Effects Of Hypoxia On The Physiological Behavior And The Expression Of Histone Deacetylase Of Human Cartilage Endplate Derived Stem Cells

BackgroundOur country and even the whole world will even be facing the aging of the population structure,the incidence of degenerative diseases of the important organs of the human body is increasing year by year,and the elder people’s living quality were badly impacted.Low back pain is a kind of typical aging related diseases,the researches showed that degenerative disc diseases play a major role in the occurrence and development of it.Our research team selected cells with stem cell properties in human degenerative cartilage endplate and named them as cartilage endplate derived stem cells.This important discovery proves that stem cells still exist in degenerative intervertebral disc,and provide new candidate seed cells for cell replacement therapy of intervertebral disc degeneration.The study of intervertebral disc derived cells will help us to understand the pathophysiological mechanism of intervertebral disc degeneration,and provide new methods for the prevention and treatment of intervertebral disc degeneration.As we all know,intervertebral disc is the largest structure of human body without vascular and nerve.It is hypoxic environment in normal intervertebral disc.Along with the degeneration of intervertebral disc,the blood vessels and nerves generated continuously in the intervertebral disc.In the degenerative intervertebral disc,the original physiological hypoxic environment has changed and the oxygen content increased.We speculate that the increased oxygen content in intervertebral disc probably plays an important role in the process of intervertebral disc degeneration.Meanwhile,we are concerned that epigenetic mechanisms play a vital role in the regulation of cell biological behavior of stem cells and tumor cells.Epigenetics is a hot research area in recent years,histone modification is one of the epigenetic mechanisms,and the changes of histone modifications may be involved in the process of intervertebral disc degeneration.In the past,most of the researches on cartilage endplate stem cells were done under normoxic environment in vitro.The effects of hypoxia on the biological behavior of CESCs in vitro and the changes of the expression of histone deacetylases in CESCs under the hypoxia in vitro have not been reported.So we designed this study aimed at understanding the effects of hypoxia on the proliferation,senescence,apoptosis and cell cycle,and the changes of gene expression of histone deacetylases of CESCs in vitro.Method1.Primary culture and selecting of CESCs.Clinical specimens were obtained from the patients with minimally invasive surgery transforaminal lumbar interbody fusion,and the cartilage endplate of intervertebral disc was isolated.The primary cells of cartilage endplate were obtained by digesting the cartilage endplate by collagenase II.Selecting the CESCs from the primary cells of cartilage endplate by agarose culture.2.Effects of hypoxia on proliferation of CESCs.CESCs was cultured in hypoxic and normoxic environment for 12 h,24 h,72 h,and the proliferation of CESCs was detected by the ways of Edu incorporation and WST-1 under different culture conditions.3.Effects of hypoxia on Senescence of CESCs.CESCs were cultured in hypoxic and normoxic environment for 12 h,24 h,72 h,respectively.The senescence of CESCs in different culture conditions was detected by β-gal staining.4.Effects of hypoxia on apoptosis of CESCs cells.CESCs were cultured in hypoxic and normoxic environment for 12 h,24 h,72 h,respectively.The apoptosis of CESCs in different culture conditions was detected by flow cytometry.5.Effects of hypoxia on cell cycle of CESCs.CESCs were cultured in hypoxic and normoxic environment for 12 h,24 h,72 h,respectively.The percentage of CESCs cell cycle in different culture conditions was detected by flow cytometry.6.Effects of hypoxia on special phenotype genes expression of osteogenic differentiation and chondrogenic differentiation in CESCs.CESCs were cultured in hypoxic and normoxic environment for 72 h,and the special phenotype genes expression of osteogenic differentiation and chondrogenic differentiation in CESCs were assayed by RT-PCR.7.Effects of hypoxia on the genes expression of HDACs isoforms in CESCsCESCs were cultured in hypoxic and normoxic environment for 12 h,24 h,72 h,respectively.The genes expression of HDACs isoforms in CESCs in different culture conditions were assayed by RT-PCR.Results1.The expression rates of CESCs’ surface markers shown that CD90 and CD73 were higher than 99%,CD105 was higher than 96%,and the expression rates of HLA-DR,CD34,CD45,CD14 and CD19 were lower than 2%.2.The proliferation activity of hypoxia stimulation group and normoxia control group was positively correlated with the culture time,and the proliferation activity of CESCs of hypoxia group was significantly higher than that of normoxia group.3.The rate of CESCs stained with beta-gal of hypoxia 72 h group was significantly less than that of normoxia 72 h group.In different culture time of hypoxia group,CESCs’ aging rates comparison found that along with the culture time prolonging,the CESCs’ aging rate was decreased.4.The apoptosis rate of hypoxia 72 h group was significantly lower than that of normoxia 72 h group.In hypoxia group,the apoptosis rate of 72 h was significantly lower than 12 h and 24 h.5.In the hypoxic stimulation group,the cell cycle distribution of CESCs showed that the ratio of G1 phase increased gradually and then decreased gradually,and the ratio of(S+G2/M)decreased gradually and then increased gradually.6.After hypoxia stimulation,the gene expression of HDACs subtype(HDAC-1,3,4,5,6,7,10,11,SIRT-1,2,5,6,7)was significantly increased in CESCs.7.The expression levels of osteogenic differentiation phenotype genes(RUNX2,COL1,ALPL)and chondrogenic differentiation phenotype genes(SOX9,COL2,Agg)were significantly increased after hypoxia stimulation in CESCs.Conclusion1.The obtained CESCs selected from the primary culture cells of human cartilage endplate specimens by agarose system had stem cell characteristics.2.Under hypoxia environment,the proliferation ability of CESCs was significantly higher than that of the control group.3.Under hypoxia environment,the CESCs resistance to cell senescence was signi ficantly higher than that of the control group.4.Under hypoxia environment,the ability of CESCs to inhibit apoptosis was significantly higher than that of the control group.5.After the CESCs was transferred from the oxygen environment to the hypoxic environment,it needs 24 h to adapt to the hypoxic environment.6.After hypoxia stimulation,the gene expression of HDACs subtype(HDAC-1,3,4,5,6,7,10,11,SIRT-1,2,5,6,7)was significantly increased in CESCs.7.The expression levels of osteogenic differentiation phenotype genes(RUNX2,COL1,ALPL)and chondrogenic differentiation phenotype genes(SOX9,COL2,Agg)were significantly increased after hypoxia stimulation in CESCs.