Establishment of a human chronic myeloid leukemia-like mouse model

AIM：To establish a new model of chronic myeloid leukemia (CML) for further studying the pathogenesis mechanisms and discovering new therapeutic targets of this disease. METHODS：The retroviral packaging technique was improved by increasing retroviral titer with higher-quality plasmid, better cell state and appropriate cell seeding density for further studying. Donor BALB/c mice were pretreated with 5-fluorouracil (5-FU), and their bone marrow cells were infected with p210 BCR/ABL retroviral supernatant (BCR/ABL group) or empty retroviral vector (MigR1) supernatant (MigR1 group). Infected bone marrow cells were transplanted into lethally irradiated recipient BALB/c mice via tail vein. The activities of the mice were observed after bone marrow transplantation (BMT). The morphology of peripheral blood cells and bone marrow cells, and the pathological changes of the liver and spleen in dying mice were also determined. RESULTS：Retroviral packaging efficiency was improved by optimizing the experimental conditions with higher-quality plasmid, better cell state and appropriate cell seeding density. All mice in BCR/ABL group died at 19 to 25 d after BMT. Their myelocytes, metamyelocytes and mature granulocytes in peripheral blood and bone marrow were abundant. Hepatosplenomegaly and granulocyte infiltration in the liver and spleen were also observed. All mice in MigR1 group were normal. CONCLUSION: We have successfully set up a mouse model of CML by increasing retroviral titer with improved retroviral packaging technique, transfecting BCR/ABL into mouse bone marrow cells in vitro and transplanting the cells to the recipients of the same lineage.     

Isolation and multilineage differentiation of mesenchymal stem cells from human umbilical cord vein in vitro

AIM: To investigate the isolation, purification, expansion and multilineage differentiation of mesenchymal stem cells (MSCs) derived from human umbilical cord vein in vitro.METHODS: By 1% collagenase Ⅱ digestion, endothelial cells were isolated from human umbilical cord vein and cultured in IMDM medium.The morphology of the cells was observed by Wright’s staining and electron microscope.Cell cycle and immunophenotype were investigated by flow cytometry.Assays of adipogenic and osteogenic differentiation were performed in vitro.von Kossa staining, Oil Red O staining and mRNA expression of osteopontin and lipoprotein lipase were studied in the induced cells.RESULTS: The cells from the cord vein displayed a fibroblast-like morphology adhering to the culture plate.FACS showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34, and HLA-DR were negative.Adipocyte and osteocyte differentiation were induced successfully.CONCLUSION: The morphology, growth characteristics, immunophenotype and pluripotentiality of the MSCs from human umbilical cord vein are similar to the MSCs from bone marrow (BM).They could potentially be an excellent source of MSCs for experiments and clinics.     

Influences of coculture with bone marrow mesenchymal stem cells on dopaminergic neurons in brain slice treated with 1-methyl-4-phenylpytidinium

AIM: To study the protective effects of bone marrow-derived mesenchymal stem cells on damaged dopaminergic neurons induced by 1-methyl-4-phenylpytidinium(MPP+).METHODS: The parkinson disease(PD) models were established in newborn rats. Bone marrow mesenchymal stem cells(MSCs) obtained from adult bone marrow were cultured, isolated and purified. MSCs were co-cultured with brain slice and the immunohistochemical technique, electron microscopy, propidium iodide staining were used to observe the changes of neurons. RESULTS: In the MPP+ treatment group, the neurites grew slowly and sparsely, dead cells were found in all regions. In the co-culture group, the neuritis grew densely, only a few cells were dead, the number of tyrosine hydroxylase(TH)-stained neurons increased and the structure of organellae was normal. CONCLUSION: MSCs may protect dopaminergic neurons against damage induced by MPP+. These results provide some data for cell transplantation therapy to Parkinsons disease.     

