1. The Time Dynamic Biological Study Of The Aging Of Human Bone Marrow Hematopoietic Cells 2. The Effect Of Ginsenoside Rg1 On The Liver Structure And Function Of D-gal Aging Mice

[Objective]To explore the dynamic biological changes of aging of bone marrow hematopoietic cells in different age groups,in order to lay a theoretical foundation for the further study of aging and its mechanism of hematopoietic stem cells,and to provide a detailed laboratory basis for the search of effective ingredients and the right time to intervene its aging.[Methods]The clinical specimens were collected and divided into 4 groups?under 30 years of age,30-45 year old group,46-60 year old group,and more than 60 years old?.Bone marrow mononuclear cells?hBMMNCs?of each group were isolated,purified and cultured,and hematopoietic cells?BMHCs?were obtained after purification.Age-related beta galactose glucoside enzyme cytochemistry staining?SA-?-gal?analysis of age-related expression strength and percentage of positive cells.The proliferation of cells in each group was determined by EdU testing,BMHCs differentiation ability was compared by culture of mixed hematopoietic cell colonies?CFU-Mix?,Superoxide dismutase?SOD?and malondialdehyde?MDA?in BMHCs of different age groups were determined by enzyme label colorimetry.Enzyme-linked immunosorbent assay?ELISA?was used to determine the content of Glutathione?GSH?in BMHCs.Protein expression levels of P16,P19,P21,P53,CyclinD1,CyclinE1,CDK2 and CDK4 were determined by Western Blot.Real-time fluorescence quantitative PCR?RT-PCR?was used to detect the mRNA expression levels of senescence related genes p16,p19,p21 and p53 in hematopoietic cells of each group.[Results]hBMMNCs can be successfully isolated by gradient centrifugation and purified to obtain BMHCs.With the increase of age,the number of BMHCs in the group over 60years old was significantly lower than that in the group under 30 years old.BMHCs with positive SA-?-gal aging staining increased in number and expression intensity with aging.The proliferation capacity of BMHCs was gradually reduced,the cell proliferation capacity of the group aged 46-60 years and the group aged 60 years and above was significantly reduced compared with the group aged under 30 years,G1phase arrest occurred in the cell cycle,and the production of CFU-Mix was also gradually reduced.The antioxidant capacity of BMHCs decreased gradually?SOD content decreased?and oxidative damage increased gradually with aging?MDA,GSH,ROS content increased significantly?.With the increase of age BMHCs was associated with increased senescence?P16,P19,P21,P53,CyclinD1?and decreased cyclin expression?CDK2,CDK4,CyclinE1?,BMHCs expression of senescence related genes increased with age?p16,p19,p21,p53 mRNA?.[Conclusion]With the growth of age,the biological changes of aging will gradually occur in human bone marrow hematopoietic cells,which are manifested as the increase in the number of aging staining positive cells,cell cycle arrest,decreased proliferation ability,and reduced differentiation ability.The aging mechanism may be related to the increased level of oxidative stress injury and the activation of p16^INK4a-Rb and p19^Arf-Mdm2-p53-p21^Cip1/Waf1signaling pathways.The present study investigated the effect and underlying mechanisms of ginsenoside Rg1?Rg1?in attenuating subacute liver injury induced by D-galactose?D-gal?in mice.Specific Pathogen Free?SPF?male C57BL/6J mice were randomly divided into 3 groups: i)D-gal-administration group?D-gal group?,where the mice were intraperitoneally administrated with D-gal?120 mg/kg/day for 42 days?;ii)D-gal+ Rg1 group where the mice were treated with 120 mg/kg/day D-gal for 42 days and with Rg1 at a dose of 20 mg/kg/day for 35 days.The first dose of Rg1 was administered on the 8th day of treatment with D-gal;and iii)the normal control group,where the mice were injected with an equal volume of saline for 42 days.The day following the final injections in all groups,peripheral blood was collected and serum was prepared to measure the contents of aspartate aminotransferase?AST?,alanine aminotransferase?ALT?,total bilirubin?TBi L?,advanced glycation end products?AGEs?and8-hydroxy-2 deoxyguanosine?8-OH-d G?.Liver tissue homogenates were prepared to measure the contents of malondialdehyde?MDA?and glutathione?GSH?,and the activities of glutathione peroxidase?GSH-Px?and superoxide dismutase?SOD?.Paraffin section were prepared to observe the microscopic structure of the liver.Transmission electron microscopy was used to observe the ultrastructure of hepatocytes.Frozen section were prepared and stained with senescence-associated ?-galactosidase to detect the relative optical density value of senescence-associated markers.Compared with the D-gal group,the contents of AST,ALT,TBi L,AGEs and MDA significantly decreased in the D?gal + Rg1 group,while the activities of SOD and GSH-Px markedly increased,and liver injury and degenerative alterations of hepatocytes were reduced.Administration of Rg1 induced a protective effect on D-gal-induced liver injury in mice by inhibiting the oxidative stress,reducing DNA damage and decreasing the AGE content.