| Objective:Adult stem cells is an important factor to sustain steady state, and hasplayed a key role in the body’s ageing process. Hematopoietic Stem cells(HSCs) are the earliest, the most indept andthe most mature study cells.carried out in the area of Stem cell research at, technology, meet the mostin-depth and as a kind of adult Stem cells are widely used in clinical.Although basic composition of hematopoietic system in the aging body ismaintained, but the number and function of HSCs gradually decline. It willlead to a recession of immune function and a variety of age-related disease.Angelica sinensis polysaccharide (ASP) that is the extraction ofmedicinal active ingredients isolated from angelica sinensis can promotehematopoietic, resist to radiation damage, confront tumor and regulateimmune, and so on. At present, it is unclear that ASP can delay HSCssenescence and its regulation mechanism. In this study, we take the research techniques of stem cell integrated with the anti-aging theory oftraditional medicine. This research aimed at building aged modelthrough replicate HSCs senescence in vitro HSCs and X-ray irradiationin vivo and discuss the possible mechanisms of ASP to delay HSCssenescence. Then it can provide for experimental basis and theoreticalbasis to search the methods for reactivating ageing HSCs and regulate itstargeting differentiationMethods:1. Sca-1~+HSCs were separated and purified by immune magnetic-activated cell sorting, MACS) technology. The cell purity was measuredby flow cytometry. And the cell activity was detected by trypan bluestaining.2. Age-related beta-galactose glucoside enzyme (SA-β-Gal) staining,methyl thiazolyl tetrazolium (MTT) colorimetry, cell cycle analysis andmethylcellulose semi-solid cultivation were observed the effect ofoxidation low density lipoprotein (ox-LDL) on aging biologicalcharacteristics in Sca-1~+HSCs by. The aim is to build the model of ox-LDLinduced aging of Sca-1~+HSCs in vitro.3. Sca-1~+HSCs were cultured with ASP an ox-LDL. SA-β-Galstaining, cell cycles analysis and methylcellulose semi-solid cultivationwere used to evaluate the changes of aging biological characteristics in Sca-1~+HSCs. The activities of SOD and GSH-Px and the levels ofmalondialdehyde（MDA）in supernatant that Sca-1~+HSCs were culturedwere detected by chemical colorimetric method. The production of ROSin Sca-1~+HSCs was evaluated by immunofluorescence.The expressions ofp16, p21, CDK2, CDK4,cyclinD and cyclinE were determined byreal-time quantitative PCR (qRT-PCR) and western blotting analysis. Thelength of telomere and the vitality of telomerase was analysed by southernblot and TRAP-PCR, respectively.The aim is to clarify the biologicalmechanisms that ox-LDL induced aging of Sca-1~+HSCs in vitro and ASPregulated aging of Sca-1~+HSCs in vitro.4. After mice were irradiated by X ray(3.0Gy/8F) total bodyirradiation(TBI), SA-β-Gal staining, cell cycles analysis andmethylcellulose semi-solid cultivation were used to evaluate the effect of Xray (3.0Gy/8F) TBI on aging biological characteristics in Sca-1~+HSCs. Theaim is to build the model of X ray induced aging of mice Sca-1~+HSCs invivo.5. The mice were treated with ASP by intragastric administrationduring X-ray(3.0Gy/8F) irradiation. SA-β-Gal staining, cell cycles analysisand methylcellulose semi-solid cultivation were used to evaluate thechanges of aging biological characteristics in Sca-1~+HSCs. The activities ofSOD and GSH-Px and the levels of malondialdehyde（MDA）in supernatantthat Sca-1~+HSCs were cultured were detected by chemical colorimetric method. The production of ROS in Sca-1~+HSCs was evaluated byimmunofluorescence.The expressions of p16, p21, CDK2, CDK4,cyclinDand cyclinE were determined by real-time quantitative PCR (qRT-PCR) andwestern blotting analysis. The length of telomere and the vitality oftelomerase was analysed by southern blot and TRAP-PCR,respectively.The aim is to clarify the biological mechanisms that Xray(3.0Gy/8F) induced aging of Sca-1~+HSCs in vivo and