| The SAM (Senescence-accelerated mouse) model has inherited early onset and irreversible advancement of senescence. One of senescence prone (P) strain, SAMP8, is found to have age-related deficits in learning and memory and immune. SAMP8 show an age-related decline in learning and memory even at 4 months of age, and a severely impaired learning and memory ability at 8-10 months of age, meantime, which do not lost in mice with normal aging characteristics such as senescence-accelerated mouse resistant/1 (SAMRl) or other strain mice (such as C57BL) at 12 month-old. Now SAM are used as a ideal animal model for study of biological mechanisms of senescence, drugs for preventing and curing Alzheimer's disease (AD) and senescence immunology in a wide range. Differing from transgenic mouse strains or gene knock-out strains, the phenotypes of senescence in SAMP8 are inherited and made no modification, so its mechanisms of aging occurrence are complicated. The cause and mechanism of advanced senescence in SAMP8 are not clear until this day.In our large number of work on AD, NIM and senescence immunology with SAM, we found that SAMP 8 may be a reasonable and useful animal model for their stable and typical pathologial phenotypes and satisfactory reacts to drugs. To find age-related pathologic proteins correlated with occurrence and development of SAMP8 senescence phenotypes, and explore posibility for using these proteins as drug targets, we have investigated technique and methods of serum proteomics at first, then researched the age-matched changes of serum proteins in SAMRl and SAMP8 with this method and identified the typical differential expressed proteins.1 Comparative proteomic study on serum of SAM1.1 Differential expression of serum proteins in SAMP8 and SAMRl.Proteins concentration of sera from 2, 6and 12 month-old SAMP8 and age-matchedSAMRl were measured by Brad-ford, and separated by two dimensionalelectrophoresis (2DE). After silver staining, the changes in sera level of each protein were estimated between SAMP8 and SAMR1 and among different month-old mice with ImageMaster 2D Elite 4.01. Differential expression of serum proteins in SAMP8 and SAMR1 mainly distributed in 5 regions:In region 1, 2DE map in the molecular mass range between 2.5-3.5 kDa and in the isoelectric point range between 4.0 and 5.0, there were no less than 5 proteins only existed in region 1 of sera from 2-, 6- and 12-month-old SAMP8, and disappewered in sera from age-matched SAMR1.In region 2, which is in the molecular mass range between 0.8-1.2kDa and the isoelectric point range between 3.5-4.5, 2 proteins were downregulated in sera from 2-, 6- and 12-month-old SAMP8.In region 3, 2DE map in the molecular mass range between 5.3-6.7 kDa and in the isoelectric point range between 6.0 and 8.5, 4 proteins were detectable in 2-month-old SAMR1 and lost in SAMP8 with same age. They also appeared in 6-month-old SAMR1 in this region, but their abundants were too high to be validated. Furthermore, 5 proteins were down-gradulated expression and more proteins were different both on pattern and abundant in region 3 of 6-month-old SAMR1 and SAMP8, the later was separated not enough to be identified by mass spectrometry. There were significant differences in region 3 between 12-month-old SAMP8 and SAMR1, but differential proteins were separated not enough to be identified by mass spectrometry.In region 4, 2DE map in the molecular mass range between 2.8-3.2 kDa and in the isoelectric point range between 6.0 and 8.5, 15 differential expressed proteins were shown between sera from SAMR1 and SAMP8 with 2months age, among them, 7 proteins were up-gradulated in SAMP8, 5 proteins only existed in SAMR1 and 3 proteins only in SAMP8, and more differential proteins between 2-month-old SAMR1 and SAMP8 were separated not enough to be identified by mass spectrometry. There also were many differential proteins separated not enough to be identified by mass spectrometry in region 4 of 6- and 12-month-old SAMR1 and SAMP8. Moreover, a differential protein in 6-month-old SAMP8, isoelectric point is about 6, was same as that in 2-month-old SAMP8.In region 5, which is in the molecular mass range between 1-2.5kDa and the isoelectric point range between 6.5-9.0, 3 proteins detected in 2-month-old SAMR1 lost in age-matched SAMP8. No significant difference was shown in region 5 between 6-month-old SAMR1 and SAMP8. At 12 months of age, aboundants of 4 proteins appewered only in SAMR1 at least were low to detected by Coomassie brilliant blue staining which was the essentials of mass spectrometry in this study.These results showed that a large number differential expressed proteins exist between sera from SAMR1 和 SAMP8, and some of them were different in expressed level, the others were appearance or disappearance. They may be correlated with senescence phenotypes and certain pathological and physiological characteristics in SAMP 8. These changes in sera of SAM might be impaortant manifestations in aging process.1.2 Age-related changes in proteins of sera from SAMR1 and SAMP8.We investigated age-related changes in sera levels of spot 1-7 from 2, 6, 12 and 15month SAMP8 and SAMR1. Spot 1-5 appeared in sera of SAMR1 with 15 months age and could not detected in sera of other month-old SAMR1 and expressed steadily in sera of 2, 6, 12 and 15month SAMP8, shows that these proteins only appear in certain aging states and indicates that they are correlated to aging degree of body. SAMP8 and SAMR1 showed no obvious changes of sera levels of spot 6 and spot 7 among mice except sera from 15-month-old SAMP8, in which spot 6 and spot 7 divided into two spots respectedly.1.3 Identification of age-matched proteins with mass spectrometry. We determined peptide mass fingerprint and peptide sequence of 7 age related proteins with Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and quadrupole-time of mass spectrometry (Q-TOF-MS). Searching database, we found that spot 3 was M-T413, defined to be immuno-globulin (Ig) kappa chain V region. M-T413 is monoclonal CD4 antibody binding to the CD4V1 region and displays a peculiar activity to inhibit HIV infectivity. M-T413 is also used to treat self-immune diseases. It maybe correlated with immuno-deficiency in SAMP8. Spot 4 was 32C2_A, which is chain A of Fab fragment,which can suppress activity of cytochrome P450 aromatase (P450 arom). P450 arom is responsible for the conversion of androgens to estrogens. 