The Preliminary Study Of Serum Differential Protein Involving In The Occurrence Of Spontaneous Breast Cancer In Mice

Objects: Using the two-dimensional gel electrophoresis(2-DE) technical to analysis the serum proteome of the normal TA2 mice, normal TA1 mice and the spontaneous breast cancer mice. Combining the technology of mass spectrometry and the methods of bioinformatics, we can search and confirm the special serum differential protein in spontaneous breast cancer. It will establish the foundation of the protein mass spectrometry which will help the early diagonosis of the breast cancer. Methods:1 We got the peripheral blood from the spontaneous breast cancer mice, normal TA2 mice and normal TA1 mice. After centrifugaling at 5000rps/min in 10 minutes, we got the upper serum to detect in 2-DE. Immobiline pH gradients isoelectric focusing was used as 1D, and vertical SDS-PAGE as 2D. Some applications, such as sample preparation and volume of loading sample, choice of IPG gel, were improved. Sliver staining, and Image Master 2D 5.0 analysis software were used to compare the sera of the spontaneous breast cancer mice, normal TA2 mice and normal TA1 mice. The result of the 2-DE were evaluated by the methods of statistics, and the ID of the differential protein spots were confirmed.2 The differential protein spots which were confirmed by 2-DE, were analyzed again by 2-DE. After coomassie blue staining, we used Electrospray Ionization Mass Spectrometry (ESI-MS) to identify the differential protein spots. Using theSEQUEST database to analyse the peptide mapping and the sequence of the amino acid which were obtained by ESI-TOF. From IPI mouse database searching, we confirmed the protein and analyzed the relationship between the proteins and breast cancer. Results:1 From comparing the sera of the spontaneous breast cancer mice, normal TA2 mice and normal TA1 mice, we successfully get the images of the 2-DE which were stained by coomassie blue staining. Analyzing by Image Master 5.0 2-DE software, we get fifteen protein spots which were three times difference in different gel. Six protein spots unexpressed in spontaneous breast cancer mice and normal TA2 mice, but expressed in normal TA1 mice;four protein spots down-regulated in spontaneous breast cancer mice , up-regulated in normal TA1 mice, unexpressed in normal TA2 mice;one protein spot down-regulated in spontaneous breast cancer mice , up-regulated in normal TA2 mice, unexpressed in normal TA1 mice;two protein spots unexpressed in spontaneous breast cancer mice , down-regulated in normal TA2 mice, up-regulated in normal TA2 mice;two protein spots down-regulated in spontaneous breast cancer mice and normal TA2 mice, up-regulated in normal TAlmice.2 The differential protein spots which were confirmed by 2-DE, were analyzed again by 2-DE. After coomassie blue staining, we got eleven spots. We used ESI-TOF to detect these eleven spots, and get nine peptide mass fingerprint (PMF). Searching in IPI mouse database, we confirmed four proteins, such as Immunoglobulin heavy chain, Serum albumin precursor, Clusterin precursor, and Apolipoprotein A-I precursor. All of these proteins were unexpressed or down-regulated in pontaneous cancer mice and normal TA2 mice, but expressed or up-regulated in normal TA1mice. Conclusions:1 Based on the different molecule and isoelectric point of the proteins, we used the 2-DE to separate the protein components of the spontaneous breast cancer micer normal TA2 mice and normal TA1 mice. Using the analyzing software, we find the differentially expressed proteins among the spontaneous breast cancer mice, normal TA2 mice and normal TA1 mice.2 Differentially expressed proteins which were identified by ESI-TOF provided reference foundation to serum diagnosis of human breast cancer. It may have the relationship between Immunoglobulin heavy chain, Clusterin precursor, and the occurrence of spontaneous breast cancer in mice. Immunoglobulin heavy chain was unexpressed in spontaneous breast cancer mice which result in immunodeficiency in mice. The immunodeficiency can make the mice susceptible infectioned by bacteria and virus which may induce cancer. Clusterin precursor may have the relation with regulation of immunity in mice. Clusterin precursor unexpressed in spontaneous breast cancer mice may have the cancer cells escaped from the immune system. We haven't found the relationship between apolipoprotein A-I precursor ,serum albumin precursor and breast cancer. Apolipoprotein A-I precursor may influence some taches of apoptosis in spontaneous breast cancer. The expression of serum albumin precursor in normal TA1 mice may because of experiment error or we unknown the relationship of breast cancer. There are a lot of work for us to validate and determinate.