| Purpose:With the increase of age,senescence has become an important vital signs of human aging and it along with our all life.With the development of human aging,it can cause the function decline of physiological and construction damage of organs and tissue,such as bone marrow,brain and so on,and finally resulting in age-related disease.Now,according to research founds,there are hundreds of theories of aging had been suggested.While stem cell aging theory has been considered as a new method to explain human aging,it is believed that there are significant relationship between stem cell and aging,and all aging of organism due to stem cells aging,it include tissue degeneration,loss function,tumor occurrence and repeated infection and so on.Those changes can reflect the level of adult stem cells aging.Moreover,it is also showed that with the stem cell recession of self-renewal and multi-directional differentiation capacity,it can lead to degeneration of organ structure and function.Some expert suggested that stem cell is the best model for studying aging and it also has an inestimable value to control and prevention age-related disease in looking for target to delay aging.Hematopoietic inductive microenvironment(HIM)is a place where HSC can differentiating,proliferating,and surviving.Bone marrow stromal cells had been considered as important component to build HIM by modern theory of hematopoietic,it was had been found that bone marrow stromal cells are composed of different cells such as fibroblasts,mast cells,endothelial cells and so on.It is not only can sustain the integrity of the HIM's ultrastructure,but also can regulate the HSC function directly or indirectly by release hematopoietic cytokine,and it also has a relationship with hematopoietic disease.So it is play an important role to research the HIM structure.It had been investigated that with the increase of one's age,hematopoietic stem cell showed a senescence phenotype and finally resulting in age-related diseases.Therefore there is a correlation between HSC and aging.Most of the BMSCs originated from bone marrow mesenchymal stem cells(BMMSCs),which is not only can differentiate into multiple types of the BMSCs,but also can regulate the HSCs function directly or indirectly by release hematopoietic cytokine and sustain the HIM's ultrastructure.So BMMSCs has become an important research objects in HIM's ultrastructure.Recent research has shown that during the passage of bone marrow mesenchymal stem cells(BMMSCs)in vitro culture,bone marrow mesenchymal cells do not grown infinitely,and had a limited number of proliferation,which is called cellular senescence and finally result in loss of cells function,but the relationship between age and the human BMMSCs are unknown,so it is important to in-depth study of bone marrow mesenchymal stem cells aging and mechanism of aging and the relationship with HSC aging,that is not only good to know biological mechanism of aging,but also benefit of understand how to delay cells senescence and prevent HSC senescence and age-related diseases.In this passage,we will combine the new technology on stem cells with aging theory,to discuss the senescence with the increase gradually of age on BMSCs.METHODS:1.The bone marrow come from human who without blood system diseases,then separate bone marrow mononuclear from bone marrow and cultured in vitro and passaged 3.Using flow cytometry to analysis the CD90,CD73,CD11b,CD14,CD19 and HLA-DR expression in the hBMSCs.2.Collect normal bone marrow,divided them into four group according to human age:Young group(10 cases,15-25 years old),middle-age group(12 cases,26~45 years old),quinquagenarian group(15 cases,45,65 years old),older group(12 cases,>65 years old).Then isolated bone marrow mononuclear cells from bone marrow and expanded from culture in vitro.(1)To detect the ability of proliferation,cells counting kit-8(CCK-8)was used to measure the ability of proliferation.Oil red O was used to stain the adipocyte and alkaline phosphatase kit was used to stain the osteoblast,all those to detect the capacity of multi-lineage differentiation.(2)The SA-?-Gal staining was used to examine the senescent cells(3)To evaluate the level of oxidative stress,the superoxide dismutase(SOD)content,total antioxidant capacity,the MDA,total glutathione peroxidase were texted by ELISA and the ROS was texted by flow cytometry(FCM).(4)E1ISA was used to detect the content of IL-2,IL-6 and TNF-? in cell culture supernate.(5)Real-time fluorescent quantitative PCR(RT-PCR)was used to detect the mRNA expression of P53 and P21.RESULT:1.Through flow cytometry analysis,it shown that it has a positive expression of CD73 and CD90 and CDllb and has a negative for CD14,CD 19 and HLA-DR.On the other hand,it also can differentiation into adipocyte and osteocyte.All those demonstrate that it is bone marrow mesenchymal stem cells(BMMSCs)2.Compare with each group,the ability of proliferation of hBMSCs declined with age increasing.The balance and the differentiation potential between differentiation to adipocytes versus osteocyte lineages was remarkably disrupted.The osteogenic potential in the hBMSCs was decreased and the adipocyte potential was increased with aging.The number of SA-?-Gal positive cells were increased with age increase.On the other hand,with aging in the hBMSCs,the cell cycle was arrested and the apoptosis and the oxidative damage was enhanced,3.According to RT-PCR,it shown that the expression of P53 and P21mRNA were up regulated.CONCLUSIONS:1.With the age increase,the studied shown that the function of hBMSCs declined and the senility also gradually take place.2.The mechanism of hBMSCs senescence may be not only related with hBMSCs DNA oxidative damage and P53-P21 pathway,but also may inflammation. |
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