BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase ...BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.展开更多
To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The ...To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 （0, 10, 20, 50 and 100 ng/mL）. After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen Ⅱ mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen Ⅱ mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.展开更多
Propoxur is a widely used dithiocarbamate insecticide. In this study, the clastogenic effect of propoxur has been evaluated using chromosomal aberration assay in mouse bone marrow cells. Single i. p. administration of...Propoxur is a widely used dithiocarbamate insecticide. In this study, the clastogenic effect of propoxur has been evaluated using chromosomal aberration assay in mouse bone marrow cells. Single i. p. administration of propoxur, at 25 mg/kg b.wt., a maximum tolerated dose (MTD) and 12 .5mg/kg b.wt (50% of MTD) have significantly induced different types of aberrations after 24 h of treatment. The aberrations were dose and time dependent and reached a maximum after 24 h of exposure. The sresult suggest a genotoxic potential of propoxur.展开更多
Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs ...Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5（100 ng/ml）, with or without 1-heptanol（2.5 la mol/L）. The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type II collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43（Cx43） protein was examined by western blotting. Results：GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type II collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion：These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF- 5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.展开更多
Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed col...Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.展开更多
AIM:To assess the capacity to isolate and expand mesenchymal stem cells(MSC)from bone marrow of CBA/Ca,ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed,cultured and maintained in supplemented D...AIM:To assess the capacity to isolate and expand mesenchymal stem cells(MSC)from bone marrow of CBA/Ca,ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed,cultured and maintained in supplemented Dulbecco’s modified Eagle’s medium.MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106,Sca-1 and CD44 and negativity to CD45,CD11b and MHC classⅡ.Differentiation capacity of MSC towards osteogenic and adipo-genic lineages were also assessed. RESULTS:MSC were successfully cultured from bone marrow of all 3 strains,albeit differences in the temporal expression of certain surface antigens.Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype(CD106-CD45+CD11b+Sca-1low)to typical MSC phenotype(CD106+CD45-CD11b-Sca-1high)with increasing passages. CONCLUSION:Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.展开更多
Objective: A murine model of mixing syngeneic and haploidentical major histocompatibility complex (MHC) matched bone marrow cells transplant was used to evaluate the effect of splenocytes in graft-versus-host disease ...Objective: A murine model of mixing syngeneic and haploidentical major histocompatibility complex (MHC) matched bone marrow cells transplant was used to evaluate the effect of splenocytes in graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR). Methods: BALB/C recipient mice were lethally conditioned with 8.5 Gy and injected with different grafts which consisted of syngeneic bone marrow cells plus splenocytes (SPLCs) and haploidentical MHC matched bone marrow cells (BMCs)plus different doses of splenocytes. Recipient mice were detected for the percentage of haploidentical MHC matched mouse origin cells in the peripheral blood cells and checked daily for the appearance of GVHD symptoms. Histopathological examination of multiple organs from moribund mice was used to evaluate the grades of GVHD. Results: Recipient mice infused with 10 × 106 haploidentical MHC matched SPLCs and 5×106 syngeneic splenocytes showed a higher level and more stable chimerism with GVHD Ⅱ degree histopathological alterations. Histopathological results of GVHD in other group's hosts were not obvious, and the levels of chimerism were unstable. All of the mice survived over 150 d. Conclusion:The proportion and dose of syngeneic and haploidentical MHC splenocytes are of importance for inducing stable engraftment on the basis of nonlethal GVHD and to balance GVHD and HVGR.展开更多