Effect of mesenchymal stem cell transplantation on papain and ［60Co］ irradiation-induced rat pulmonary emphysema

AIM: To investigate the effect of bone marrow mesenchymal stem cells(MSCs) transplantation on papain and ［⁶⁰Co］ irradiation-induced rat pulmonary emphysema and the underlying mechanisms.METHODS: Female rats were randomly divided into three groups: control group, emphysema group, emphysema+MSCs transplantation group. Rats were exposed to ［⁶⁰Co］ irradiation and instilled with papain intratracheally. Bone marrow MSCs from male rats were infused through tail veins. Morphologic analysis of the lung tissue was performed. The engraftment of male bone marrow MSCs in female recipient lung was determined by PCR of Sry gene and Y chromosome fluorescence in situ hybridization (Y-FISH). Sry gene was amplified by PCR using the genomic DNA from the lungs as template. Surfactant protein C (SP-C) immunofluorescent staining and Y-FISH were performed on the same lung section to determine whether the engrafted bone marrow MSCs differentiated into type II pneumocytes. RESULTS: Destruction of alveolar walls was observed in rat lungs from emphysema group and emphysema+MSCs transplantation group. MSCs transplantation significantly ameliorated the emphysematous changes. Significant differences in the mean linear interval (MLI), the mean alveoli number (MAN) and mean alveoli area (MAA) between emphysema group and emphysema+MSCs transplantation group were observed. DNA fragment of Sry gene was amplified in the genomic DNA from the rat lungs in emphysema+MSCs transplantation group. Y chromosome positive cells were observed in the lungs tissue from emphysema+MSCs transplantation group. Some of the Y chromosome positive cells also expressed SP-C, the marker of type II pneumocytes. CONCLUSION: Bone marrow MSCs transplantation improves papain and irradiation-induced pulmonary emphysema. The underlying mechanisms may include the engraftment of bone marrow MSCs in the lungs and differentiation of MSCs into type II pneumocytes.     

Differentiation of embryonic-like stem cells derived from human bone marrow into multinucleated fibers in vitro

AIM: To compare the capacity of in vitro differentiation into multinucleated fibers between embryonic-like stem cells (ELSCs) and mesenchymal stem cells (MSCs) derived from human bone marrow. METHODS: To isolate ELSCs, human bone marrow mononuclear cells were cultured in gelatin-coated flask with serum-free Knockout-DMEM medium designed for the expansion of human embryonic stem cells. MSCs were isolated from the same bone marrow by the traditional method. The morphological characters of both ELSCs and MSCs were observed under inverted phase-contrast microscope, and the expression of their multipotent antigen markers was identified by immunofluorescent staining. ELSCs and MSCs were cultured in myogenic differentiation medium. The protein levels of muscle-specific antigen markers myosin heavy chain (MHC), myogenin and MyoD were detected by the method of immunostaining. The mRNA expression of MHC, myogenin and MyoD was detected by RT-PCR. The capacity of in vitro differentiation into multinucleated fibers was compared between ELSCs and MSCs by calculating the proportion of MHC-positive multinucleated fibers. RESULTS: ELSCs, which weakly expressed the multipotential markers Oct-4, Nanog-3 and Sox-2, were isolated from bone marrow by the method of serum-free medium. ELSCs appeared smaller, slenderer and more homogeneous, and were morphologically different from MSCs derived from the same marrow. No multipotential marker in MSCs was expressed. ELSCs and MSCs were induced into long multinucleated fibers expressing MHC and myogenin at mRNA and protein levels by culturing in the myogenic differentiation medium. However, on the 10th day after induction, the proportion of the MHC-positive fibers in ELSCs was (25.7?4.1)%, and the proportion in MSCs was (15.8?7.6)%.The capacity for differentiation into muscle in ELSCs was significantly higher than that in MSCs (P<0.05). CONCLUSION: Bone marrow ELSCs are induced into multinucleated fibers and have the stronger myogenic differentiation capacity than MSCs derived from the same marrow. ELSCs are a more ideal candidate for muscular disease therapy.     

