Study on the biological characteristics of rat bone marrow mesenchymal stem cells

AIM: To investigate the basic biological characteristics of adult rat bone marrow mesenchymal stem cells(rBMMSCs), and compare to that of human BMMSCs (hBMMSCs). METHODS: rBMMSC and hBMMSCs were separated from bone marrow with the difference of adherence and Ficoll-Paque reagent, and expanded in culture medium in vitro, respectively. The proliferation and growth characteristics of the primary and different passage culture of rBMMSCs and hBMMSCs were analysed. The neural differentiation capacity of rBMMSCs with passages were observed. To detect the surface antigens of rBMMSCs, the labeled cells were analysed on a FACScan flow cytometer. The karyotype of rBMMSCs were detected by blocking cellular fission with colchicines. RESULTS: rBMMSCs and hBMMSCs have a strong self-renewal capacity. Approximately (4-8)×10¹² and (3-4)×10¹² cells were obtained after passage 15 in vitro, respectively. The ability of proliferation, CFU-Fs, and neural differentiation of rBMMSCs and hBMMSCs were decreased gradually with passages, but the ability of proliferation and CFU-Fs of rBMMSCs were higher than that of hBMMSCs at different passage. FACScan result showed rBMMSCs were uniformly positive for CD29 and CD44, and negative for CD11b, CD45, CD61, CD71, CD80, CD86,MHCⅠ and MHCⅡ. rBMMSCs had an normal karyotype, which had an average of 37.0±4.0 to 40.5±2.5 chromosomes. CONCLUSION: Adult rBMMSCs have strong self-renewal and neural differentiation capacity, and have an normal karyotype. So rBMMSCs can be used as the seed cells for tissue engineering.     

Wnt signaling pathway is involved in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells

AIM：To investigate the changes of Wnt signaling pathway in catalpol-induced proliferation of rat bone marrow mesenchymal stem cells (BMSCs). METHODS：The BMSCs were isolated from SD rats, purified by differential time adherent method and divided into control group and catalpol (1.0 mg/L) group. Flow cytometry was used to detect the proliferation index of BMSCs. The mRNA levels of Wnt3a, Wnt5a, Wnt11 and β-catenin was evaluated by real-time PCR. In addition, the protein expression level of β-catenin was determined by Western blotting. RESULTS：Prolife-ration index was increased from 8.90%±0.46% to 17.93%±1.68% after treatment with catalpol (P<0.01). Compared with control group, the mRNA expression of Wnt5a, Wnt11 and β-catenin was all increased with catalpol treatment. No difference of Wnt3a mRNA expression between control group and catalpol group was observed. Meanwhile, the protein expression of β-catenin was increased in catalpol group compared with control group. CONCLUSION：Catalpol promotes BMSCs going into the cell cycle. Classical and non-classical Wnt signaling pathways are activated in this process.     

Age-related changes of bone marrow mesenchymal stem cells in senile osteoporosis

AIM：To investigate the different functions of bone marrow mesenchymal stem cells (BMMSCs) in age-related osteoporosis. METHODS：The senescence-accelerated mice (SAMP6) were used in the experiment. The BMMSCs were isolated from femora and tibiae by flushing. Flow cytometric analysis was performed with MSCs-related monoclonal antibodies. The expression of differentiation genes was tested by real-time RT-PCR. RESULTS：In the progress of age-related osteoporosis, BMMSCs exhibited a decrease in osteogenesis and an increase in adipogenesis. Transforming growth factor β(TGF-β) signaling was significantly changed along with aging in SAMP6 mice. CONCLUSION：The functional changes of BMMSCs may play an important role in senile osteoporosis. The alteration of TGF-β-related gene expression may be the molecular mechanism of dysfunction in BMMSCs.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

miR-193 regulates proliferation of rat marrow mesenchymal stem cells by cell cycle-related proteins

AIM: To investigate the effects of microRNA-193 (miR-193) on the proliferation and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS: Cultured rat MSCs were transfected with pre-miR-193 or anti-miR-193 to regulate the expression of miR-193. The proliferation of the MSCs after transfection was evaluated by MTS assay, colorimetric BrdU cell proliferation assay and Ki-67 immunostaining. Cell apoptosis was analyzed by flow cytometry with Annexin V/PI staining. The effect of miR-193 on the expression of cell cycle-related proteins was evaluated by qRT-PCR. RESULTS: Transfection of pre-miR-193 or anti-miR-193 regulated the expression of miR-193 in MSCs effectively. Over-expression of miR-193 significantly promoted the proliferation of MSCs (P<0.05), and inhibition of miR-193 reduced the proliferation of MSCs (P<0.05). miR-193 had no significant effect on the apoptosis of MSCs (P>0.05). The result of qRT-PCR indicated miR-193 promoted the expression of cyclin-dependent kinase 2 (CDK2) significantly (P<0.01). CONCLUSION: miR-193 promotes the proliferation of MSCs possibly through the CDK2 pathway.     