AIM: To identify the key cytokines involved in hepatic differentiation of mouse bone marrow mesenchymal stem cells (mBM-MSCs) under liver-injury conditions. METHODS: Abdominal injection of CCl4 was adopted to duplicat...AIM: To identify the key cytokines involved in hepatic differentiation of mouse bone marrow mesenchymal stem cells (mBM-MSCs) under liver-injury conditions. METHODS: Abdominal injection of CCl4 was adopted to duplicate a mouse acute liver injury model. Global gene expression analysis was performed to evaluate the potential genes involved in hepatic commitment under liver-injury conditions. The cytokines involved in hepatic differentiation of mBM-MSCs was function-ally examined by depletion experiment using specifi c antibodies, followed by rescue experiment and direct inducing assay. The hepatic differentiation was characterized by the expression of hepatic lineage genes and proteins, as well as functional features. RESULTS: Cytokines potentially participating in hepatic fate commitment under liver-injury conditions were initially measured by microarray. Among the up-regulated genes determined, 18 cytokines known to closely relate to liver growth, repair and development, were selected for further identif ication. The f ibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF) and oncostatin M (OSM) were fi nally found to be involved in hepatic differentiation of mBM-MSCs under liver-injury conditions. Hepatic differentiation could be dramatically decreased after removing FGF-4, HGF and OSM from the liver-injury conditioned medium, and could be rescued by supplementing these cytokines. The FGF-4, HGF and OSM play different roles in the hepatic differentiation of mBM-MSCs, in which FGF-4 and HGF are essential for the initiation of hepatic differentiation, while OSM is critical for the maturation of hepatocytes. CONCLUSION: FGF-4, HGF and OSM are the key cytokines involved in the liver-injury conditioned medium for the hepatic differentiation of mBM-MSCs.展开更多
Objective：To explore the effect of Renshen Yangrong Decoction（人参养荣汤,RYD） in protecting bone marrow from radiation injury.Methods：One hundred and eighty Kuming mice were subjected to the three tests for anti-r...Objective：To explore the effect of Renshen Yangrong Decoction（人参养荣汤,RYD） in protecting bone marrow from radiation injury.Methods：One hundred and eighty Kuming mice were subjected to the three tests for anti-radiation injury effect evaluation,i.e.the test of peripheral white blood cell（WBC） count, the test of bone marrow nucleated cell count,and the bone marrow micronucleus test,using 60 mice for each test.The mice in each test were divided into 6 groups：the blank control group,the model control group,the positive control group treated by Shiyiwei Shenqi Tablet（十一味参芪片,1.0 g/kg）,and three RYD groups treated with high（42.0 g/kg）,moderate（21.0 g/kg）,and low（10.5 g/kg） doses of crude drugs of RYD,with 10 mice in each group.The treatment was given by gastrogavage perfusion continuously for 7-14 days before mice received ^（60）Co-γray radiation and continued until the end of the experiment.The body weights of the mice were monitored,the changes in peripheral WBC and bone marrow nucleated cells were counted,and the variation in bone marrow micronucleated cells was observed on the respective appointed days.Results：A significant decrease in body weight,peripheral WBC count,and bone marrow nucleated cell count,as well as marked changes in bone marrow micronucleated cells were observed in the mice after radiation,indicating that the radiation injury model was successfully established.As compared with the model control group,the decrease in body weight,peripheral WBC count,and bone marrow nucleated cell count,as well as the increase in bone marrow micronucleus cell count in the high dosage RYD treated group were obviously inhibited or lessened （P0.05 or P0.01）.Conclusion：RYD showed obvious protective effect in mice with bone marrow injury induced by radiation.展开更多
Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripher-al nerve defects in rodents. I...Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripher-al nerve defects in rodents. In this study, we established a standardized experimental model of radial nerve defects in primates and evaluated the effect of repair on peripheral nerve injury. We repaired 2.5-cm lesions in the radial nerve of rhesus monkeys by transplantation of autografts, acellular allografts, or acellular allografts seeded with autologous bone marrow stem cells. Five months after surgery, regenerated nerve tissue was assessed for function, electrophysiology, and histomorphometry. Postoperative functional recovery was evaluated by the wrist-extension test. Compared with the simple autografts, the acellular allografts and allografts seeded with bone marrow stem cells facilitated remarkable recovery of the wrist-extension functions in the rhesus monkeys. This functional improvement was coupled with radial nerve distal axon growth, a higher percentage of neuron survival, increased nerve fiber density and diameter, increased myelin sheath thickness, and increased nerve conduction velocities and peak amplitudes of compound motor action potentials. Furthermore, the quality of nerve regeneration in the bone marrow stem cells-laden allografts group was comparable to that achieved with autografts. The wrist-extension test is a simple behavioral method for objective quantification of peripheral nerve regeneration.展开更多
