| Hematopoietic stem cell(HSC) is a kind stem cell of self-renewal and multi-directional differentiation potential, and can maintain the physiological functions undering the regulation of the hematopoietic microenvironment(HM) and variety internal machanism. Literature reported physiological aging and radiation therapy and chemotherapy can cause bone marrow stromal cells senescence, but it is unclear the effect of senescence hematopoietic microenvironment on hematopoietic stem cells and its mechanism. The level of reactive oxygen species(ROS) plays an important role in physiological activities of hematopoietic stem cell. The low level of reactive oxygen species in hematopoietic stem cells have long-term self-renewal ability, but excessive accumulation of ROS in cells can cause oxidative stress, induce DNA damage and cause sress-induced premature senescence( SIPS). Cx43, connexin 43, mainly express in the bone marrow stromal cells. It is the basic structure and function protein of gap junction between BMSCs and HSCs. ROS can be transferred from hematopoietic stem cells to bone marrow stromal cells by Cx43. Connexin43 was a necessary condition that it can regulate the level of reactive oxygen species between bone marrow stromal cells and hematopoietic stem cells, reduce oxidative stress of hematopoietic stem cell and promote normal hematopoiesis. We established the model that hematopoietic stemcells and hematopoietic progenitors cells(HSCs/HPCs) extracted from femur bone marrow were co-cultured with D-Gal-indued aging BMSCs in vitro, then observe the biology characteristics of bone marrow stromal cells aging and the effects of BMSCs on hematopoietic stem/progenitor cells.Angelica sinensis polysaccharide(ASP) is the effective medicinal ingredients which separated and extracted from traditional Chinese medicine-angelica, ASP can regulate hematopoiesis, resistance to radiation damage, anti-tumor and immune regulation and so on. Preliminary study showed that angelica sinensis polysaccharide can delay the bone marrow stromal cells senescence in the aging rat model and promote hematopoietic cell proliferation differentiation by delaying bone marrow stromal cells senescence. But it has not yet been elucidated that angelica sinensis polysaccharide regulate the relationship between bone marrow stromal cells and hematopoietic cells. This study further investigate that angelica sinensis polysaccharide regulate the effect of bone marrow stromal cells,for example, oxidative stress, cell components, Cx43 expression, secretion cell active factor. We studied that angelica sinensis polysaccharide regulate the effect of low oxygen hematopoietic microenvironment on hematopoietic stem cell oxidative stress, and provide experimental foundation and theoretical basis that angelica sinensis polysaccharide regulates hematopoietic stem/progenitor cell senescence from bone marrow microenvironment degree, by establishing the model that hematopoietic stem cells and hematopoietic progenitors cells(HSCs/HPCs) extracted from femur bone marrow were co-cultured with D-Gal-indued aging BMSCs in vitro.Methods: 1. D-Gal induced to establishing aging BMSCs model,explored the change of oxidative stress, cellular component, gap junctionprotein Cx43 expression and secretion activity factor in BMSCs, and the regulation of angelica sinensis polysaccharide on senescence bone marrow stromal cells : After the femora and tibia bones were separated out of the C57 mouse body, we abstracted whole bone marrow cell suspension under sterile conditions and cultured the third passages BMSCs in vitro and carried out relevant experimental detection. Experimental group : the control group was cultured as usual for 96 hours; the aging group was cultured with D-galactose(D-Gal) 30mg/m L for 96 hours; the ASP group was cultured with ASP 100μL/m L for 96 hours; the ASP delaying aging group was cultured with D-galactose(D-Gal) 30mg/m L for 48 hours and then was cultured with ASP 100μL/m L for 48 hours. Senescence relevant bological indicators were tested by SA-β-Gal staining and cell cycle analysis, the level of ROS was detected by immunofluorescence and flow cytometry(FCM), the gene expression of RUNX2 and PPARγ in BMSCs were tested by RT-PCR, the expression of Cx43 protein and SDF-1 was checked by immunofluorescence and the function of Cx43 was checked by fluorescence yellow scratch transmission technique, the amount of IL-1β,Il-6, TNF-ɑ and RANTES in BMSCs culture supernatant were detected by ELISA.2. The model of BMSCs and BMNCs co-cultured was established to observe the oxidative stress effect of BMSCs senescence on hematopoietic cells and the regulation function of angelica sinensis polysaccharide: After extracting BMNCs were cultured 6 hours, we removed the BMSCs in the BMNCs and the suspending BMNCs were collected. BMNCs(1×106/m L)with the above four groups of BMSCs model were co-cultured. After 48 h,we tested relevant indicators by collecting cells. The number of cell activity was tested by using