| Aging is a multi-level, multi-dimensional, complex progressive changing process happening in vivo involving multiple organs. Its consequence is the recession or disorder of body function. The basis of organism aging is cell aging, which is regulated by genetic and environmental factors together. At present, although there are a variety of theories explain the happening of aging from different aspects, its key mechanism and regulation mode are not clear.Silent mating type information regulation 2 homolog 1(SIRT1) is NAD+ dependent histone deacetylase. It affects different downstream molecules such as P53, P16, FOXO, NF-κB and PGC-1α through deacetylation, thus regulating different process of cells such as aging, apoptosis and metabolism. Research shows that SIRT1 plays a key role in caloric restriction(CR) induced extension of life cycle, thus attracting widespread interest of the researchers. SIRT1 can inhibit aging of endothelial cell and stem cells that is induced by oxidative stress and high sugar in vitro; studies about rodent also shows that SIRT1 plays an important role in aging and the decrease of age-dependent organ function. For role of SIRT1 on some downstream molecules, or even in physiological or pathological process, however, there are often some contradictory reports. This suggests that there may be more complex mechanism and regulatory factors of SIRT1 function.SIRT1 sequence is highly conserved, and splice variant is not considered absent. But Lynch et al. in 2010 found there was alternative splicing in SIRT1 pre-m RNA, namely generation of SIRT1 with the absent of exon 8(SIRT1-ΔExon8). Recent studies have found that there are large differences between biological function and regulation mechanism of human and mouse SIRT1-ΔExon8 with silent mating type information regulation 2 homolog 1-Full Long(SIRT1-FL). During the study, we also found that there was SIRT1 with the absent of exon 7(SIRT1-ΔExon7) which had similar biological function and regulation mechanism as SIRT1-ΔExon8 in mice. SIRT1 plays an important role in aging, but whether its splicing variant plays a role has not been reported yet.V Based on the above content, we design the following topic to attempt to explore the role of SIRT1 and its alternative splicing variants in the aging process and possible mechanisms.Part I The effect of SIRT1 and SIRT1-ΔExon8 on the senescence model of the 293 T cells in vitro.Objective:To study the effect of SIRT1 and SIRT1-ΔExon8 on the aging process of 293 T cells in vitro by observing the m RNA level of SIRT1 and SIRT1-ΔExon8 in the D-gal induced cells senescence model.Methods:1. Counting Kit-8 was used to identify the effect of D-gal on 293 T cells viability at different concentrations(0,5,10,15,20 g/L). 2. The 293 T cells were cultured in DMEM medium containing 10 g/L D-gal at the third passage and amplified to sixth passage in vitro, the senescence model of the 293 T cells, SA-β-gal staining was used to determine the percentage of the positive aging cell. 3. The m RNA level of SIRT1 and SIRT1-ΔExon8 were measured in both the induction group and control group of the 293 T cells by RT-PCR assay.Results:1. The CCK-8 assay showed that the 10 g/L D-gal could induce 293 T cells senescence and the cell viability was higher than the others.2. The percentage of the SA-β-gal positive cell in induction group(74.25% ? 3.62%) is higher than control group(7.68% ? 1.43%)(P <0.05). 3.The m RNA expression level of SIRT1 and SIRT1-ΔExon8 in the induction group of 293 T cells significantly increased by 78.26% ? 0.05% and 88.03% ? 0.03% compared to the control group(P <0.05).Summary:1. D-gal could induce 293 T cells senescence model successfully.2.The SIRT1 and SIRT1-ΔExon8 may act as the key regulator in 293 T cells senescence.Part II Changes of SIRT1-ΔExon7 expression in rat aging and the significanceObjective:To confirm the alternative splicing variants of SIRT1 in rat hippocampus; To observe the changes of SIRT1-FL and SIRT1-ΔExon7 expression in hippocampus of rats in different age groups, thus preliminarily discussing the changes of SIRT1-FL and SIRT1-ΔExon7 expression in aging process.Methods:1. Through agarose gel electrophoresis and c DNA sequencing for PCR product to detect whether the amplified gene is SIRT1-ΔExon7; 2. SA-β-gal staining was used to observe rat hippocampus of different day age(P7, P90, P720); 3. RT-PCR was used to detect expressions of SIRT1-FL and SIRT1-ΔExon7 m RNA in hippocampus of rats in different age groups.Results:1. The agarose gel electrophoresis and sequencing showed there were expressions of SIRT1-ΔExon7 in rat hippocampus; 2. SA-β-gal staining results showed that aging cells with positive SA-β-gal can accidentally be seen in newborn mice, while aging cells with positive SA-β-gal that a large amounts of cytoplasm was blue stained can be seen in aging rats. It was promoted that with the age increased, the activity of brain SA-β-gal was also increased and aging cells were gradually increased; 3. RT- PCR results showed that expressions of SIRT1-FL m RNA in P90 group and P720 group were not statistically different compared to that of P7 group; but expression of SIRT1-ΔExon7 m RNA were increased by 77.40 ± 7.38% and 89.28 ± 9.23% respectively compared to P7 group(P <0.05).Summary:1. There is alternative splicing variant of SIRT1 with the absence of exon 7 in rats; 2. SIRT1-ΔExon7 is highly expressed in aging hippocampus.Part III Expression of SIRT1-ΔExon7 in inducted rat MSCs aging cells and significanceObjective:Rats MSCs that were induced to form aging cell models were used to verify thefindings in rats, and HDAC activity of SIRT1 was tested to study its function, in order to further study the roles of SIRT1 and SIRT-Δ7 in the aging process.Methods:1. The 18 th generation of MSCs were stained by β- galactosidase(SA-β-gal) to observe the positive rate of aging cells. 3. RT-PCR was used to detect the expressions of SIRT1-FL and SIRT1-ΔExon7 m RNA of cells in aging group and the control group.Results:1. Along with the continuous passage, positive SA-β-gal aging cells were increased. Positive rate of aging group were 57.64 ± 7.39%, which were higher than that of the control group(16.35 ± 2.17%)(P <0.05). 3. RT-PCR results showed that SIRT1-FL m RNA expression in the aging group were lower than the control group by 20.09 ± 2.74%(P <0.05); And expressions of SIRT1-ΔExon7 m RNA was higher than the control group by 132.85 ± 11.07%(P <0.05). 4. The analysis results on acetylation activity of SIRT1 showed that.Summary:1. The significant increase of SIRT1-ΔExon7 expression in aging cells promotes that SIRT1-ΔExon7 may have a promotion role in aging process;Conclusions:1. It is for the first time to confirm the existence of SIRT1 alternative splicing in rat. It forms the alternative variant SIRT1-ΔExon7, instead of the previously reported SIRT1-ΔExon8.2. Changes of SIRT1-ΔExon7 expression in aging process are observed in vivo animals and in vitro experiment(cells of two origin),which suggested that alternative splicing of SIRT1 may participate in the aging process.3. It is for the first time to confirm the existence of SIRT1 alternative splicing in rat. It forms the alternative variant SIRT1-ΔExon7, instead of the previously reported SIRT1-ΔExon8. |
PDF Full Text Request