Meglumine cyclic adenylate induces differentiation of bone marrow mesenchymal stem cells into cardiomyocytes in vitro

AIM: To study the effect of meglumine cyclic adenylate (MCA) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into cardiomyocytes in vitro. METHODS: The whole bone marrow adherent culture method was used to isolate, culture and amplify the BMSCs. The surface markers of BMSCs were determined by flow cytometry analysis. MCA at concentrations of 10^-2 mol/L, 10^-3 mol/L, 10^-4 mol/L, 10^-5 mol/L, 10^-6 mol/L and 10^-7 mol/L was added to the culture medium containing the second generation of BMSCs.5-Azacytidine(5-Aza) was used as a positive control. The cell viability was measured by MTT method.The cAMP content in BMSCs was detected by ELISA. The mRNA expression of GATA-4, Cx43 and β-MHC in MCA group and MCA+H89 (a PKA inhibitor) group was measured by SYBR-RT-PCR. The differentiation effects of MCA and 5-Aza were compared by flow cytometry. RESULTS: Most of the BMSCs expressed CD44 and CD71, and did not express CD45. MCA inhibited the viability of BMSCs in a time-and dose-dependent manner, and MCA atthe concentration of 10^-2 mol/L showed particularly remarkable effect. MCA significantly increased intracellular cAMP level in BMSCs in a concentration-dependent manner. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group were significantly higher than that in blank group (P<0.05), and the highest effect was under the condition of MCA induction at the concentration of 10^-3 mol/L for 3 days. The mRNA expression of GATA-4, β-MHC and Cx43 in MCA group was higher than that in 5-Aza group and H89+MCA group (both P<0.05). Differentiation rate in MCA group was slightly higher than that in 5-Aza group (20.24%±1.02% vs 18.39%±0.58%, P<0.05). CONCLUSION: MCA stimulates BMSCs to increase intracellular cAMP production and inhibits the viability of BMSCs, thus promoting the mRNA expression of GATA-4, β-MHC and Cx43 through the cAMP/PKA signaling pathway.     

Trichostatin A promotes mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells

AIM：To investigate whether trichostatin A (TSA), a new revulsant,can induce mouse mesenchymal stem cells to differentiate into insulin-secreting cells and to explore the appropriate concentration of TSA. METHODS：The mesenchymal stem cell line from C57BL/6 mice was cultured in vitro and divided into 5 groups before treated with different concentrations of TSA, (group A: DMSO; group B~E: treated with 25 nmol/L, 50 nmol/L, 100 nmol/L and 200 nmol/L of TSA, respectively). After exposed to different cultured media for 10 d during the 2 stages, the cells were detected by the following methods: the insulin-secreting cells in each group were identified by dithizone staining and the results were calculated with immunohistochemical half quantitative analysis. The insulin secreted by insulin-secreting cells in each group was identified by immunofluorescence, and the mean fluorescence intensity of insulin was compared. The content of insulin in each group was quantified by ELISA. The appropriate concentration of TSA was determined according to the above results. RESULTS：TSA treatment for 10 d promoted the mouse bone marrow mesenchymal stem cells to differentiate into insulin-secreting cells which produced insulin. The immunohistochemistry and immunofluorescence imaging analysis of insulin-secreting cells showed that the insulin staining positive area, positive ratio, total density of insulin expression and mean fluorescence intensity of insulin in group B were significantly higher than those in the other TSA-treated groups. When the concentrations of TSA gradually increased, the content of insulin reduced accordingly. The content of insulin in group B was significantly higher than that in the other TSA-treated groups. CONCLUSION：TSA treatment for 10 d promotes bone marrow mesenchymal stem cells from C57BL/6 mice to differentiate into insulin-secreting cells and the appropriate concentration of TSA is 25 nmol/L.     

