Establishment Of The Aging Model Of Sca-1~+ Hematopoietic Stem Cell In Vivo And Its Relative Biological Mechanism

Objective: Birth, aged, sickness and death is the natural order of life. Aging is irreversible process of decline in overall of structure and function from all kinds of body cells, tissue and organ which accompanied by the body after development and maturation over time. New study suggests that: body's aging is actually the stem cells'aging, such as tissue and organ degeneration, cancer incidence and repeated infection are the concrete expression of the level of stem cells'aging. Therefore, the in-depth study of the mechanism of stem cells aging has important theoretical and clinical value to prevent and treat the senile diseases.Experimental results show that HSC senescence is closely related to body aging. HSC senescence is cell growth arrest, irreversible remain in G1 phase, self-renewal and differentiation capacity reduced, the number of cells decreased and apoptosis increased. HSC senescence can lead to the ability of blood and immune system decline, increased incidence of cancer and fail of the body structure and function of important organs. Currently, we have not catch the mechanism of HSC aging, if we find the ways to delay the aging, it will undoubtedly that have important theoretical and clinical value to prevent senile diseases. Constructed and detected biological information of aging model is an important foundation work to study the mechanism of HSC aging, but now, there is still not ideal and accepted aging model of HSC in vivo. C57 mice as the research object in our study, we using the continuity of the male donor mice HSC transplantation to establish the replicating HSC aging model of mice in vivo, and testing the biological characteristics of HSC aging on overall, cell and molecular level. The purpose is provided theoretical and experimental evidence to study the mechanism of HSC aging and find the ways to delay the aging.Methods:1. Sca-1+HSC was isolated and purified from mice bone marrow mono-nucleated cell by magnetic activated cell sorting (MACS). The purity of separated cells was analyzed by flow cytometry (FCM), the expression of Sca-1+ antigen was detected by immunofluorescence and the activity of separated cells was detected by Trypan blue staining.2. The replicating HSC aging model of mice in vivo was established through the HSC serial transplantation from the male donor mice to the female receptor by tail vein. The expression of Y chromosome gene was analyzed by PCR, which was used to identify whether hematopoietic cells of recipient mice come from male donor. After transplantation, we determined the hematopoietic function of reconstruction by observed the number of CFU-S, detected spleen index and thymus index, observed the recovery indicator in peripheral blood (WBC, RBC and PLT).3. Biological characteristics (self-renewal and differentiation capacity) of aging HSC by serial transplantation was evaluated by mixed hematopoietic progenitor cell culture in vitro, cell cycle assay and senescence-associatedβ-galactosidase (SA-β-gal) cytochemical staining.Results:1. Mice Sca-1+HSC purification and identificationFCM showed that the purity of separated Sca-1+HSC from MNCs was only 1.7% before MACS; but the purity of separated Sca-1+HSC from MNCs was more than 87.2% after MACS. Immunofluorescence showed that only a little cells expressed Sca-1+antigen before MACS, but almost all of the cells expressed Sca-1+antigen after MACS. The survival of Sca-1+HSC which detected by Trypan blue staining was 96%～99%. These results suggest that the separated Sca-1+cells have a higher purity and activity.2. The detection of CFU-S, spleen index, thymus index and peripheral blood indicator after donor HSC serial transplantation2.1 After twice transplantation, the ability of donor HSC to form CFU-S decreased.2.2 After male donor HSC serial transplantation, the weight of spleen and thymus, spleen index and thymus index showed a clear decline.2.3 After twice transplantation, receptor mice WBC, RBC, PLT and HCT were decreased, suggested that the ability of HSC such as self-renewal and hematopoietic reconstruction reduced during serial transplantation.3 The detection of Y chromosome geneWe analyzed the Y chromosome gene by PCR, the result showed that the Y chromosome gene of 342bp (Sry sequence) was detected in hematopoietic cells from receptor mice. It's means that HSC of male donor mice recovering hematopoietic of female receptor.4 Biological characteristics of aging HSC4.1 With the HSC serial transplantation, the number of CFU-Mix and cells from CFU-Mix decreased evidently.4.2 With the HSC serial transplantation, cells stopped in G1 phase of cell cycle, the percentage of G0/G1 phase of cell cycle was increased and S phase was decreased.4.3 With the HSC serial transplantation,? the percentage of cells expressed SA-β-gal was increased evidently.Conclusion:1. It is successful that isolated and purified Sca-1+HSC from male C57 mice bone marrow mono-nucleated cells by MACS, and cells have a higher activity.?2. It is effective that Sca-1+HSC from male donor could reconstruct hematopoietic of female receptor.3. It is successful to establish the replicating HSC aging model of mice in vivo through the Sca-1+HSC serial transplantation.