If induced pluripotent stem(iPS)cells are to be used to treat damaged tissues or repair organs in elderly patients,it will be necessary to establish iPS cells from their tissues.To determine the feasibility of using t...If induced pluripotent stem(iPS)cells are to be used to treat damaged tissues or repair organs in elderly patients,it will be necessary to establish iPS cells from their tissues.To determine the feasibility of using this technology with elderly patients,we asked if it was indeed possible to establish iPS cells from the bone marrow(BM)of aged mice.BM cells from aged C57BL/6 mice carrying the green fluorescence protein(GFP)gene were cultured with granulocyte macrophage-colony stimulating factor(GM-CSF)for 4 days.Four factors(Oct3/4,Sox2,Klf4 and c-Myc)were introduced into the BM-derived myeloid(BM-M)cells.The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low.However,we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice,which carried GFP.Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining.The iPS cells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice,and can be differentiated to three germ layers in vitro.By co-culturing with OP9,the BM-M-iPS cells can be differentiated to the myeloid lineage.The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF,and lost expression of Nanog and Pou5f1,at least in part,due to methylation of their promoters.On the contrary,Tnf and Il1b gene expression was upregulated and their promoters were hypomethylated.展开更多
The mutagenicity of the protein-modified Enterococcus faecalis was evaluated by a mouse bone marrow micronucleus test and a mouse sperm abnormality test.The test substance was designed with three dose groups (1,2 and ...The mutagenicity of the protein-modified Enterococcus faecalis was evaluated by a mouse bone marrow micronucleus test and a mouse sperm abnormality test.The test substance was designed with three dose groups (1,2 and 5 g/kg·bw) and intragastrically administrated,with cyclophosphamide as a positive control and normal saline as a normal control.The micronucleus rate and sperm abnormality rate were measured.The results showed that the micronucleus rates and sperm abnormality rates in the different dose groups were not significantly different from those in normal control group ( P >0.05),while the positive control group was significantly higher than the normal control group ( P <0.01).The conclusion is that the protein-modified E.faecalis was tested to be negative in both the mouse bone marrow micronucleus test and mouse sperm abnormality test,suggesting that it has no mutagenicity in vivo.展开更多
The genotoxic potentiality of the crude leaf extract of Casearia tomentosa, a medicinal preparation, has been evaluated in Swiss albino mice. The extract significantly induced the division_disruptive chromosomal c...The genotoxic potentiality of the crude leaf extract of Casearia tomentosa, a medicinal preparation, has been evaluated in Swiss albino mice. The extract significantly induced the division_disruptive chromosomal changes in bone marrow cells as well as in primary spermatocytes; the latter also exhibited marked increase in synaptic disruptions. A significant decrease in sperm count was noted. The incidence of structural damages in chromosomes, however, remained within the range of control level frequency. This herbal preparation, therefore, appears to be primarily spindle_poisoning in its action, but not clastogenic. The probable mechanism of this differential genotoxicity is discussed.展开更多
基金Supported by Natural Science Foundation of Shandong Province,China,No.ZR2020MH014,No.ZR2021QH179 and No.ZR2021MH182.
文摘BACKGROUND The low survival rate of mesenchymal stem cells(MSCs)caused by anoikis,a form of apoptosis,limits the therapeutic efficacy of MSCs.As a proapoptotic molecule,mammalian Ste20-like kinase 1(Mst1)can increase the production of reactive oxygen species(ROS),thereby promoting anoikis.Recently,we found that Mst1 inhibition could protect mouse bone marrow MSCs(mBMSCs)from H 2 O 2-induced cell apoptosis by inducing autophagy and reducing ROS production.However,the influence of Mst1 inhibition on anoikis in mBMSCs remains unclear.AIM To investigate the mechanisms by which Mst1 inhibition acts on anoikis in isolated mBMSCs.METHODS Poly-2-hydroxyethyl methacrylate-induced anoikis was used following the silencing of Mst1 expression by short hairpin RNA(shRNA)adenovirus transfection.Integrin(ITGs)were tested by flow cytometry.Autophagy and ITGα5β1 were inhibited using 3-methyladenine and small interfering RNA,respe-ctively.The alterations in anoikis were measured by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling and anoikis assays.The levels of the anoikis-related proteins ITGα5,ITGβ1,and phospho-focal adhesion kinase and the activation of caspase 3 and the autophagy-related proteins microtubules associated protein 1 light chain 3 II/I,Beclin1 and p62 were detected by Western blotting.RESULTS In isolated mBMSCs,Mst1 expression was upregulated,and Mst1 inhibition significantly reduced cell apoptosis,induced autophagy and decreased ROS levels.Mechanistically,we found that Mst1 inhibition could upregulate ITGα5 and ITGβ1 expression but not ITGα4,ITGαv,or ITGβ3 expression.Moreover,autophagy induced by upregulated ITGα5β1 expression following Mst1 inhibition played an essential role in the protective efficacy of Mst1 inhibition in averting anoikis.CONCLUSION Mst1 inhibition ameliorated autophagy formation,increased ITGα5β1 expression,and decreased the excessive production of ROS,thereby reducing cell apoptosis in isolated mBMSCs.Based on these results,Mst1 inhibition may provide a promising strategy to overcome anoikis of implanted MSCs.