Effects of naringin on MAPK signal pathway in rat bone marrow mesenchymal stem cells during osteogenic differentiation

AIM: To study the effects of Chinese herbal monomer naringin (NG) on the MAPK signal pathway in bone marrow mesenchymal stem cells (MSCs) derived from SD rats during the differentiation into osteoblasts in vitro . METHODS: The changes of evaluating indicators alkaline phosphatase (ALP), bone gla protein (BGP) and type I collagen (Col I) in MSCs were observed under the conditions of normal, adding p38 pathway inhibitor SB203580, adding extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059, adding c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125, and adding SB203580, PD98059 and SP600125 together. The protein phosphorylation of p38, ERK1/2 and JNK was measured by Western blotting. The mRNA expression levels of transforming growth factor beta 1 (TGF-β₁), bone morphogenetic protein 2 (BMP-2) and core binding factor α1 (Cbfα1) were measured by fluorescence quantitative PCR. RESULTS: The most effective concentration of NG to promote the differentiation of MSCs into osteoblasts was 10^-7 mol/L. The highest expression levels of both ALP and BGP were observed in NG group (P<0.05), while the expression of Col I did not reveal significant difference (P>0.05). Compared with NG group, the expression levels of ALP, BGP and Col I decreased differently after adding different inhibitors. Compared with control group, the protein phosphorylation of JNK was increased (P<0.05), and the phosphorylation of p38 was decreased (P<0.05), while the phosphorylation of ERK1/2 did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the protein phosphorylation of p38, ERK1/2 and JNK showed fluctuation with some increasing and others decreasing. Compared with control group, the expression of BMP-2 was increased (P<0.05), and the expression of Cbfα1 was decreased(P<0.05), while the expression of TGF-β₁ did not reveal significant difference (P>0.05) in NG group. Compared with NG group, the mRNA expression levels of TGF-β₁, BMP-2 and Cbfα1 decreased differently after adding different inhibitors. CONCLUSION: Activation of ERK/JNK signaling and up-regulation of BMP-2 expression may be the main mechanism of NG to promote the differentiation of MSCs into osteoblasts. NG has strong impact on p38 pathway to improve the expression of BMP-2 in MSCs.     

Lentivirus-mediated calcitonin gene-related peptide transfection enhances endothelial differentiation of rat bone marrow mesenchymal stem cells

AIM：To study the effect of calcitonin gene-related peptide (CGRP) gene transfection mediated by lentivirus on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to endothelial cells. METHODS：Rat bone marrow MSCs were isolated by density gradient centrifugation combined with adherence method. Recombinant lentivirus vector carrying CGRP gene (Lenti-CGRP) was transfected into the MSCs. The secretion of CGRP in culture supernatants of the transfected MSCs was detected using ELISA method. The cells at passage 3 were divided into three groups: CGRP group (MSCs transfected with Lenti-CGRP), CGRP+CGRP8-37 (an antagonist of CGRP receptor) group and control group (MSCs transfected with PBS). The differentiation of the MSCs was detected by immunocytochemical staining for CD31 and factor Ⅷ-related antigen. The proliferation of the cells was measured by cell counting, and the angiogenic ability of the cells was analyzed using Matrigel assay. RESULTS：The proportion of CD31-and factor Ⅷ-related antigen-positive cells in CGRP and CGRP+CGRP8-37 groups was larger than that in control group (P<0.05). The numbers of the cells in CGRP and CGRP+CGRP8-37 groups were significantly increased compared with control group (P<0.05). Lumen-like structures were observed in CGRP and CGRP+CGRP8-37 groups. The above indexes in CGRP+CGRP8-37 group were reduced compared with CGRP group. CONCLUSION: Transfection with CGRP gene induces rat bone marrow MSCs to differentiate into endothelial cells and enhances their proliferation, suggesting that CGRP may play a role in the regulation of angiogenesis.     