32C2_A inhibiting its activity may result in decreased estrogen level in SAMP8 and old SAMR1, which accelerated the aging process. Spot 6 was apolipoprotein A2 (apoA2). Three variants of apoA2 (type A, B, and C) with different amino acid substitutions at four positions are present in mice. The SJL/J, A/J, LLC, SAMP1, SAMP2, SAMP7, SAMP9, SAMP 10 and SAMP11 strains with a high incidence and severe senile amyloidosis have type C Apo A2 with glutamine at position 5, whereas the SAMR1, SAMR4, SAMP6, BALB/c, CBA/N, C3H/He, DDD and NZB strains with a very low incidence of amyloidosis have type BApo A2 with proline at position 5. SAMP8, AKR/J, BIOBR, C57BL/6J and NSYstrains with type AApo A2, which has methionine at position 26, showed a mild amtloid deposition in several organs. Spot 5 was alpha-fetoprotein. No correlation was found between alpha-fetoprotein and aging, which might give a new view to aging process. No matching results of spot 1, 2 and 7 were found out, they might be novel proteins not to be submitted to database.In addition, obvious difference existed in region 3 and region 4 between sera protein patterns from SAMP8 and SAMR1, and two regions are Ig heavy chain region and Ig light chain region respectively based on database. It revealed that immune was closely associated to aging. Moreover, these Igs with various functions deal with the other systems such as nervous system and endocrine system. For example, 32C2_A may reduce estrogen level, which maybe affects nurishment of central nerves and result in cognitive deficits. Fifferences of sera proteins in region 3 and region 4 between SAMR1 and SAMP8 indicated that alteration of kind and quantity of Ig might closely related to accelerated senescence.2. The changes of M-T413 mRNA expressed level in splenocytes from SAMP8 Identified by mass spectrometry, M-T413, which abundant significantly increased in sera of SAMP8, is immuno-globulin (Ig) kappa chain V region. M-T413 is endogenetic moiioclonal CD4, thus it may be come from immune system. To show M-T413 mRNA expressed level in splenocytes from SAMP8, we studied it with RT-PCR. The obvious changes of M-T413 mRNA showed in aging processes of SAMR1 and SAMP8. The value of M-T413 mRNA was lowst in SAMR1 at 2 monthsage, then, it increased gradually and reached to peak at 6 months age which followed by a gradual decrease until 15 months age whose M-T413 mRNA value was higher than that at 2 months age. M-T413 could not be detected in serum of 6-month-old SAMR1, although its mRNA level was highest.Unlike SAMR1, the value of M-T413 mRNA was highest at 2 months age in SAMP8, and decreased gradually to bottum at 12 months age, which resembled 15 month in M-T413 mRNA level. It was coincident to that the abundant of M-T413 was higher in serum from 2-month-old SAMP8. However, no obvious differences of mRNA expression levels were observed between SAMP8 and SAMR1 at 6, 12 and 15 months of age.It can be comformed that splenocyte is one of important resources of M-T413. M-T413 mRNA expressed level changed obviously related to age in splenocyte from SAMR1 and SAMP8. M-T413 mRNA level was lowest at 2 months followed a gradual increase to 6 months, then declined. Meantime, highest M-T413 mRNA level was displayed and then discreased. Compared to age-matched SAMR1, M-T413 mRNA level was distinctly enhanced in splenocyte from 2-month-old SAMP8.3. Age-related changes in CD4+ cell number of whole blood fromSAMP8.It is possible that M-T413, an endogenetic monoclonal CD4 antibody, may effect the quantity of CD4+ cell number in whole blood. So we observed dynamic changes of CD4+ cell number of SAMP8 during aging process.The age-associated decreased CD4+ cell number was found both in SAMR1 and SAMP8. It was higher at 2-, 6-month-old SAMR1 and fall clearly at 12- and 15-month-old SAMR1. Age-related alteration of CD4+ cell number in SAMP8 conformed to that of SAMR1, just it was more evident. Otherwise, all CD4+ cell number of 2, 6, 12 and 15 months SAMP8 were inferior that of age-matched SAMR1.Combined with analysis of Age-related changes in M-T413 in sera from SAMR1 and SAMP8, it can be found that both level of M-T413 mRNA in splenocyte and CD4+ cell number of whole blood from 6-month-old SAMR1 was highest, and M-T413 appeared in serum from SAMP8 since 2 months after birth, both level of M-T413 mRNA in splenocyte and CD4+ cell number of whole blood from 2-month-old SAMP8, then markedly declined.The age-related change trends of M-T413 mRNA in splenocyte and level of M-T413 niRNA in splenocyte and CD4+ cell number of whole blood were basically similar which meaned closed relation between them. As a endogenetic monoclonal CD4 antibody, M-T413 might be one of factors resulted in decreaded number of CD4+ cells in whole blood of SAM.In conclusion, serum proteins changed distinctly when age-associated cognitive deficit appeared. So we can start to investigate pathological and physiological mechanism of aging and find drug targets with serum proteins in order to accelerate the experimental results transfermation to clinical applications. Our studies revealed that form of selfantibodies play an important role in aging process. These selfantibodies with various functions can affect the functions of the other systems such as nervous system and endocrine system. Meantime, they also indicate that studies of differently expressed protein function are keys of more applications of these proteins.
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