基金This project was supported by a grant from the National Natural Sciences Foundation of China (No 30471753)
文摘To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 （0, 10, 20, 50 and 100 ng/mL）. After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen Ⅱ mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen Ⅱ mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.
文摘Propoxur is a widely used dithiocarbamate insecticide. In this study, the clastogenic effect of propoxur has been evaluated using chromosomal aberration assay in mouse bone marrow cells. Single i. p. administration of propoxur, at 25 mg/kg b.wt., a maximum tolerated dose (MTD) and 12 .5mg/kg b.wt (50% of MTD) have significantly induced different types of aberrations after 24 h of treatment. The aberrations were dose and time dependent and reached a maximum after 24 h of exposure. The sresult suggest a genotoxic potential of propoxur.
基金supported by the National Natural Science Foundation of China(30471753)
文摘Objective：To investigate the effect of the gap junction blocker 1-heptanol on the in vitro chondrogenic differentiation of mouse bone marrow mesenchymal stem cells（MSCs） following induction by GDF-5. Methods：MSCs were isolated from mouse bone marrow and cultured in vitro. After 3 passages cells were induced to undergo chondrogenic differentiation with recombinant human GDF-5（100 ng/ml）, with or without 1-heptanol（2.5 la mol/L）. The effect of 1-heptanol on MSCs proliferation was investigated using the MTT assay. Type II collagen mRNA and protein were examined by RT-PCR and immunocytochemistry respectively, and the sulfate glycosaminoglycan was assessed by Alcian blue dye staining. Connexin43（Cx43） protein was examined by western blotting. Results：GDF-5 induced proliferation and chondrogenic differentiation of MSCs. While 1-heptanol treatment had no effect on this proliferation, it inhibited the expression of both type II collagen mRNA and protein. The Alcian blue staining revealed that 1-heptanol also inhibited the deposition of the typical cartilage extracellular matrix promoted by recombinant GDF-5. Western blotting demonstrated that 1-heptanol had no effect on the expression of Cx43. Conclusion：These results suggest that mouse bone marrow MSCs can be differentiated into a chondrogenic phenotype by GDF- 5 administration in vitro. While the gap junction blocker, 1-heptanol, did not reduce gap junction Cx43, these intercellular communication pathways clearly played an important functional role in GDF-5-induced cartilage differentiation.
基金supported by the National Natural Science Foundation of China,No.30471836
文摘Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.
基金Supported by The Research University Grant Scheme UPM,04-02-10-0924RUExploratory Research Grant Scheme,Ministry of Higher Education,ERGS/1/2012/5527106
文摘AIM:To assess the capacity to isolate and expand mesenchymal stem cells(MSC)from bone marrow of CBA/Ca,ICR and Balb/c mice. METHODS:Bone marrow of tibia and femur were flushed,cultured and maintained in supplemented Dulbecco’s modified Eagle’s medium.MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106,Sca-1 and CD44 and negativity to CD45,CD11b and MHC classⅡ.Differentiation capacity of MSC towards osteogenic and adipo-genic lineages were also assessed. RESULTS:MSC were successfully cultured from bone marrow of all 3 strains,albeit differences in the temporal expression of certain surface antigens.Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype(CD106-CD45+CD11b+Sca-1low)to typical MSC phenotype(CD106+CD45-CD11b-Sca-1high)with increasing passages. CONCLUSION:Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.