Notch signaling in bone marrow mesenchymal stem cells induced by lipopolysaccharide

AIM: To investigate the roles of Notch signaling in lipopolysaccharide (LPS)-induced proliferation and secretion of interleukin-6 (IL-6) and chemokine CXCL1 in bone marrow mesenchymal stem cells (BMSCs).METHODS: BMSCs were isolated by whole bone marrow culture. The expression levels of Notch signaling pathway receptors and ligands in the BMSCs treated with LPS were measured by qPCR and Western blot. The proliferation of BMSCs was analyzed by MTT assay and viable cell counting. The secretion levels of IL-6 and CXCL1 induced by LPS were measured by ELISA.RESULTS: Treatment with LPS at 1 mg/L effectively induced the proliferation of BMSCs and the secretion of IL-6. Obvious expression of Notch receptors and ligands in the BMSCs was observed, and LPS had little effect on the mRNA and protein levels of Notch receptors and ligands, but LPS increased the protein levels of Hes1 and Hey1, the target genes of Notch signaling. LPS at 1 mg/L increased the proliferation of BMSCs, whereas DAPT (Notch signal inhibitor) reduced the basal and LPS-induced proliferation of BMSCs (P<0.01). LPS treatment robustly increased the secretion of IL-6 and CXCL1 as assessed by ELISA. However, inhibition of Notch signaling almost completely abolished LPS-induced secretion of IL-6 and CXCL1 (P<0.05).CONCLUSION: Inhibition of Notch signaling reduced not only the proliferation of BMSCs but also IL-6 and CXCL1 secretion induced by LPS.     

Mechanisms of catalpol-induced osteogenic differentiation in SD rat bone marrow mesenchymal stem cells

AIM: To investigate the mechanisms for catalpol-induced osteogenic differentiation of SD rat bone marrow mesenchymal stem cells (BMSCs) in vitro. METHODS: The cells were divided into control group, osteoinduction group and catalpol group. The activity of alkaline phosphatase (ALP) was measured at 7 d, 14 d and 21 d after catapol treatment, meanwhile ALP positive cell numbers and calcium nodes were counted at 14 d and 21 d after catapol treatment,respectively. The mRNA expression of Runx2, osteocalcin, Wnt3a, β-catenin, Wnt5a and Wnt11 was detected at 7 d, 14 d and 21 d after catapol treatment by real-time PCR. RESULTS: Catalpol at 2.0 mg/L increased ALP activity and ALP positive cell numbers significantly(P<0.05), meanwhile, it also increased calcium nodes numbers in cultured BMSCs (P<0.05). Compared with control group, catalpol increased the mRNA expression of Runx2 significantly at 14 d, but not at the 7 d and 21 d. Catapol also promoted the mRNA expression of osteocalcin significantly from 7 d to 21 d. Compared with control group, the mRNA expression of Wnt3a and β-catenin in catalpol group was increased at 14 d and 21 d. In addition, the mRNA expression of Wnt5a and Wnt11 in catalpol group was higher than that in control group at 14 d, but that was decreased at 21 d. CONCLUSION: Catalpol induces differentiation of BMSCs into osteoblast by increasing the mRNA expression of Runx2, and promotes the differentiation and mature of these osteoblasts by increasing ALP secretion, osteocalcin mRNA expression and calcium deposition. The activation of Wnt signaling pathway may be involved in this pro-osteogenic differentiation process.     

Effect of bone marrow mesenchymal stem cells on engraftment of hematopoietic stem/progenitor cells in sensitized mice

AIM: To evaluate the effects of bone marrow-derived mesenchymal stem cells (MSCs) on engraftment of hematopoietic stem/progenitor cells in sensitized mice. METHODS: Mouse bone marrow-derived MSCs were cultured by adherent culture method. MSCs combined with or without hematopoietic stem/progenitor cells were implanted into the sensitized mouse model, which was established by allogeneic splenocyte transfusion, and were divided into 6 groups: MSC intervention groups, including sensitized mice with MSCs on day 11, sensitized mice with MSCs on day 0 and sensitized-mice with MSCs both on day 11 and day 0; control groups, including sensitized mice without MSC intervention, non-sensitized mice without MSC intervention and non-sensitized mice without MSCs or transplantation of hematopoietic stem/progenitor cells. The survivors were assessed after transplantation and hematopoietic recovery was monitored weekly including hematological change, immune function reconstruction, bone marrow cell recovery, chimera analysis and graft-versus-host disease development. RESULTS: Compared with different control groups, MSC intervention did not prolong the survival rates of the sensitized model mice after lethal irradiation. CONCLUSION: Under the experimental conditions, MSC combined with C57BL/6 bone marrow hematopoietic stem/progenitor cells fail to promote the growth of engraftment in C57BL/6 allogeneic splenocyte-sensitized BALB/c mice in vivo.     

