Research Of Differential Expression Profile Of Total Protein Between Wild Type MEF And MEF Deficient Of PLC-γ1

phopholipase C- γ1(PLC-γ1) is widely known to play an important role in regulating cell proliferation and differentiation,development of the organisms,cell transformation and oxidative stress.Till now,the mechanism how phopholipase C- γ 1 acts can not be thoroughly illustrated,nor has the interaction between PLC- γ 1 pathway and other signal pathways been systematically reported. This research chose 2-DE+MS as the basic method from all kinds of proteomics strategies and compared the total protein expression map of MEF genetically deficient in PLC- γ 1 (PLC-γ1-/-) to that of wild type MEF (PLC-γl+/+) aimed to find some protein spots differentially expressed,thus we can discuss the impact of knockout of PLC- γ1 on signal transduction initiated by growth factors such as EGF comprehensively.In this way,we can study the biological function of PLC- γ1 and mechanism of PLC- γ1 pathway indirectly,which will contribute a lot to further analysis.This study optimized some methods at first,namely,selecting the best protein extraction method,bulding clear and high-resolution composite 2-DE map with IPG strips of different pH gradients,and picking out the silver staining method which is compatible with further PMF analysis,with less background staining and high sensitivity.We compared the 2-DE map of PLC-γ1+/+ and PLC-γ1-/-24h after the EGF stimulation.Good reproducibility and high comparability was achived within three independent experiments .About 1450 ±100 spots were detected both in the wide type group( PLC-γl+/+) and the knokout group (PLC-γ1-/-) by MELANIE 3.02 software after manual correction. Correlation analysis was done using the %vol of matched spots from the two group,which is a useful function offered by the software.Consequently,the correlation coefficients of the PLC-γ1+/+ group were 0.858,0.867,0.875,yet the other were 0.842,0.846,0.863.Wecut out 7 bad-matched spots(exist in one group and out of the other) after analysis by auto matching and manual correction,three of which has been analyzed by PMF up to now.Of the three analyzed,one has been idendified as vimentin definitely,while the other two can not be identified. Vimentin is a kind of type IIIinterfilament and has high structural homogenity with c-fos,c-jun and CREB. Vimentin also modulates translocation of some signal molecule. Whether vimentin is a downstream event of PLC-γ1 pathway and Whether vimentin is an interchange site between PLC-γ1 pathway and other signal pathways needs further confirmation by molecular biology analysis such as yeast two-hybrid system and western blot.