Effect of ovariectomy on biological characteristics of MSCs from SD rats

AIM: To investigate the effects of ovariectomy on the biological characteristics of the mesenchymal stem cells (MSCs) derived from the osteoporotic (OP) SD rats and normal rats by comparing the morphology, growth curve and cell cycle. METHODS: Female SD rats of 10-month old were randomly divided into model group and sham operation group (10 rats in each group). The osteoporotic model was established by ovariectomy. Twelve weeks after ovariectomy, the MSCs were isolated from normal and OP rats, and purified by differential time adherent method. The morphological difference of the MSCs between normal and OP rats was observed under inverted phase contrast microscope, transmission electronic microscope (TEM) and scanning electronic microscope (SEM). The growth curves of the MSCs were detected by MTT method. The cell cycles of the MSCs were determined by flow cytometry. RESULTS: Compared to the MSCs from normal rats, the morphology of the MSCs form OP rats became wider and flatter, the cell organelles were decreased and the cell viability was descended. The differentiation of the MSCs from OP rats into adipocytes appeared and the cell multiplication capacity was declined. CONCLUSION: The cell morphology of the MSCs derived from rats with ovariectomy changes obviously and the cell multiplication capacity is decreased.     

Experimental study on inducing bone mesenchymal stem cells to differentiate into cardiomyocytes in vitro

AIM: To study the differentiation of rat bone mesenchymal stem cells (MSCs) into cardiomyocytes in vitro. METHODS: MSCs were isolated and purified from the bone marrow of rats by density gradient centrifugation and adhering to the plastic culture. The third passage MSCs were treated by 5-azacytidine (5-aza). The induced cells were evaluated by immunocytochemistry staining and RT-PCR analysis. RESULTS: After being induced by 5-aza, some MSCs became bigger and longer. The connection of the cells were formed on day 14.The direction of the cells arraying was similar gradually. The induced cells were stained positively for desmin, α-actin and troponin I. RT-PCR showed that these cells expressed β myosin heavy chain. CONCLUSION: 5-aza can induce MSCs to differentiate into cardiomyocytes in vitro.     

Mechanism of telomere mainteance in human bone marrow mesenchymal stem cells

AIM： To study the telomere maintenance mechanism in mesenchymal stem calls (MSCs).〖WT5"HZ〗 METHODS：MSCs were isolated from healthy human bone marrow by their adherence to plastic and then were checked with CD14-FITC,CD45-FITC,CD44-FITC,HLA-DR-FITC,CD34-PE,CD29-PE and CD166-PE.Telomere length and ECTR DNA in MSCs were detected by Southern blotting.The localization of TRF1 and promyelocytic leukemia (PML) in MSCs were detected with immunofluorescence staining.TRAP protocol was performed to detect the telomerase activity in MSCs and MSCs-derived adipocytes.Western blotting and TRAP protocol were applied to measure telomerase activity of MSCs,which were synchronized by serum starvation and aphidicolin treatment.〖WT5"HZ〗RESULTS：The telomere in length seemed shorter and relatively more homogeneous in MSCs and HeLa cells than that in WI-38-2RA cells.TRF1 did not concide with PML nuclear body in MSCs and HeLa cells while it exclusively did in WI-38-2RA cells.ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38-2RA cells.Telomerase was negative in MSCs but it was positive in MSCs-derived adipocytes detected by TRAP.Moreover,a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and aphidicolin treatment.Untreated MSCs expressed very low level of telomerase probed by Western blotting with 2C4 mAb,but the telomerase level had significantly increased when these cells were trapped in S phase.〖WT5"HZ〗CONCLUSION：The telomere of MSCs is maintained by telomerase pathway instead of alternative lengthing of telomere(ALT) and the level of telomerase expression is associated with cell cycle stage.     

Biological characteristics and induced differentiation of bone marrow-derived mesenchymal stem cells from sensitized mice in vitro

AIM: To evaluate the biological characteristics and differentiating potentials of bone marrow-derived mesenchymal stem cells (MSCs) from sensitized mice by allogeneic splenocyte transfusion in vitro. METHODS: Adherent culture method was applied for culturing the bone marrow-derived MSCs from sensitized mice. The cell morphology was examined and the surface marker profiles were analyzed by flow cytometry. The differentiating potentials of the MSCs into osteogenic, adipogenic and myogenic lineages were explored. The bone marrow-derived MSCs from the normal mice were collected and served as controls. RESULTS: Both the bone marrow-derived MSCs from sensitized and normal mice were exhibited a homogeneous distinctive morphology and were positive for the surface markers CD29, CD105, CD44 and Sca-1, negative in CD 34 and CD11b. The abilities of both MSCs to differentiate into osteogenic, adipogenic and myogenic pathways in the same condition were also observed. CONCLUSION: There is no difference in the biological characteristics and induced differentiating potentials between the sensitized mouse bone marrow-derived MSCs by allogeneic splenocytes transfusion and the MSCs from normal mice.     

