Establishment Of The Model Of Deficiency Qi-Blood And Aging Hsc/Hpc

Objective:With the acceleration of the process of the world andChina’s aging population, the longer life expectancy of population agingand anti-aging research have become a hot issue for the life sciences andsocial sciences. Aging and death is inevitable laws of nature. Latestresearch think that, the aging adult stem cells/progenitor cells in agingorganism is an important reason. So, investigating the hematopoietic stemcell biological characteristics in different state of life is a very importantstep to explore the aging biosome. hematopoietic stem/progenitor cells(hematopoietic stem cell/progenitor of the cell, the HSC/HPC)is one kindof important stem cells, which have the ability of self-renewal anddifferentiation potentialand and the ancestor cell of all blood cells. Inspecial conditions, it can be the formation of some non-hematopoieticcells. New study has proved that the aging of the HSC/HPC has a relationwith the aging organism and age-related diseases.The traditional chinese medical “Qi and Blood Deficiency” is acommon clinical disease, and build the “Qi deficiency” model is one ofthe focus of attention of many scholars of Chinese medicine academics. Qideficiency in clinical practice is often manifested as immune function, thepresent study show that: the deficiency of immune cell function is theprimary factor to cause immune system dysfunction, most of the original immune cells come from bone marrow. The inhibition of bone marrow(hematopoietic system) is the nature of qi deficiency syndrome. Thetraditional Chinese medical blood deficiency and Western medicine"anemia" is similar in nature. In fact, the blood deficiency is anemia. Weuse χ-ray and phenyl hydrazine hydrochloride to built the “qi and blooddeficiency” model, by the way of using total body irradiation (TBI) andsingle intraperitoneal injection of phenyl hydrazine hydrochloride toestablish “qi and blood deficiency” and explore the biologicalcharacteristics of hematopoietic stem cell aging.Methods1Model group: one-time total body χ-ray irradiation, dose of6.5GY6hafter intraperitoneal injection of40mg/Kg phenyl hydrazine hydrochloride.Control group: with false exposure, injection of saline40mg/Kg.2Observe the behavior of the experimental group in the3,7,14,28-dayrespectively; calculate the number of bone marrow mononuclear cells,hematopoietic stem cells; detect mononuclear cell cycle, peripheralhemogram, the spleen index, spleen pathology chip and other indicators.3Immunomagnetic separation was used to isolate and purificate Sca-1~+HSC/HPC, flow cytometry, immunofluorescence staining and Taiwanphenol blue staining.4Senescence-associated beta-galactosidase(SA-β-gal) staining, mixedcells colony culture(CFU-Mix) and cell cycle were used to detect thebiological characteristics of aging cells.5The aging genes testing: PCR detect the aging-related genes, includingp16INK4a, p53.Results1The model groups of C57mice show apathetic and sluggish; the peripheral blood like white blood cells, red blood cells, hemoglobin andplatelet significantly reduced. The level of red blood cells and hemoglobinat their lowest at the7th day, such state last to the28d, then it graduallyrestored to normal. The number of bone marrow mononuclear cellsdrastically reduced to the lowest level at the3d, and slowly began torecover at the7th day, returning to the50%of the control group’s level atthe14d, recovery to65.7%of the control group at the28d; the Sca-1~+HSC at the3d was a sharp decline to almost zero level,the depletion ofSca-1~+HSC to the lowest level, at the7d is still quite low, slow recovery.The14d recovery to14.5%of the control group, at the28d return to21.3%. Spleen index were significantly reduced.2The percentage of Sca-1~+cell of bone marrow mononuclear cells (MNCs)before the MACS is (1.81.2)%; the percentage of the separation andpurification of Sca-1~+cell of MNCs is (87.331.25)%.Immunofluorescence showed that the expression of Sca-1+was only avery small number before the separation and purification of MNCs, afterthe separation and purification of the labeled cells, we can get the largenumber of cells expressing Sca-1~+. Trypan blue dye detect activity ofSca-1~+HSC/HPC96%to99%.3The senescent cells stained dark blue by β-galactosidase(SA-β-gal),which suggesting that hematopoietic stem/progenitor cells are insenescence; the number of CFU-Mix less than control groups, and the sizeis also less than the control groups; the phase of G1in G0/G1wassignificantly increased in bone marrow mononuclear cells; the proportionof S phase reduced significantly.4Aging-related genes on gray value in model group: p16INK4a，p53, weresignificantly higher. Conclusions1Dose of6.5χ-ray and phenylhydrazine hydrochloride can successfullyestablished Qi and blood deficiency animal models.2Using immunomagnetic beads magnetic separation method (MACS) cansuccessfully and effectively separate, purificate the Sca-1~+HSC/HPC, andkeep high cells activity.3The characteristics of aging of hematopoietic stem/progenitor cell in Qiand Blood Deficiency existing.