Analysis Of Epigenetic Enzyme Screened During HPDLSCs Aging

BackgroundUnlike adult cells, msesenchymal stem cells(MSC) appear to possess the proliferative capacity and multipotent differentiation capacity to form bone tissue, adipose tissue, cartilage tissue and so on. So far, MSC has been found from several tissues and organs, including bone tissue, periodontal ligament tissues, intestinal tissues, cardiac muscle tissues and so on. MSC, with sevral advantages in proliferation and differentination, has become siginificant seed cells to to reestablish lost tissues because inflammation, injury and surgical operation throughout adulthood in tissue engineering field and been paid more attention to by future life sciences and medical sciences. During the process of application of MSC therapy, however, there are a lot of problems, including how to obtain the MSC, how to induce MSC to differentate to form the needed tissues and how to solve several fuctional weakness during aging and expansion in vitro, which weaken the curative effect of MSC threapy lagely, especially the phenomenon following MSC aging. Aging is characterized by a gradual reduction in the ability to cope with physiological challenges to the external environment stimulation, which ultimately induces disease susceptibility and eventually leads to death. Thus, due to pathological factors, environmental factors and so on, the self-regulation mechanism of mesenchymal stem cel s is gradually abnormal, whose biological characteristics is unstable during aging.Aging, as a complex life process, is caused by a variety of factors, including gene mutations in signaling pathways(including insulin signaling pathway and WNT signaling pathways, etc.), abnormal regulation of transcription factor, unusual chromosome, and the change of external environment factors. Effect of environmental factors on aging is often accomplished by changing the state of chromosome. This change in state of chromosome is due to the change of physiological conditions or environmental stimuli or in some pathologic condition. Compared to simple genetic mutations and abnormal regulation of transcription factors, this change in state of chromosome is more persistent and more sustainable in the body is, and can be inherited to the next generation. At present more attention on the study of aging in the change of the gene expression level. Epigenetics is a corresponding concept to genetics. The development and maintenance of an organism is orchestrated by the difference of the expression leve of the related gene between different individuals is based on base pair sequence in term of genetics; Conversely, during aging, The development and maintenance of an organism is orchestrated by a set of chemical reactions that switch parts of the genome off and on, in term of epigenetic. In other words, epigenetics is not based on the genetic sequence but base on the state of chromatin modified by DNA methylation, histone modifications and so on; From this point, epigenetic play an enssential role in various life, including growth, differentiation, stress, aging and some pathological conditions etc.. There are many regulaltion ways to regulate the gene expression level, including DNA methylation, histone modification, genomic imprinting, X-chromosome inactivation, non coding RNA. So far, more attention to the aging related research of the aging changes of gene expression levels, especially the epigenetic regulation of gene expression of genetics in the aging process. AimAfter clarifying the change of h PDLSCs biological characters changes during aging, epigenetic enzyme is screened and analyzed including histone acetyltransferase, histone deacetylase, histone methyltransferases, histone demethylase, some of which with high flux changes are considered as target enzyme. Try to search the possible relationship between them and explain the possible mechanism in epigenetics. Methods1. Study design: The obtained teeth were divided into Groups A, B, and C according to the donors’ age(aged 18-35, 35-55, and 55-62 respectively).2. Isolation and characterization of h PDLSCs: The activity of PDLSCs was first determined based on their colony- forming ability, surface markers, proliferative/differentiative potentials, senescence-associated β- galactosidase(SA-βG) staining.3. Screening and analysis of epigenetic enzyme regulating h PDLSCs aging by RT-PCR: Screen the epigenetic enzyme among different groups during aging, including histone acetyltransferase, histone deacetylase, histone methyltransferases, histone demethylase, by RT-PCR. Western blot verify the screening results and anlyze the level of histone H3 acetylation among different groups. Meanwhile, determine the interest proteins Gcn5 and p300.4. The influence of Gcn5 and p300 to h PD LSCs osteogenesis capacity: After disturbing histone acetylation transferase the expression level of Gcn5 and p300 by si RNA, h PDLSCs were induced osteogenic differentiation. Analyze the expression level of related osteogenic gene ALP, RUNX2 and OCN after 4 weeks and 7 days. Investigate osteogenic activity between experimental group and control group by analyzing alizarin red stain after 4-week induction.5. The influence of Gcn5 and p300 to h PDLSCs proliferation capacity : After disturbing histone acetylation transferase the expression level of Gcn5 and p300 by si RNA, MTT assay and flow cytometry analyze the proliferation capacity; meanwhile, investigate proliferation activity between experimental group and control group by MTT assay and flow cytometry, after treating using p300 activator.6. Statistical analysis: All values are presented as the mean ± standard deviation(SD). Each cell line was tested in triplicate in independent experiments. Comparisons between groups were analyzed by Student’s t-test or two-way analysis of variance(ANOVA) with SPSS software. A P value of < 0.05 was considered to indicate a significant difference. Results1. It was found that human PDLSCs could be isolated from the PDL tissue of different-aged subjects. However, the ability of the PDLSCs to proliferate and to undergo osteogenic differentiation displayed age-related decreases. In addition, these cells exhibited an age-related increase in SA-βG expression(P<0.05). All cells were negative for the hematopoietic markers CD31 and CD45 and were highly positive for the mesenchymal-associated marker CD90; there was no significant difference between the 3 test groups(P>0.05). The rate of CD146 positivity in Group A was much higher than that in Groups B and C(P<0.05).2. In this study, epigenetic enzyme is screened and analyzed including histone acetyltransferase, histone deacetylase, histone methyltransferases, histone demethylase. The result shows histone acetyltransferase takes on a sustained downward trend during aging, among which, Gcn5 and p300 exhibit notable reduction(P<0.05); Meanwhile, the Western-blot shows the level of histone H3 acetylation also takes on a sustained downward trend during aging.3. This study shows si RNA can inhibit the express ion level of Gcn5 and p300. After disturbing histone acetylation transferase the expression level of Gcn5 and p300 by si RNA, h PDLSCs were induced osteogenic differentiation. The related results show the osteogenic differentiation capacity has not been affected(P>0.05).4. After disturbing histone acetylation transferase the expression level of Gcn5 and p300 by si RNA, MTT assay and flow cytometry analyze the proliferation capacity. The results show the proliferation capacity of experimental group decline; meanwhile, proliferation activity exists enhancement, after treating using p300 activator. Conclusion1. Some biological character of h PDLSCs(proliferation, osteogenic differentiation andβ-gal activity) exist age dependency; but age has little influence on the capacity of fat differentiation.2. During aging process, histone acetylation level shows a downward trend, besides, the expression level of the corresponding histone acetyltransferase participating in this process decrease then.3. This study demonstrates that Gcn5 and p300 have little influence on the capacity of osteogenic differentiation during aging process.4. This study also suggestes that Gcn5 and p300 mainly have an influence on the function of h PDLSCs in aging process in proliferation.