Biological characteristics of mesenchymal stem cells from different resources in green fluorescent protein transgenic mice

AIM：To compare the biological characteristics, surface markers and multi-differentiation potential of the mesenchymal stem cells (MSCs) derived from the umbilical cord and bone marrow in the green fluorescent protein (GFP) transgenic mice. METHODS：Umbilical cord MSCs (UCMSCs) were isolated by collagen type II enzymatic digestion and bone marrow MSCs (BMSCs) were isolated by density gradient centrifugation. The growth of the 2 types of MSCs was observed under inverted microscope. The cell proliferation was detected by determining the growth curve and MTT assay. The Trypan blue method was performed to analyze the cell viability rate. The cell cycle and cell surface markers were measured by flow cytometry. The differentiation potentials of the 2 types of MSCs were tested by the differentiation kits toward adipocytes and osteoblasts. RESULTS：The UCMSCs attached to the culture surface 1 d after the isolation, and the cells showed spiral shape with notable growth and proliferation after 2 d of culture. After 3 d, the cell arrived sub-confluent and was ready for passage. BMSCs still showed circular shape and started to attach to the surface 4 d after culture. They formed the small colony shape only after 5 d with obvious proliferative potential. The cells became confluent 7 d after the culture. The original generation of cultivating UCMSCs growth curve was shown typically an “S” shape. But the BMSCs growth was slower than the UCMSCs. The cell proliferation was obvious for UC-MSCs in 3~5 d. BMSCs proliferated significantly only after 7 d. The viability rate arrived more than 96% for both types of MSCs. The cell cycle of both MSCs did not show significant difference (G0/G1 phases were above 85%, P>0.05). Both MSCs positively expressed CD44, CD90 and CD105 (60.7%±2.3%) but the expression of CD45, CD19, CD14 and CD79 was negative (less than 25.6%±4.8%, P>0.05). More than 90% of the MSCs from the umbilical cord and bone marrow differentiated towards the adipocytes and osteoblasts without significant difference (P<0.05). CONCLUSION：UCMSCs have stronger ability of proliferation and multi-directional differentiation potentials. UCMSCs in GFP transgenic mice as a high-quality tracer can serve for tracking the stem cells in vivo.     

Dexamethason enhanced aorta-derived CD105+ mesenchymal stem cells to differentiate into adipocytes

AIM: To investigate whether aorta-derived CD₁₀₅⁺ cells show characteristics of mesenchymal stem cells, and if dexamethason enhances this kind of CD₁₀₅⁺ cells to differentiate into adipocytes. METHODS: The distribution of CD105 in aorta was assessed by imunohistochemistry. The aorta wall cells were isolated and immunophenotypes were identified by FACS. CD₁₀₅⁺ cells were sorted using MACS CD105 micromagnetic beads. The differentiation of CD₁₀₅⁺ cells into adipocytes and osteoblasts was induced under different conditions and indicated by staining of Oil red O, detecting of alkaline phosphatase activity, calcium accumulation stained with silver nitrate and transmission electron microscope analysis, respectively. RESULTS: The endothelial cells, a part of medial smooth muscle cells and adventital fibrblasts were CD105 positive. The isolated aortic arch cells were positive for CD105, CD106, CD44, CD29, and negative for CD45, CD11a, CD11b and HLADR. The CD₁₀₅⁺ cells differentiated into adipocytes contained Oil-Red-O-positive lipid droplets, the osteocytes with calcium deposition and alkaline phosphatase activity. Ultrastructurally, it was observed that some needle-shaped crystal calcium deposition similar to bone spicules was inside the cytoplasm of induced osteocytes. When the dexamethason was absent in the adipogenic medium, there were no adipocytes with lipid droplets. CONCLUSION: The results demonstrated that CD₁₀₅⁺ cells showed characters of MSCs reside in aortic wall, and was able to differentiate into adipocytes and osteocytes in vitro. Dexamethason enhanced aorta-derived CD₁₀₅⁺ with characters of MSCs to differentiate into adipocytes. These suggested that MSCs might be related with atherosclerosis.     