High expression of PDX-1 in bone marrow mesenchymal stem cells mediated by superfect

AIM: To construct a eukaryotic expression vector containing pancreatic duodenal homebox-1 (PDX-1) and to elevate the expression efficiency of exogenous gene in rat bone marrow mesenchymal stem cells (MSCs). METHODS: Recombinant vector containing PDX-1 was constructed. Flow cytometry was used to identify the cell cycle of bone marrow mesenchymal stem cells (MSCs) cultured in vitro. Recombinant vector containing PDX-1 was transfected into bone marrow MSCs using superfect in medium. After being selected by G418, RT-PCR and Western blotting were used to investigate the expression of PDX-1 in MSCs. RESULTS: Restricted enzyme analysis and sequencing showed that PDX-1 gene segment was consistent with that in GenBank. Flow cytometry showed that there were about 85.9% cells at the cell cycle of G0/G1. The whole cells transfected emitted green fluorescence under flow cytometry. The efficiency of transfection was above 40%. RT-PCR and Western blotting demonstrated that there was expression of PDX-1 in transfected bone marrow MSCs. CONCLUSION: Recombinant vector containing PDX-1 was constructed successfully. Superfect mediated expression of exogenous gene in bone marrow MSCs in a high efficiency, and bone marrow MSCs containing exogenous gene are an ideal cells for gene therapy.     

The preliminary study on differentiation of mesenchymal stem cells into corneal epithelium like cells

AIM: To observe the differentiation and development of human mesenchymal stem cells (MSCs) transplanted onto corneal stroma of rabbits and investigate the feasibility of MSCs differentiated into corneal epithelium like cells.METHODS: 24 New Zealand albino rabbits were randomly divided into 2 groups: The human MSCs combined with amniotic membrane were transplanted onto the experimental animals, and the controls were transplanted with the amniotic membrane only. The MSCs were cultured on preserved human amniotic membrane for 4 days and labeled with 5’-Bromo-2’-deoxyuridine (BrdU), then the cells were transplanted onto the surface of the corneal stroma of the rabbits. The eyeballs were taken off after 1, 2, 3, 4, 6 and 8 weeks. The growth and differentiation of human MSCs were observed by histopathological and immunohistochemical examination.RESULTS: When the MSCs cultured on amniotic membrane were transplanted onto the surface of the corneal stroma of rabbits, the corneal epithelium were positive in CK3/CK12 staining and negative in CK 13 staining, revealed by immunohistochemical examination. The BrdU positive cells in the reconstructive corneal epithelium were found and showed positive in CK3/CK12 staining. CONCLUSION: After transplanted onto the corneal stroma of rabbits with human amniotic membrane, the MSCs survive, proliferate and differentiate into corneal epithelium like cells.     

Strontium ranelate promotes differentiation of bone marrow mesenchymal stem cells to osteoblasts by increasing expression of bone morphogenetic protein 2

AIM: To explore whether strontium ranelate (Sr) promotes differentiation of rat bone marrow mesenchymal stem cells (BMSCs) to osteoblasts by increasing the expression of bone morphogenetic protein 2 (BMP-2). METHODS: Rat BMSCs were isolated, purified and cultured, then were induced to differentiate into osteoblasts. The cells were treated with different concentrations of Sr or noggin (an inhibitor of BMP-2) according to the experimental purposes. The activity of alkaline phosphatase (ALP) was measured by colorimetry. Mineralized nodules were measured by alizarin red staining. The expression of BMP-2 was detected by Western blotting. RESULTS: Treatment with Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d obviously increased the activity of ALP,and Sr at concentration of 3 mmol/L produced the maximum effect. Exposure of the cells to Sr at concentration of 3 mmol/L for 21 d significantly increased mineralized nodules. Exposure of the cells to Sr at concentrations of 0.1 mmol/L to 7 mmol/L for 7 d markedly increased the expression of BMP-2. Preconditioning with noggin at concentration of 100 μg/L for 2 h not only inhibited Sr-induced expression of BMP-2, but also antagonized the increase in the activity of ALP and mineralization induced by Sr in BMSCs. CONCLUSION: Up-regulation of the expression of BMP-2 may be one of the mechanisms by which Sr promotes differentiation of rat BMSCs to osteoblasts.     

