| Background:The problem of population aging is becoming more serious in China as the great country develops rapidly in its economy. On the authority of national Statistical Bureau, of the total population of 1.36 billion in China,0.202 billion people are more than 60 years old, taking 14.9% of the population when the data was published in Jan.,2014. The data recently reported in authoritative media revealed that China would get into super aging countries in the year of 2020, that is, at that time one elderly would be supported only by 1.36 non-elderly. All the data mentioned above present a growing importance in aging-related researches in life science.The results of recent studies demonstrate that the direct and prime cause leading to aging of organism is none other than the aging of adult stem cells. Hence, related researches become important in search of mechanism of stem cell aging and of the biological indicators and protocols to regulate and control aging process. Proliferation and differentiation of hematopoietic stem cells (HSC), hematopoiesis and its release, maintenance of dynamic equilibrium of hemocyte amount, and aging and death of blood cells are all under precise regulation and control of hematopoietic inductive microenvironment (HIM). The functional status of hematopoietic microenvironment can be reflected in biological characteristics of bone marrow stromal cells (BMSCs) which are the core ingredient of HIM. Aging of organism can be presented as functional decline of HSC, which leads to deficiency of hematopoiesis and immunity in company with diseases related with aging.Ginseng with ginsenoside as its key medical ingredient is an important Chinese traditional medicine for "bu-qi" (air make-up). Studies show that ginsenoside Rgl, with a regulative and controlling function in elongation of the life of organism and that of cells, is one of the most active ingredient in ginseng. Previous studies also demonstrate that ginsenoside monomer Rgl has an obvious function in anti-aging for bone marrow progenitor stem cell, the key ingredient in medullary hematopoiesis of SD rats, but there is no report found on studies of BMSC in anti-aging as key ingredient of HIM.Objective:In our research, a model was established of in vitro and vivo aging of rat BMSCs induced by D-galactose. An exploration was done into the characteristics of biological indexes related with Rgl’s function of delaying BMSCs aging and its impact on hematopoiesis to make clear the mechanism of Rgl in delaying aging of BMSCs in organism which leads to decline of hematoposis, and to search for a possible biological mechanism for a new protocol to delay aging of hematopoietic microenvironment.Methods:1.Method of whole bone marrow adherent culture:marrow stroma cells (BMSCs) are separated and purified, and adherently cultured to the 3rd generation (P3) with the cellular morphology observed under the light microscope and cell activity checked with trypan blue stain. Bone marrow mononuclear cells (BMNCs) were separated and extracted from whole bone marrow cells with the method of density gradient centrifugation after lymphocyte separation medium were put in. After adherently cultured for 4 hours, the upper suspension cells were co-cultured with adherent-cultured marrow stroma cells (BMSCs).2.The impact of D-gal on biological characteristics of BMSC aging was observed with aging-associated SA-β-Gal stain, colorimetric method of cell count kit 8 (CCK8) and cell cycle and apopotosis of flow cytometry and observation of subcellular morphology to establish a D-gal induced in vitro aging model for BMSC.3.Suspension BMNCs, were co-cultured with BMSCs and then the impact of senile BMSCs on the biological characteristics was observed with aging-associated SA-β-Gal staining, CCK8 assay,cell cycle and apoptosis analysis of flow cytometry and CFU-Mix forming test, all above were to explore the impact of aged BMSCs to BMNCs in co-cultured system.4.BMSCs were cultured with Rgl and D-gal. The changes in characteristic indexes of biology of senescence in BMSCs were observed with following methods:aging-associated SA-β-Gal staining, observation of subcellular morphology by transmission electron microscope, and colorimetric method of CCK8, cell cycle and apoptosis analysis of flow cytometry.Enzyme-linked immuno sorbent assay (ELISA) was adopted to detect the levels of IL-2, IL-6, IL-1β, TNF-α,SCF and GM-SCF in cell culture supernatant. Cell lysis buffer was prepared, protein concentration determined and then the total content of intracellular superoxide dismutase (SOD) as well as the content of mallondialdehyde (MDA) was detected with WST-8 and TBA methods. Chemifluorescence method and laser CONFOCAL observation was adopted to detect the content of intracellular reactive oxygen specie (ROS) of BMSCs, and changes in expressions of BMSCs cycle regulatory proteins of P16, P21, P53, CDK2, CDK4, cyclinD and cyclinE were detected by Western blot. All above were to illustrate biological mechanism of anti-aging effect of Rgl in vitro aged BMSCs induced by D-gal.5.BMSCs, after worked upon with Rgl and D-gal, were cultured together with BMNCs. Methods of aging-associated SA-β-Gal staining, colorimetric method of CCK8, CFU-Mix test, cell cycle and apoptosis analysis of flow cytometry were adopted; and the intracellular contents of SOD and MDA were determined with WST-8 and TBA methods in BMNCs; intracellular content of ROS was detected by flow cytometry (FCM) and laser CONFOCAL.6.To establish aged rat model, subcutaneous injections of D-gal were done in the neck of rats, after isolation and purification of BMSCs and BMNCs, SA-β-Gal stain, cell cycle and apoptosis assay and CCK8 test were adopted to observe the effect of the D-gal injections on characteristics of biology of senescence in BMSCs.7. SA-β-Gal staining, CFU-Mix forming test and cell cycle assay were detected to observe the aging related biological characteristics of BMNCs in aged rat model.8. After the injections of Rgl was administrated and isolation and purification of BMSCs and BMNCs, SA-β-Gal stainnig, cell cycle and apoptosis analysis of flow cytometry, and colorimetric method of CCK8 were adopted to observe index changes of biological characteristics for aging; the total content of SOD as well as content of MDA and GSH-Px were detected with enzyme method in cell culture supernatant fluid; FCM was adopted to determine content of reactive oxygen specie (ROS); Changes in expressions of BMSCs cycle regulatory proteins of P16, P21, P53, CDK2, CDK4, cyclinD and cyclinE were detected by Western blot; ELISA was adopted to detect the levels of IL-2, IL-6, IL-1β, TNF-α, SCF and GM-SCF in cell culture supernatant fluid to illustrate biological mechanism of D-gal in inducing in vitro BMSCs aging and that of Rgl in controlling aging of BMSCs.9.The SA-β-Gal staining, flow cytometry cell cycle analysis and CCK8 cell proliferation test methods to observe the characteristic aging biology changes in BMNCs of aged rat model; flow cytometry method observe the level of ROS and after the total protein was extracted, the enzymic method to detect the level of total SOD and MDA. The protein expresstion of P21 and P53 in BMNCs was tested by WB method, the potential relationship between BMSCs and BMNCs in aged rat model and the anti-aging regulation processed by Rgl among these two cells above were demonstrated.Results:1. When BMSCs were adherently cultured to 3rd generation,80% cell fusion could be observed with morphology of cells presented as fusiformis or polypody and growing in lines of swirls. Activity of BMSCs was 96±0.68% detected by trypan blue stain, which suggested that BMSCs, when purified by whole bone marrow adherent culture, would have high degree of purity and activity.2. The presentation of typical expression of aging biology of BMSCs by in vitro induction of D-gal:a. The ratio of SA-β-Gal-stained positive cells has been obviously increased. b. Electrical microscope ultrastructure of BMSCs of rats of the aging group in compare with the control group:cells become biger and flatter, mitochondria swell, golgi body pond becomes larger, aging-related metachromatins of cell nuclears get together, more apoptosis bodies of typical structure of apoptosis are observed, typical characteristics of aging morphology are presented in cells, proliferative capacity of CCK8 is obviously weakened, ratio of cells in G1 period is obviously increased and that in S period decreased. D-gal can induce decrease of content of intracellular SOD of senile BMSCs and increase of content of ROS and MDA. It also induces increase of expression of protein of P16, P21,P53 and decrease