文摘Objective: A murine model of mixing syngeneic and haploidentical major histocompatibility complex (MHC) matched bone marrow cells transplant was used to evaluate the effect of splenocytes in graft-versus-host disease (GVHD) and host-versus-graft reaction (HVGR). Methods: BALB/C recipient mice were lethally conditioned with 8.5 Gy and injected with different grafts which consisted of syngeneic bone marrow cells plus splenocytes (SPLCs) and haploidentical MHC matched bone marrow cells (BMCs)plus different doses of splenocytes. Recipient mice were detected for the percentage of haploidentical MHC matched mouse origin cells in the peripheral blood cells and checked daily for the appearance of GVHD symptoms. Histopathological examination of multiple organs from moribund mice was used to evaluate the grades of GVHD. Results: Recipient mice infused with 10 × 106 haploidentical MHC matched SPLCs and 5×106 syngeneic splenocytes showed a higher level and more stable chimerism with GVHD Ⅱ degree histopathological alterations. Histopathological results of GVHD in other group's hosts were not obvious, and the levels of chimerism were unstable. All of the mice survived over 150 d. Conclusion:The proportion and dose of syngeneic and haploidentical MHC splenocytes are of importance for inducing stable engraftment on the basis of nonlethal GVHD and to balance GVHD and HVGR.
基金Supported by The Grant of Medicine and Health Key Projects of Zhejiang Province, Science and Technology Fund of Ministry of Health of the People’s Republic of China, No. WKJ2007-2-037Shaoxing Key Project for Science and Technology, No. 2007A23008the Natural Science Foundation of Zhejiang Province, China, No. Y2090337
文摘AIM: To identify the key cytokines involved in hepatic differentiation of mouse bone marrow mesenchymal stem cells (mBM-MSCs) under liver-injury conditions. METHODS: Abdominal injection of CCl4 was adopted to duplicate a mouse acute liver injury model. Global gene expression analysis was performed to evaluate the potential genes involved in hepatic commitment under liver-injury conditions. The cytokines involved in hepatic differentiation of mBM-MSCs was function-ally examined by depletion experiment using specifi c antibodies, followed by rescue experiment and direct inducing assay. The hepatic differentiation was characterized by the expression of hepatic lineage genes and proteins, as well as functional features. RESULTS: Cytokines potentially participating in hepatic fate commitment under liver-injury conditions were initially measured by microarray. Among the up-regulated genes determined, 18 cytokines known to closely relate to liver growth, repair and development, were selected for further identif ication. The f ibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF) and oncostatin M (OSM) were fi nally found to be involved in hepatic differentiation of mBM-MSCs under liver-injury conditions. Hepatic differentiation could be dramatically decreased after removing FGF-4, HGF and OSM from the liver-injury conditioned medium, and could be rescued by supplementing these cytokines. The FGF-4, HGF and OSM play different roles in the hepatic differentiation of mBM-MSCs, in which FGF-4 and HGF are essential for the initiation of hepatic differentiation, while OSM is critical for the maturation of hepatocytes. CONCLUSION: FGF-4, HGF and OSM are the key cytokines involved in the liver-injury conditioned medium for the hepatic differentiation of mBM-MSCs.
基金Supported by the Scientific and Technologic Items of Traditional Chinese Medicine Administration of Beijing City(No.2004 JingZhongZhongⅣ15)
文摘Objective：To explore the effect of Renshen Yangrong Decoction（人参养荣汤,RYD） in protecting bone marrow from radiation injury.Methods：One hundred and eighty Kuming mice were subjected to the three tests for anti-radiation injury effect evaluation,i.e.the test of peripheral white blood cell（WBC） count, the test of bone marrow nucleated cell count,and the bone marrow micronucleus test,using 60 mice for each test.The mice in each test were divided into 6 groups：the blank control group,the model control group,the positive control group treated by Shiyiwei Shenqi Tablet（十一味参芪片,1.0 g/kg）,and three RYD groups treated with high（42.0 g/kg）,moderate（21.0 g/kg）,and low（10.5 g/kg） doses of crude drugs of RYD,with 10 mice in each group.The treatment was given by gastrogavage perfusion continuously for 7-14 days before mice received ^（60）Co-γray radiation and continued until the end of the experiment.The body weights of the mice were monitored,the changes in peripheral WBC and bone marrow nucleated cells were counted,and the variation in bone marrow micronucleated cells was observed on the respective appointed days.Results：A significant decrease in body weight,peripheral WBC count,and bone marrow nucleated cell count,as well as marked changes in bone marrow micronucleated cells were observed in the mice after radiation,indicating that the radiation injury model was successfully established.As compared with the model control group,the decrease in body weight,peripheral WBC count,and bone marrow nucleated cell count,as well as the increase in bone marrow micronucleus cell count in the high dosage RYD treated group were obviously inhibited or lessened （P0.05 or P0.01）.Conclusion：RYD showed obvious protective effect in mice with bone marrow injury induced by radiation.
