本文選題：微波輻射　切入點：鳥槍法蛋白質組學　出處：《浙江大學》2012年博士論文

【摘要】：背景 隨著移動通訊設備的大面積普及引用,大眾越來越關心生活環(huán)境中手機微波輻射可能產生的有害健康的效應。到目前為止,手機微波輻射對晶狀體造成損傷的機制以及引發(fā)白內障病理過程是眼科學研究討論的重點之一,也是爭論的焦點。近幾年手機微波輻射所致蛋白組學改變引起人們的關注。以鳥槍法為代表的新興蛋白質組學技術在生命科學研究中的廣泛應用,使得人們從整體水平研究蛋白質組成和調控活動規(guī)律成為可能。如果能從人晶狀體上皮細胞的蛋白質組譜中篩選出與微波輻照密切相關的標志性蛋白,將有助于深入研究手機微波輻射所致晶狀體損傷的機制。 目的 探討1.8GHz微波輻射對體外培養(yǎng)的人晶狀體上皮細胞蛋白表達的影響。 方法 體外培養(yǎng)人晶狀體上皮細胞(hLECs)置于sXc-1800細胞輻照系統(發(fā)射217Hz脈沖調制的1.8GHz微波)內連續(xù)輻照2小時,輻照組輻照強度(specific absorption rate, SAR)為4W/kg,假輻照為0 W/kg。輻照后立即提取全蛋白裂解液,經過溶液內酶解所得樣本直接進入Ettan多維液相色譜系統進行分離,而后通過LTQ-Orbitrap質譜儀進行質譜分析。運用化學計量法對質譜結果做相對定量分析,比較輻照前后蛋白質表達譜差異,篩選出微波輻照相關標志性蛋白。 運用實時定量RT-PCR技術在轉錄組水平進一步對質譜分析的結果進行篩選。 人晶狀體上皮細胞分別接受SAR分別為2,3,4 W/kg的1.8GHz手機頻率微波連續(xù)輻照2小時,提取總蛋白后用Western blot試驗對上述篩選出的標志性蛋白進行驗證。 結果 鳥槍法蛋白質譜比較分析提示4W/kg的微波輻射組與假輻照組之間人晶狀體上皮細胞蛋白質表達譜存在差異。8個差異蛋白經過質譜分析和化學計量法統計,獲得初步鑒定。實時定量RT-PCR結果顯示VCP,USP35和SRP68的1nRNA表達輻照組與假輻照組之間有明顯差異(P0.05)。Western blot試驗顯示4W/kg和3W/kg的微波輻射2小時組VCP和USP35蛋白表達明顯高于假輻照組(P0.05),SRP68蛋白表達明顯低于假輻照組(P0.05),但2W/kg輻照組與假輻照組之間這3個蛋白的表達均無明顯差異(P0.05)。 結論 鳥槍法蛋白質譜分析是篩選人晶狀體上皮細胞微波輻照相關差異蛋白表達的有效方法。微波輻射可導致人晶狀體上皮細胞蛋白質表達譜發(fā)生改變。經驗證的3個蛋白中VCP和USP35可能參與了微波輻照所致人晶狀體上皮細胞蛋白質表達質量控制過程,SRP68的改變提示微波輻照對蛋白分泌有影響。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R776.1

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本文編號：1698025