trypan blue staining. Senescence relevant bological indicators of BMNCs were tested by SA-β-Gal staining and cell cycleanalysis; immunofluorescence and flow cytometry(FCM) detected the level of ROS; the changes of aging biology indexs(P53, P21, P38, P16)were observed by western blotting.3.The model of BMSCs with Sca-1~+HSC/HPCs co-cultured was established to study the oxidative stress effect of ASP regulating senescence BMSCs on hematopoietic stem cell: We extracted Sca-1~+HSC/HPCs by immune magnetic bead separation. Sca-1~+HSC/HPCs with the above four groups of BMSCs model were co-cultured. After 48 h,we tested relevant indicators by collecting suspending Sca-1~+cells. The proliferation and differentiation function of HSC/HPCs was tested by CFU-Mix cultured. Immunofluorescence observed the level of ROS; flow cytometry(FCM) detected γH2AX of DNA damage indicator; the amount of 8-OHd G in Sca-1~+HSC/HPCs culture supernatant were detected by ELISA. The expression of gene(P53 、 P21 、 P38 、 P16) were tested by RT-PCR. The expression of P38 was observed by immunofluorescence.Results: 1. The cell cycle of senescence BMSCs blocked in G1 phase;the positive ratio of SA-β-Gal stained BMSCs also obviously increased; the level of ROS significantly increased; adipogenic differentiation related gene(PPARγ) expression was significantly up-regulated and osteogenic differentiation related gene(Runx2) was significantly down-regulated; the expression of Cx43 and SDF-1 were obviously down-regulated, the function of fluorescence dye transfer was decreased. The secretion of IL-1β,Il-6, TNF-ɑ and RANTES significantly increased in senescence BMSCs.BMSCs which were injected angelica sinensis polysaccharide, cell cycle arrest was alleviated; the positive ratio of SA-β-Gal stained also obviously decreased; the level of ROS also significantly decreased; the ratio of osteogenic differentiation/adipogenic differentiation increased, the expression and function of Cx43 rised again. The expression of SDF-1 wasalso obviously up-regulated. The secretion of IL-1β, Il-6, TNF-ɑ and RANTES decreased.2. Compared to aging group, ASP delaying aging group BMSCs showed that levels of SA-β-gal + cell %, G1 phase %, secretion number of IL-1β,Il-6, TNF-ɑ and RANTES, and adipogenic differentiation related gene(PPARγ) expression significantly decreased, but levels of S-phase %,osteogenic differentiation related gene(Runx2), SDF-1 protein and connexin protein(Cx43) expression significantly increased.3. After BMNCs co-cultured with D-gal-administration group BMSCs,the number of live cells decreased, cell cycle of BMNCs blocked in G1 phase, the positive ratio of SA-β-Gal stained and levels of ROS obviously increased, the expression of aging biology protein(P53, P21, P38, P16)were up-regulated. Compared to the BMNCs co-cultured D-gal-administration group BMSCs, the BMNCs co-cultured with ASP delaying aging group BMSCs showed that the number of live cells increased, SA-β-gal + cell %, G1 phase % and levels of ROS decreased, the expression of aging biology protein(P53, P21, P38, P16) were down-regulated.4. The Sca-1~+HSCs/HPCs co-cultured with D-gal-administration group BMSCs showed that the CFU-Mix forming number of multipotential progenitor cell obviously decreased, the expression levels of reactive oxygen species(ROS) 、 γH2AX and 8-OHd G obviously increased,expression of senescence-associated fluorescence protein P38 and genes(P53, P21, P38, P16) were up-regulated. Compared to the Sca-1~+cells co-cultured D-gal-administration group BMSCs, the Sca-1~+cells co-cultured with ASP delaying aging group BMSCs showed that the ability of CFU-Mix forming was enhanced, the expression levels of reactiveoxygen species(ROS) 、 γH2AX and 8-OHd G significantly decreased, the expression of protein P38 and genes(P53, P21, P38, P16) were down-regulated.Conclusion:1. Senescence bone marrow stromal cells can enhance oxidative stress and induce the change of bone marrow microenvironment: the cellular composition of heterogeneous cell population can be changed; expression and function of Cx43 can be reduced, ability of senescence bone marrow stromal cells relieving oxidative stress in HSC/HPCs can be decreased;secretory activity factor was changed in bone marrow stromal.2. Senescence bone marrow microenvironment can inhibit proliferation and differentiation of HSC/HPCs. This mechanism could be the enhancing of oxidative stress in senescence bone marrow microenvironment and the lower ability of relieving hemopoietic cells oxidative stress, induce the occurrence of stress-induced premature senescence of HSC/HPCs, cause HSC/HPCs DNA damage and cell aging.3. ASP can improve hematopoietic microenvironment and delay the aging of hematopoietic stem/progenitor cells, and its mechanism may be related to reduce the oxidative stress of BMSCs and HSC/HPCs and up-regulate gap junction Cx43 protein, reduce the DNA oxidative damage, inhibit aging related protein expression. |
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