Study on Cx43 communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells in vitro

AIM: To approach the gap junction distribution and communication function of cardiomyocyte-like cells derived from rat bone marrow mesenchymal stem cells (MSCs) in vitro.METHODS: MSCs were isolated by extraction of bone marrow specimens,gradient centrifugation and the adherence of culture plates.MSCs were culture in vitro,treated with 5-azacytidine and incubated for 24 h.The induced MSCs,which had been incubated for 2,3 and 4 weeks,were divided into group Ⅰ,Ⅱ and Ⅲ.In addition,the normal cardiomyocyte cells were used as control group.The distribution of connexin 43(Cx43) and the mean fluorescence redistribution rate were detected in every group with the laser scanning confocal microscope.RESULTS: Cx43 protein grain density in induced MSCs was increased with the lasting of incubation by quantitive analysis of Cx43 distribution.After 4 weeks,the Cx43 protein density in induced MSCs was nonsignificant deviation with control group (63.87±12.43,64.87±12.15,P>0.05).The diversify tendency of the mean fluorescence redistribution rate was approximation with the result of Cx43 in every group.The results showed that groupⅠ was 19.59%±6.08%,groupⅡ was 37.17%±3.84%,groupⅢ was 46.82%±2.69%,and control group was 49.71%±5.53%.CONCLUSION: MSCs can be differentiated to cardiomyocyte-like cells,which have been induced and incubated for 4 weeks in vitro.The communicational function of those MSCs is similar to the normal cardiomyocyte cells.     

Inhibitory effects of Ginkgolide A on stress ulceration in rats

AIM: Studying the relation between telomerase activity and chronic myelocytic leukemia(CML) courses by detect telomerase activity(TMA) of mononuclear cells of bone marrow samples in different CML phases. METHOD: Using telomerase PCR-ELISA method. RESULTS: Telomerase activity of normal bone marrow is 9.85±0.68, CML chronic phase is 24.48±12.73 which is significantly higher than that of normal bone marrow(P＜0.05). CML accelerated phase TMA is 76.76±21.84 and blast crisis is 90.62±25.41, both are significantly higher than that of chronic phase(P＜0.001, P＜0.001)but there is no significant difference between accelerated phase and blast crisis(P＞0.05), at remisson TMA is 18.12±6.27 which is still higher than normal group(P＜0.05).CONCLUTIONS: Telomerase activity in different phases of CML is significantly higher than normal bone marrow group. When telomerase is extremely activated , it means that CML patients were entering the accelerated phase. Telomerase activity might be used as an useful new marker in detecting and curing CML.     

Relation between telomerase activity and chronic myelocytic leukemia course

AIM: Studying the relation between telomerase activity and chronic myelocytic leukemia(CML) courses by detect telomerase activity(TMA) of mononuclear cells of bone marrow samples in different CML phases. METHOD: Using telomerase PCR-ELISA method. RESULTS: Telomerase activity of normal bone marrow is 9.85±0.68, CML chronic phase is 24.48±12.73 which is significantly higher than that of normal bone marrow(P＜0.05). CML accelerated phase TMA is 76.76±21.84 and blast crisis is 90.62±25.41, both are significantly higher than that of chronic phase(P＜0.001, P＜0.001)but there is no significant difference between accelerated phase and blast crisis(P＞0.05), at remisson TMA is 18.12±6.27 which is still higher than normal group(P＜0.05).CONCLUTIONS: Telomerase activity in different phases of CML is significantly higher than normal bone marrow group. When telomerase is extremely activated , it means that CML patients were entering the accelerated phase. Telomerase activity might be used as an useful new marker in detecting and curing CML.     