Lentivirus mediated CCN1 gene on growth and migration of rat bone marrow mesenchymal stem cells

AIM：To investigate the role of cysteine-rich 61 (Cyr61/CNN1) in proliferation and migration of bone marrow mesenchymal stem cells (BMSCs). METHODS：The lentiviral vector carrying CCN1 (Lenti-GFP-CCN1) was constructed and then transfected into the rat BMSCs. The cells were divided into non-transfection group, transfection group (transfected with Lenti-GFP-CCN1) and negative control group (Lenti-GFP). The fluorescence intensity of the transfected BMSCs was observed under inverted fluorescence microscope. The effects of CCN1 on the proliferation and migration of BMSCs were detected by MTT assay and scratch wound healing assay. RESULTS：The proliferation of BMSCs transfected with Lenti-GFP CCN1 had no significant difference compared with negative control group and control group. The width/thickness ratio of migrated BMSCs in wound healing was significantly higher in Lenti-GFP-CCN1 group than that in negative control group and control group (P<0.05). CONCLUSION：Exogenous CCN1 promotes the migration of BMSCs.     

Proliferation and differentiation of bone marrow mesenchymal stem cells in process of bone loss in ovariectomized rats

AIM: To study the function of proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) for bone loss in the pathogenesis of osteoporosis (OP) in ovariectomized rats. METHODS: Animal model of OP was established by ovariectomy (OVX,bilateral ovarian resection) in 10-week-old healthy female Sprague-Dawley (SD) rats.BMSCs were isolated, cultured and purified by the combination of density gradient centrifugation, adhesion separation and limited dilution method, and cultured in vitro to the 3rd~4th passage in all experiments. The BMSCs phenotype appraisal was studied by flow cytometry. Colony-forming assay was applied to detect the BMSCs proliferation ability. The MTT method was used to analyze the growth curves of BMSCs. After adipogenic induction (ADI), lipid drops were observed by oil red O staining to compare the adipogenic potential between the 2 kinds of BMSCs. After osteogenic induction (OSI), calcium nodules were observed by alizarin red staining (ARS). The mRNA expression levels of BMSCs osteogenesis-related proteins, for instance, Runx2, osteocalcin (OCN) and osteopontin (OPN) were measured by RT-PCR. RESULTS: Compared with sham group, the colony-forming ability of BMSCs in OVX group became decreased, the proliferation capacity was declined, the osteogenic potential was decreased, and the adipogenic potential was increased(P<0.05). CONCLUSION: In ovariectomized OP rats, the proliferation and osteogenesis of BMSCs decrease, and the adipogenesis of BMSCs increases, which may cause rapid bone loss and play an important role in the pathogenesis of OP.     

Effects of azathioprine on proliferation,cell cycles and apoptosis of me-senchymal stem cells from rat bone marrow in vitro

AIM: To investigate the effects of azathioprine (AZA) on the proliferation, cell cycle and apoptosis of mesenchymal stem cells (MSCs) from the bone marrow of Sprague-Dawley rats in vitro. METHODS: MSCs were cultured in low-glucose DMEM containing 10% FBS,and treated with AZA at concentrations of 50 mg/L, 100 mg/L, 200 mg/L and 300 mg/L for 24 h, 48 h and 72 h. The effects of AZA on the growth curve and proliferation of MSCs were tested by cell counter under microscope. The apoptosis and cell cycle was determined by flow cytometry. RESULTS: Pure MSCs were gained by 3 times of passages. No significant effect of AZA at concentration of less than 100 mg/L on the proliferation, cell cycle and apoptosis of MSCs was observed (P>0.05). Under the condition of more than 200 mg/L for 72 h, AZA inhibited the growth of MSCs.The inhibitory rate was more than 66%, and the rate of apoptosis was increased (P<0.05). However, at the concentration of 300 mg/L for 72 h, AZA decreased the apoptotic rate and the necrotic rate of MSCs was obviously increased (P<0.05). Using AZA at concentration of more than 200 mg/L, as the action time prolonged, the MSCs in G₀/G₁ phase were increased, and those in S phase were decreased (P<0.05). CONCLUSION: At some concentrations, AZA significantly affects the proliferation, apoptosis and cell cycle of MSCs. Large dose of AZA may cause MSCs to death.