ASP regulatedaging of HSCs in vitvo.Results:1. The purity of Sca-1~+cells was only（86.216.17）%before MACS.But the percentage of separated Sca-1~+cells from MNCs was（11.451.87）%after MACS. The survival of Sca-1~+HSCs which detected by trypan bluestaining was （93.65±4.54）%. These results suggest that the separatedSca-1~+HSCs have a higher purity and good activity.2. Sca-1~+HSCs induced by ox-LDL in vitro appeared the typicalbiolodical feature of senescence as follow: The percentage of SA-β-Galpositive cells was significantly increased; the ratio of G1stage wasstrikingly increased, while the ratio S stage was decreased; the number ofCFU-Mix was also notably decreased. The content of ROS was increasedsignificantly and the activity of SOD and GSH-Px was decreased in agedSca-1~+HSCs, while the content of MDA in supernatant that aged Sca-1~+HSCs cultured was increased. The length of telomere wasremarkedly Shortening in aged Sca-1~+HSCs, and the activity of telomeraseof aged Sca-1~+HSCs was also reduced. ox-LDL could remarkedlyupregulate the expression of p16and p21in mRNA and protein levels,decrease the protein expression of CDK4, cyclinD and cyclinE inSca-1~+HSCs. There was no significant difference in the protein expressionof CDK2in Sca-1~+HSCs.3. ASP could decreased the the percentage of SA-β-Gal positive cellsand the ratio of G1stage and the expression of P16protein, increased theratio of S stage and the expression of CDK4and cyclinD protein andinhibit damage of telomere DNA in aged Sca-1~+HSCs. There was nosignificant difference in the number of CFU-Mix,the content of ROS, theactivity of SOD and GSH-Px and the content of MDA in supernatant thataged Sca-1~+HSCs cultured, the expression of p21, CDK2and cyclinE andthe activity of telomerase in Sca-1~+HSCs.4. Sca-1~+HSCs of mice induced by X ray (3.0Gy/8F) TBI appeared thetypical biolodical feature of senescence as follow: The percentage ofSA-β-Gal positive cells was significantly increased; the ratio of G1stagewas strikingly increased, while the ratio S stage was decreased; the numberof CFU-Mix was also notably decreased. The content of ROS wasincreased and the level of T-AOC was decreased in aged Sca-1~+HSCs. Thelength of telomere was remarkedly Shortening in aged Sca-1~+HSCs, and the activity of telomerase of aged Sca-1~+HSCs was also reduced.X raycould remarkedly upregulate the expression of p16and p21in mRNA andprotein levels, decrease the protein expression of CDK4, cyclinD andcyclinE in Sca-1~+HSCs. There was no significant difference in the proteinexpression of CDK2in Sca-1~+HSCs.5. ASP could decreased the the percentage of SA-β-Gal positive cellsand the ratio of G1stage, increased the ratio of S stage and the number ofCFU-Mix in aged Sca-1~+HSCs. The content of ROS was decreased and thelevel of T-AOC was increased in aged Sca-1~+HSCs. ASP could inhibitdamage of telomere DNA and increase the activity of telomerase of agedSca-1~+HSCs, downregulate the expression of p16and p21in mRNA andprotein levels, upregulate the protein expression of CDK6, cyclinD andcyclinE in aged Sca-1~+HSCs. There was no significant difference in theprotein expression of CDK2in Sca-1~+HSCs.Conclusions:1. MACS can be effective seperated Sca-1~+HSCs in mice.Theseparated Sca-1~+HSCs have higher purity and good activity.2. ox-LDL could be used as a effective failure agent that couldreplicate aged model of Sca-1~+HSCs in vitro.3. ASP could delay Sca-1~+HSCs aging induced by ox-LDL whichmaybe ascribed to the regulation of cell cycle regulatory protein expression and the inhibition of damage in telomere DNA.4. X-ray(3.0Gy/8F) TBI could induce Sca-1~+HSCs aging in mice andit would be used for building the aged model of mice Sca-1~+HSCs in vivo.5. ASP could delay Sca-1~+HSCs aging induced by X-ray TBI micewhich maybe ascribed to the inhibition of damage of oxidative stress andtelomere DNA, the regulation of cell cycle regulatory protein xpression andthe addition of telomerase activity. |
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