Generation of insulin-producing cells from PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells

AIM: To differentiate bone marrow mesenchymal stem cells (BMSCs) into functional insulin-producing cells to produce sufficient pancreatic islet cells for transplantation. METHODS: Recombinant adenovirus vectors carrying PDX1 and NKX6.1 genes were constructed and the bone marrow mesenchymal stem cells were infected by the recombinant adenovirus combined with several cytokines for differentiation. The expressions of PDX1, NKX6.1 and insulin and C-peptide in the differentiated bone marrow mesenchymal stem cells were detected by RT-PCR and Western blotting. After the differentiated bone marrow mesenchymal stem cells were transplanted into subrenal capsule of diabetic mice, cell morphology of the grafts as well as their secretion of insulin and C-peptide were detected. Besides, regulating capacities of grafts on the blood glucose level of the diabetic mice were also detected. RESULTS: BMSCs induced by recombinant adenovirus (pAdxsi-CMV-PDX1/CMV-NKX6.1) and several cytokines showed positive dithizone staining and the expressions of β-cell related molecule such as insulin and glucose transporter 2 were detected by RT-PCR, which showed a sustaining and stable expression. The similar results were showed by Western blotting, immunohistochemical staining and indirect immunofluorescence. The insulin secretions in the cells stimulated with glucose at concentrations of 5.5 and 25 mmol/L in the experimental group were (1 240.4±109.3) mU/L and (3 539.8±245.1) mU/L, respectively, and were significantly higher than those in control group. Moreover, transplantation of the cells to STZ mice in treatment group made serum glucose recover to normal level. CONCLUSION: PDX1 and NKX6.1 gene-modified bone marrow mesenchymal stem cells differentiate into insulin-producing cells in vitro. When these cells transplanted into STZ induced diabetic mice, their serum glucose could return to the normal level and they could live well. Thus this is a promising method for diabetes treatment.     

Microparticles derived from bone marrow mesenchymal stem cells promote angiogenesis in rat myocardial infarction model

AIM: To observe the effects of microparticles derived from bone marrow mesenchymal stem cells (MSC-MPs) on angiogenesis and cardiac function in a rat myocardial infarction model. METHODS: MSCs were obtained from Sprague-Dawley rats. MSCs were treated under serum-free condition in hypoxia for 72 h, and the microparticles were isolated from the supernatants. The phenotypic profile of MSC-MPs was determined by bead-based flow cytometry and the morphology was observed under a transmission electron microscope. The rat myocardial infarction model was established. The cardiac function was evaluated by echocardiography after the intramyocardial injection of MSC-MPs. The myocardial infarct size was observed by Masson staining. The blood vessel density in the peri-infarcted area was measured using immunohistochemical staining for von Willebrand factor and α-smooth muscle actin. The expression of vascular endothelial growth factor (VEGF) was analyzed by real-time PCR. RESULTS: Apoptotic MSCs released a large quantity of microparticles which were phenotypically similar to the parent MSCs and 100~1 000 nm in diameter. The cardiac functions of myocardial infarction rat model were improved at 7 d and 28 d after intramyocardial injection of MSC-MPs compared with control group. The myocardial infarct size was reduced and angiogenesis was promoted significantly in the infarcted heart injected with MSC-MPs 28 d after treatment. MSC-MPs treatment also increased the expression level of VEGF within 7 d.CONCLUSION: MSC-MPs protect cardiac tissue from ischemic injury and improve cardiac function by promoting angiogenesis after myocardial infarction.     

The correlation between differentiation and apoptosis in the process of differentiation from adult rat mesenchymal stem cells into neuron-like cells

AIM:To supply the theoretic evidences of elongating the lifetime of neuron-like cells differentiated from adult rat mesenchymal stem cells, we investigated the relationship between the differentiation and apoptosis in the process of induction. METHODS: The mesenchymal stem cells(MSCs) were isolated primarily from rat bone marrow, and purified by passage culture. The 5th passage of MSCs was induced by β-mercaptoethanol and all-trans-retinoic acid (ATRA). After 1 h, 3 h and 5 h of induction, the cells were stained immunocytochemically with anti-MAP-2 and anti-GFAP antibodies, respectively. In addition to counting the ratio of neuron-like cells in MSCs, DAPI staining was employed to identify whether the differentiated cells have an apoptotic morphological changes. The ratio of apoptotic cells at 1 h, 3 h and 5 h after induction were detected by flow cytometry (FCM). CONCLUSIONS:1. β-mercaptoethanol and ATRA had the different ability that induced MSCs to differentiate to neuron-like cells. 2. Apoptosis was also initiated in the process of differentiation, and there is positive correlation between the ratio of differentiation and apoptosis.     

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.