The Effects Of TSA Or SAHA On Differentiation Of Adipocytes Or Neuron In Arbas Cashmere Goat Adipose-Derived Stem Cells

Recently, the somatic cell nuclear transfer is important techonology for generating transgenic mammals and protecting endangered animals. However, somatic cells as terminally-differentiated cells are difficult to be reprogrammed by oocytes, and lead to low-level efficiency of producing cloned animals. Previous studies confirmed that epigenetic defection of cloned embryos had pivotal role to result in low cloned efficiency. Therefore, pluripotent stem cells were deemed to be desired donor cells. However, livestock embryonic stem cells have not been established in vitro, so they could not be used to produce cloned livestock yet. Mesenchymal stem cells (MSCs) as a kind of adult stem cells that had opening chromosome status and multipotency were more reliable donor cells than somatic cells using cloned livestock production.MSCs that derived from adipose tissue were considered to be useful for cellular therapy, tissue engineering and generating transgenic mammals. Previously, adipose-derived stem cells (ADSCs) as donor cells significantly improved cloned embryonic development. In this study, cashmere goat ADSCs (gADSCs) were treated with histone deacetylase inhibitors, Trichostatin A (TSA) and Vorinostat (SAHA), and their histone acetylation modifications, multipotency and differentiation were analysized after treatment. The results of this study will benefit for using gADSCs to produce transgenic and cloned cashmere goats.1. Effects of TSA and SAHA on H3K9 acetylation of gADSCs1) Proliferative curve of gADSCs in vitroAfter continuously counting cell numbers during 7 days cultivation, the proliferative curve of gADSCs showed S-like, revealed that the cells had robust ability to proliferation.2) Effects for TSA and SAHA treatment on proliferation of gADSCsHircine ADSCs were treated with 500 nM TSA or 16 μM SAHA for 24h,48h and 72h, respectively, and the cellular proliferation was determined by MTT methods. The results showed that the cellular proliferation was significant inhibition after 24h treatment.3) H3K9 acetylation level analysis by immunofluorescence and western blotting.The level of H3K9 acetylation was significant increase when the gADSCs were treated with 500 nM TSA or 16 μM SAHA for 24h. Compared to the level of acetylated H3K9 after SAHA treatment, it was higher in gADSCs that were treated with TSA (P<0.01).4) Expression of HDACs in gADSCs after HDACi treatmentHDAC1, HDAC6 and SIRT1 in gADSCs were detected after HDACi treatment. Compared to un-treated cells, mRNA and protein of HDAC1 and HDAC6 was significant up-regulated after TSA and SAHA treatment. Level of SIRT1 mRNA was down-regulated in SAHA treated cells, but up-regulated in TSA gADSCs. However, its protein was down-regulated in treated cells (P<0.01).2. Effects of increasing H3K9 acetylation by HDACi treatment on proliferation, apoptosis and pluripotency of gADSCs1) Effects of TSA and SAHA treatment on cell cycle and apotosis of gADSCsAfter teated gADCS with TSA and SAHA for 24h, the cell cycle was stagnated at G0/G1 stage, and apotosis was induced.2) Expression of proliferation, apoptosis and pluripotency related genes in treated and un-teated gADSCs.Expression of proliferation, apoptosis and pluripotency related genes in treated and un-teated gADSCs were analyzed by Q-PCR. After HDACi treatment, expression of pluripotent genes NANOG, OCT4 and SOX2 was significant up-regulated. Expression of proliferation related genes TERT was down-regulated, whereas, PCNA was upregulated. Expression of P53 and BAX that regulated cellular apoptosis was down-regulated significantly.3) Protein level of proliferative, apoptosis and pluripotent factersProtein level of proliferative, apoptosis and pluripotent facters was analyzed by western blotting. The results showed that contrary to up-regulated mRNA level of these genes, protein level of NANOG, OCT4, and SOX2 was reduced in protein level. TERT, PCNA and P53 were also down-regulated, BAX was up-regulated. The results indicated that cellular proliferation, apoptosis and pluripotency were affected by H3K9 acetylation.3. Effects of TSA and SAHA treated gADSCs on differentiating into adipocyte and nerve cells1) Effect of TSA and SAHA treated gADSCs on differentiating into adipocytesCompared to control group, after treating gADSCs with TSA and SAHA for 3 days, the key adipogenesis factor PPARy2 was significant up-regulated, suggested that the treated cells improved adipocyte differentiation.2) Effects of TSA and SAHA treated gADSCs on differentiating into nerve cellsCompared to control group, after treating gADSCs with TSA, the key neurogenesis factors ENO2 and NEUN was significant up-regulated, but SAHA-treated gADSCs only up-regulated expression of ENO2, suggested that the treated cells improved nervous differentiation.In conclusion, TSA and SAHA treatment significantly inhibited activity of HDAC, up-regulated level of acH3K9, activated gene expression and induced opening chromatin status in gADSCs. It improved gADSCs differentiate into adipocyte and nerve cells. The cells with treatmenting by TSA and SAHA improved reprogramming ability will be more useful as donor cells for generating cloned goats.