of that of CDK2,CDK4 and cyclinD and cyclinE wasn’t altered obviously.3.The ratio of SA-β-Gal-stained positive cells obviously increases in suspension BMNCs in co-culture system of the aging group in compare with that of the control group. The number of CFU-Mix of suspension BMNCs is obviously reduced in aging microenvironment compared with that of control group.Ratio of cells in G1 period is obviously increased and that in S period decreased. After BMNCs in aging microenvironment for 24 hours, its proliferative capacity decreases keeping lower than the control group up to 120 hours. Contents of intracellular ROS and MDA co-cultured in aging co-culture were obviously increased while content of SOD decreased,and ROS level observed by laser confocal was higher.4.1n vitro impact of Rgl on senile BMSCs can decrease the ratio of SA-β-Gal-stained positive cells, reduce aging-related morphologic expression of cells, ratio of G1 phase cells, apoptosis rate and intracellular ROS, and increase ratio of S phase cells. It also reduces secretion of IL-2 and TNF-α and protein expression of P16, P21,and P53. It still increases secretion of IL-6, IL-1β, GM-CSF and SCF, intracellular content of SOD, and protein expression of CDK2 and CDK4. But under in vitro impact of Rg1, there are no obvious changes of MDA content and protein level of cyclinD and cyclinE.5. BMSCs, after worked upon with Rgl and D-gal, were cultured together with BMNCs, the ratio of SA-β-Gal-stained positive cells decreased, reduction of ratio of G1 phase cells, apoptosis rate and intracellular ROS, and increase of ratio of S phase cells were observed. It were also observed the reduction of secretion of IL-2 and TNF-a. It was still observed the increases of secretion of SCF, intracellular content of SOD.But under in vitro impact of Rgl, there are no obvious changes of MDA content.6. After neck subcutaneous injection of 120mg/kg D-gal is administrated for rats, BMSCs present typical expressions of aging biology: obvious increase of SA-β-Gal-stained positive cells; obvious increase of ratio of G1 phase cells and decrease that of S phase cells; increase of apoptosis rate and decrease cell proliferation capacity tested by CCK8; obvious higher protein expression of P53, P21 and P16 and decrease of protein expression of CDK2; increase of contents of ROS and MDA in cell supernatant and of secretion of IL-2 and TNF-a; decrease of SOD and reduction of IL-6 and SCF; and there were no obvious change was observed in protein expression of cyclinD.7. In aged rat model, BMNCs present typical expressions of aging biology:obvious increase of SA-β-Gal-stained positive cells; obvious increase of ratio of G1 phase cells and decrease that of S phase cells; obvious higher protein expression of P53 and P16; increase of contents of ROS and MDA intracellularly; decrease of SOD and CUF-Mix forming ability.8. In vivo impact of Rgl, after acting on BMSCs, can decrease rates of SA-β-Gal stained positive cells and of G1 phase cells, promote cell reproductive capacity determined by CCK8, lower apoptosis rate, increase SOD in cell supernatant while reduce ROS and MDA, regulate up secretion of IL-6 and SCF in supernatant and regulate down that of TNF-a and IL-2, and lower protein expression of P16, P21 and P53. No obvious changes in protein expression of cyclinE and CDK2 and GSH-Px is observed.9. In BMNCs of aged rat model, it is observed that the decreases in the rates of SA-β-Gal stained positive cells and of G1 phase cells, promotion of ratio of S phase cells and CFU-Mix forming ability, also the intracellular increase of SOD and the reduction of ROS,the lower protin expression of P53 and P16 is also been observed.Conclusions:1. D-gal can built the aging model of BMSCs equally in vitro and vivo.2.Aging of BMNCs can be induced by aged BMSCs.3. Ginsenoside Rgl can inhibit the aging of BMSCs induced by D-gal equally in vitro and vivo and through the approach above the hematopoietic function of BMNCs can be recovered.4.It is through down-regulation of expressions of aging-related proteins and inhibition of level of oxidative stress injury and chronic inflammation to delay the aging processes in BMSCs which induced by D-gal,and the origin of these positive effects is direct function of ginsenoside Rg1. |
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