质谱结果做相对定量分析,比较辐照前后蛋白质表达谱差异,筛选出微波辐照相关标志性蛋白。运用实时定量RT-PCR技术在转录组水平进一步对质谱分析的结果进行筛选。人晶状体上皮细胞分别接受SAR分别为2,3,4 W/kg的1.8GHz手机频率微波连续辐照2小时,提取总蛋白后用Western blot试验对上述筛选出的标志性蛋白进行验证。结果鸟枪法蛋白质谱比较分析提示4W/kg的微波辐射组与假辐照组之间人晶状体上皮细胞蛋白质表达谱存在差异。8个差异蛋白经过质谱分析和化学计量法统计,获得初步鉴定。实时定量RT-PCR结果显示VCP,USP35和SRP68的1nRNA表达辐照组与假辐照组之间有明显差异(P0.05)。Western blot试验显示4W/kg和3W/kg的微波辐射2小时组VCP和USP35蛋白表达明显高于假辐照组(P0.05),SRP68蛋白表达明显低于假辐照组(P0.05),但2W/kg辐照组与假辐照组之间这3个蛋白的表达均无明显差异(P0.05)。结论鸟枪法蛋白质谱分析是筛选人晶状体上皮细胞微波辐照相关差异蛋白表达的有效方法。微波辐射可导致人晶状体上皮细胞蛋白质表达谱发生改变。经验证的3个蛋白中VCP和USP35可能参与了微波辐照所致人晶状体上皮细胞蛋白质表达质量控制过程,SRP68的改变提示微波辐照对蛋白分泌有影响。
[Abstract]:Background. With the widespread use of mobile communication devices, the public is increasingly concerned about the harmful health effects of microwave radiation from mobile phones in the living environment. The mechanism of lens damage caused by microwave radiation of mobile phone and the pathological process of cataract are one of the focuses of ophthalmology research. In recent years, the proteomics changes caused by microwave radiation of mobile phones have attracted people's attention. The new proteomics technology, represented by bird gunshot, has been widely used in life science research. This makes it possible to study protein composition and regulation activities at the global level. If the iconic proteins closely related to microwave irradiation can be screened from the proteome spectrum of human lens epithelial cells, It will be helpful to study the mechanism of lens damage induced by microwave radiation. Purpose. To investigate the effect of 1.8GHz microwave irradiation on protein expression in cultured human lens epithelial cells. Method. Cultured human lens epithelial cells (hLECs) were exposed to sXc-1800 irradiation system (1.8GHz microwave with 217Hz pulse modulation) for 2 hours. In the irradiation group, the irradiation intensity was 4 W / kg and the false irradiation was 0 W / kg. The whole protein lysate was extracted immediately after irradiation. The samples obtained by enzymatic hydrolysis in the solution were separated directly into the Ettan multidimensional liquid chromatography system. Then the LTQ-Orbitrap mass spectrometer was used to analyze the mass spectrometry. The results of mass spectrometry were analyzed by chemical metrology. The differences of protein expression profiles before and after irradiation were compared and the related iconic proteins were screened out by microwave irradiation. The results of mass spectrometry were further screened by real-time quantitative RT-PCR at transcriptome level. Human lens epithelial cells were exposed to two consecutive hours of microwave irradiation of 1.8GHz cell phone with SAR of 2 ~ 3 ~ 3 ~ 4 W/kg, respectively. The total protein was extracted and verified by Western blot test. Results. The comparative analysis of protein spectrum of bird gunshot showed that there were differences in protein expression profiles of human lens epithelial cells between microwave radiation group and false irradiation group of 4W/kg. Eight differentially expressed proteins were analyzed by mass spectrometry and chemometrics. The results of real-time quantitative RT-PCR showed that there was a significant difference between the irradiated group and false irradiation group in 1nRNA expression of VCP-USP35 and SRP68. Western blot test showed that the expression of VCP and USP35 protein in 4W/kg and 3W/kg 2 hours microwave irradiation group was significantly higher than that in false irradiation group. The expression of SRP68 protein in 2W/kg group was significantly lower than that in false irradiation group, but there was no significant difference in the expression of these three proteins between 2W/kg irradiation group and false irradiation group. Conclusion. It is an effective method to screen differentially expressed proteins related to microwave irradiation in human lens epithelial cells by using bird gunshot protein spectrum analysis. Microwave irradiation can result in changes in protein expression profile of human lens epithelial cells. VCP and USP35 may be involved in the process of protein expression quality control in human lens epithelial cells induced by microwave irradiation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R776.1

【参考文献】