Protome Analysis Of The Human Lens Epithelial Cells Exposed To Microwave Of Mobile Phone-type

Cataract is any opacity in the lens, and is the most common cause of visual impairment in clinic and the leading cause of permanent blindness in the elderly. Among the forty to forty-five millions of cataract, about forty six percent was caused by cataract. Aging is the most common cause of cataract, but many other factors was involved, including glaucoma, metabolic diseases (such as diabetes mellitus), toxins, trauma, systemic diseases, and heredity diseases. But presently, another risk factor called electromagnetism radiation has been regard more and more important, and microwave is one kind of the electromagnetism radiation. Anyway, controversy exists concerning the health risks from exposures to electromagnetism /microwave radiation. Research on the mechanisms of lens opacification has revealed a number of individual, identifiable cataractogenic stresses in human. Microwave radiation is one of them. Low power microwave radiation higher than 10 mW/cm2 can affect the proportion of water soluble protein (WSP) in cultured rabbit lens, cause changes of lens hydration and induce lens opacity. The signal transduction mechanisms in lens epithelial cellls after exposure to microwave radiation may related to increasing of PKC-α, c-fos and c-jun expression.The analysis of cellular proteins expressed by a genome, by a cell or by a tissue, termed proteomics, represents a powerful analytic technology to enhance the study ofthe diagnosis, treatment and prevention of human disease. The techniques includs two dimensional gel electrophoresis (2-DE), mass spectrometry (MS), and bioinformatics. Proteomics can be expected to show the changes in the protein expression profile, thus lead to the identification of new molecular markers and potential therapeutic targets. So, we used proteomics-based techniques to analyze protein expression patterns of human lens epithelial cells. It is hoped that this information will yield further insight into the molecular mechanisms and signaling pathways underlying lens cataract development and lead to the identification of key proteins that can be used for diagnostic, prognostic and therapeutic purposes.In the first, we constructed stable protein maps of human lens epithelial cells and then established the proteomics experiment system. In further studies, the aim was to evaluate the possible effects of in vitro 1800 MHz GSM-like exposure on human lens epithelial cells, using analytic technology of proteomics.PartiThe establish of proteome system of human lens epithelial cellsObjective The aim of the research is to establish the techniques in the proteomeresearch of Human lens epithelial cells, including the techniques of Two-dimensionalelectrophoresis and Mass spectrometry.Methods Total protein of cultured human lens epithelial cells was extracted withtwo kinds of different methods. The proteins were separated differently usingimmobilized pH gradients 2-DE and visualized by silver staining. The digitizedimages which got with GS-800 scaner were then analyzed with PDQuest software inorder to establish the differential expression profiles. The differential protein spotswere cut from the gels using proteomework spot cutter and subjected to in-geldigestion with trypsin. The digested peptides' separation was conducted by aFinnigan LCQ MS coupled with a Surveyor HPLC system.Results A high resolution and reproducible 2-ED image is successfully obtained.The maps of 2-DE showed that lens proteins were in the section of pH 4-7 and therelative molecular weight was 17kD~72kD. While relative molecular weight of moreabundant proteins was localized at 19kD~50kD, as well as the isoelectric points werefound to lie between 5~7. Two of these proteins were identified by massspectrometry and database queries.Conclusions We have constructed stable protein maps of human lens epithelialcell, the technique will be used in human lens research to characterize physiologicalprocesses and disease.Part IIProteome analysis of the human lens epithelial cells exposed to microwave Objective The aim of this study was to evaluate the possible effects of in vitro 1800 MHz GSM-like exposure on human lens epithelial cells. Methods In three separate experiments, human lens epithelial cells were exposed and sham-exposed (6 dishes each) to 1800 MHz GSM-like radiation for 2 hours. The SAR is 1.0, 2.0, and 3.5w/kg respectively. At the moment of the radiation finished, proteome were extracted from lens epithelial cells immediately and Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE), silver staining and PDQuest 2-DE analysis software were used to separate and analyze the proteome of lens epithelial cells exposed and sham-exposed. The differential expressed protein spots were cut from the gels using proteome work spot cutter and subjected to in-gel digestion with trypsin. The digested peptides' separation was conducted by a Finnigan LTQ MS coupled with a Surveyor HPLC system. And protein identification was performed by application of the SEQUEST search program to the SWISS PROT human non-redundant protein database. To further confirm the results of HSP70, Western-blot analysis and semi-quantitative RT-PCR were performed. Results Compared the protein profiles of cells exposed with those of sham-exposed, 7 proteins were detected as dysregulated. Five differentially expressed protein spots were selected and identified with electrospray ionization tandem MS (ESI-MS-MS) . Being analyzed by ESI-MS-MS and database searching, HSP-70 and hnRNP K was up-regulated in cells exposed. The results were confirmedby RT-PCR, and western blot.Conclusions The 2-DE combined with MS is one of powerful tools for screeningpotential electromagnetism-reaction markers. HSP-70 and hnRNP K might inviole inthe stress reaction of human lens epithelial cells exposed to microwave. Theseresponses of cells are the non-thermal effects of electromagnetic field.