基金supported by the National High-Technology Research and Development Program of China(863 Program),No.2006AA02A130the National Natural Science Foundation of China,No.81372041,31070869,30700847
文摘Current research on bone marrow stem cell transplantation and autologous or xenogenic nerve transplantation for peripheral nerve regeneration has mainly focused on the repair of peripher-al nerve defects in rodents. In this study, we established a standardized experimental model of radial nerve defects in primates and evaluated the effect of repair on peripheral nerve injury. We repaired 2.5-cm lesions in the radial nerve of rhesus monkeys by transplantation of autografts, acellular allografts, or acellular allografts seeded with autologous bone marrow stem cells. Five months after surgery, regenerated nerve tissue was assessed for function, electrophysiology, and histomorphometry. Postoperative functional recovery was evaluated by the wrist-extension test. Compared with the simple autografts, the acellular allografts and allografts seeded with bone marrow stem cells facilitated remarkable recovery of the wrist-extension functions in the rhesus monkeys. This functional improvement was coupled with radial nerve distal axon growth, a higher percentage of neuron survival, increased nerve fiber density and diameter, increased myelin sheath thickness, and increased nerve conduction velocities and peak amplitudes of compound motor action potentials. Furthermore, the quality of nerve regeneration in the bone marrow stem cells-laden allografts group was comparable to that achieved with autografts. The wrist-extension test is a simple behavioral method for objective quantification of peripheral nerve regeneration.
基金This work was supported by the Ministry of Education,Science,Technology,Sports and Culture,Japan。
文摘If induced pluripotent stem(iPS)cells are to be used to treat damaged tissues or repair organs in elderly patients,it will be necessary to establish iPS cells from their tissues.To determine the feasibility of using this technology with elderly patients,we asked if it was indeed possible to establish iPS cells from the bone marrow(BM)of aged mice.BM cells from aged C57BL/6 mice carrying the green fluorescence protein(GFP)gene were cultured with granulocyte macrophage-colony stimulating factor(GM-CSF)for 4 days.Four factors(Oct3/4,Sox2,Klf4 and c-Myc)were introduced into the BM-derived myeloid(BM-M)cells.The efficiency of generating iPS cells from aged BM cultured in GM-CSF was low.However,we succeeded in obtaining BM-M-iPS cells from aged C57BL/6 mice,which carried GFP.Our BM-M-iPS cells expressed SSEA-1 and Pou5f1 and were positive for alkaline phosphatase staining.The iPS cells did make teratoma with three germ layers following injection into syngeneic C57BL/6 mice,and can be differentiated to three germ layers in vitro.By co-culturing with OP9,the BM-M-iPS cells can be differentiated to the myeloid lineage.The differentiated BM-M-iPS cells proliferated well in the presence of GM-CSF,and lost expression of Nanog and Pou5f1,at least in part,due to methylation of their promoters.On the contrary,Tnf and Il1b gene expression was upregulated and their promoters were hypomethylated.
基金Supported by Top-notch Academic Programs Project of Jiangsu Higher Education Institutions(PPZY2015C230)
文摘The mutagenicity of the protein-modified Enterococcus faecalis was evaluated by a mouse bone marrow micronucleus test and a mouse sperm abnormality test.The test substance was designed with three dose groups (1,2 and 5 g/kg·bw) and intragastrically administrated,with cyclophosphamide as a positive control and normal saline as a normal control.The micronucleus rate and sperm abnormality rate were measured.The results showed that the micronucleus rates and sperm abnormality rates in the different dose groups were not significantly different from those in normal control group ( P >0.05),while the positive control group was significantly higher than the normal control group ( P <0.01).The conclusion is that the protein-modified E.faecalis was tested to be negative in both the mouse bone marrow micronucleus test and mouse sperm abnormality test,suggesting that it has no mutagenicity in vivo.
文摘The genotoxic potentiality of the crude leaf extract of Casearia tomentosa, a medicinal preparation, has been evaluated in Swiss albino mice. The extract significantly induced the division_disruptive chromosomal changes in bone marrow cells as well as in primary spermatocytes; the latter also exhibited marked increase in synaptic disruptions. A significant decrease in sperm count was noted. The incidence of structural damages in chromosomes, however, remained within the range of control level frequency. This herbal preparation, therefore, appears to be primarily spindle_poisoning in its action, but not clastogenic. The probable mechanism of this differential genotoxicity is discussed.
