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
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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(P<0.05)。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(P<0.05),SRP68蛋白表达明显低于假辐照组(P<0.05),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(P>0.05)。
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。
Background
The widespread use of mobile telecommunications has aroused public concern with potential health risks associated with exposure to microwave eletromagnetic fields from these novel products of mobile phones. Aging is one of the most common causes for cataract, but many other factors are involved. Microwave eletromagnetism radiation has been regard as one of the much more important risk factors. Also there is concern about microwave radiation-induced proteomic changes. The shotgun method, as the representative of emerging proteomic technologies, has been widely used in life science research, which can be expected to show the changes in protein expression profile. The potential molecular targets obtained by proteomic analysis might be applied in further investigations to ascertain the possible alteration of protein expression levels which are related to the biological effects induced by microwave radiation and to understanding the possible pathogenic mechanism of cataract.
Objective
To investigate the impact of 1.8GHz microwave radiation on the protein expression of human lens epithelial cells (hLECs) in vitro.
Methods
The hLECs were exposed and sham-exposed to 1.8GHz microwave radiation for 2 hours. The specific absorption rate (SAR) was 4 W/kg. After exposure, the proteome extracted from LECs were loaded on the Ettan MDLC system connected to the LTQ-Orbitrap MS. Chemometrics method was used to analyze the differences of MS/MS between exposed- and sham exposed-groups, and to screen the candidate microwave-induced biomarker proteins.
QRT-PCR was used to screen the candidate biomarkers on mRNA level in hLECs exposed to 1.8GHz microwaves (SAR,4 W/kg) for 2 hours.
After the hLECs were exposed to 1.8GHz microwaves (SAR,4,3 and 2 W/kg) for 2 hours, the western blot assay was utilized to detect the expression of microwave-induced biomarker proteins.
Each experiment was repeated at least three times.
Results
The results of shotgun proteomic analysis indicated that there were 8 proteins with the differential expression between exposure group and sham-exposure group. The results of qRT-PCR showed that there were the expressional differences of 3 genes (VCP, USP35 and SRP68) between exposure group and sham-exposure group. The results of western blot assay exhibited that the expressional levels of two proteins (VCP and USP35) significantly increased and the expressional level of protein SRP68 significantly decreased in hLECs exposed to 1.8GHz (SAR,4 and 3 W/kg) microwave radiation for 2 hours, as compared with sham groups (P<0.05), but there were no significant expressional differences of VCP, USP35 and SRP68 proteins between the exposure group and sham-exposure group when SAR was 2 W/kg (P>0.05).
Conclusion
The shotgun method can be applied to screen the differentially expressed proteins in hLECs exposed to 1.8GHz microwaves, and 3 protein biomarkers associated with microwaves radiation were validated by western blot assay. VCP and USP35 proteins might involve in the protein quality control reaction of hLECs exposed to microwave. The alteration of SRP68 protein indicated that microwave may affect the protein secretion.
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(P<0.05)。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(P<0.05),SRP68蛋白表达明显低于假辐照组(P<0.05),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(P>0.05)。
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。
Background
The widespread use of mobile telecommunications has aroused public concern with potential health risks associated with exposure to microwave eletromagnetic fields from these novel products of mobile phones. Aging is one of the most common causes for cataract, but many other factors are involved. Microwave eletromagnetism radiation has been regard as one of the much more important risk factors. Also there is concern about microwave radiation-induced proteomic changes. The shotgun method, as the representative of emerging proteomic technologies, has been widely used in life science research, which can be expected to show the changes in protein expression profile. The potential molecular targets obtained by proteomic analysis might be applied in further investigations to ascertain the possible alteration of protein expression levels which are related to the biological effects induced by microwave radiation and to understanding the possible pathogenic mechanism of cataract.
Objective
To investigate the impact of 1.8GHz microwave radiation on the protein expression of human lens epithelial cells (hLECs) in vitro.
Methods
The hLECs were exposed and sham-exposed to 1.8GHz microwave radiation for 2 hours. The specific absorption rate (SAR) was 4 W/kg. After exposure, the proteome extracted from LECs were loaded on the Ettan MDLC system connected to the LTQ-Orbitrap MS. Chemometrics method was used to analyze the differences of MS/MS between exposed- and sham exposed-groups, and to screen the candidate microwave-induced biomarker proteins.
QRT-PCR was used to screen the candidate biomarkers on mRNA level in hLECs exposed to 1.8GHz microwaves (SAR,4 W/kg) for 2 hours.
After the hLECs were exposed to 1.8GHz microwaves (SAR,4,3 and 2 W/kg) for 2 hours, the western blot assay was utilized to detect the expression of microwave-induced biomarker proteins.
Each experiment was repeated at least three times.
Results
The results of shotgun proteomic analysis indicated that there were 8 proteins with the differential expression between exposure group and sham-exposure group. The results of qRT-PCR showed that there were the expressional differences of 3 genes (VCP, USP35 and SRP68) between exposure group and sham-exposure group. The results of western blot assay exhibited that the expressional levels of two proteins (VCP and USP35) significantly increased and the expressional level of protein SRP68 significantly decreased in hLECs exposed to 1.8GHz (SAR,4 and 3 W/kg) microwave radiation for 2 hours, as compared with sham groups (P<0.05), but there were no significant expressional differences of VCP, USP35 and SRP68 proteins between the exposure group and sham-exposure group when SAR was 2 W/kg (P>0.05).
Conclusion
The shotgun method can be applied to screen the differentially expressed proteins in hLECs exposed to 1.8GHz microwaves, and 3 protein biomarkers associated with microwaves radiation were validated by western blot assay. VCP and USP35 proteins might involve in the protein quality control reaction of hLECs exposed to microwave. The alteration of SRP68 protein indicated that microwave may affect the protein secretion.
引文
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