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.     

Effects of rat mesenchymal stem cells modified by CGRP on proliferation and phenotype transformation of vascular smooth muscle cells in vitro

AIM：To explore the effects of rat mesenchymal stem cells (MSCs) modified by calcitonin gene-related peptide (CGRP) on the proliferation and phenotype transformation of rat vascular smooth muscle cells (VSMCs) in vitro. METHODS：Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein (EGFP) and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin (α-SMA) and osteopontin (OPN) were assessed by Western blotting. RESULTS：Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased (P<0.01). The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups (P<0.05). Furthermore, cell viability was >90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups (P<005). CONCLUSION：CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy 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.     

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.     

Biological characteristics of bone marrow mesenchymal stem cells from GFP transgenic mice transfected with human β-nerve growth factor gene

AIM:To investigate the changes of biological characteristics of GFP transgenic mouse bone marrow mesenchymal stem cells (MSCs), which were transfected with human β-nerve growth factor (β-NGF) gene in a recombinant eukaryotic expression vector. METHODS:MSCs obtained from GFP transgenic mice were isolated, cultured and purified by the whole bone marrow adherence methods. Human β-NGF gene was transfected into the MSCs by a recombinant eukaryotic expression vector. The β-NGF expression in the MSCs was detected by the method of immunocytochemistry. Hippocampal neurons from neonatal mice were cultured with culture supernatant of the MSCs transfected with pcDNA3-β-NGF and the biological characteristics of the MSCs were investigated 3 d after culture under inverted phase-contrast microscope. RESULTS:The β-NGF positive rate of MSCs in pcDNA3-β-NGF transfection group ［(37.12±2.14)%］ was significantly higher than that in MSCs control group ［(2.36±0.62)%］ and blank control group ［(1.43±0.76)%］.The neurite length of neonatal mouse hippocampal neurons cultured with culture supernatant from pcDNA3-β-NGF-transfected MSCs ［(31±3)μm］ was significantly longer than that in negative control group ［(23±4)μm］, suggesting that MSCs transfected with β-NGF gene maintained better biological characteristics. CONCLUSION: The constructed recombinant eukaryotic expression vector of human β-NGF gene can be transfected into MSCs efficiently and NGF can be effectively expressed in MSCs. MSCs transfected with β-NGF gene are capable of stable expression and secretion of β-NGF, and maintenance of better biological characteristics.     

Transfusion of mesenchymal stem cells improve the hematopoiesis in mice with aplastic anemia by regulating immune cells

AIM: To study the possible immunologic mechanism of mesenchymal stem cells (MSCs) by which the hematopoiesis of mice with aplastic anemia (AA) is improved. METHODS: The mice model of immuno-mediated aplastic anemia was established. Thirty BALB/c mice were divided into 3 groups: radiation group, AA model group and MSCs group. The mice in radiation group only had processing of 5 Gy ［60Co］γ radiation. The mice in AA model group were intravenously injected with the cell suspension of thymocyte and lymph-node cell mixture (prepared from DBA/2 mice, 1×106 cells) after 5 Gy ［60Co］γ radiation. The animals in MSCs group received the same treatment as the mice in AA model group did, and afterwards was injected with 1×106 MSCs intravenously at 3 days. All the mice in the 3 groups were observed and analyzed the following parameters: peripheral blood cells, pathological features of bone marrow, the relation between the changes of peripheral CD4+, CD8+, CD4+/CD8+, NK cells and the hematopoiesis. RESULTS: After 5 Gy ［60Co］γ radiation, the peripheral blood cells in all groups decreased at 7 days, while at 21 days those in MSCs group and radiation group restored to normal but those in AA model group was still at lower level. At 7 days, the cells of CD4+, CD8+ and CD4+/CD8+ in each group were similar without statistical difference, but the number of NK cells in MSCs group was quite lower than that in the other 2 groups (P<0.05). At 14 days, the CD4+ cells in all groups were obviously decreased, while the CD8+ cells in MSCs group and AA model group were significantly higher than those in radiation group. At 21 days, the CD8+ and NK cells in MSCs group were quite the same as those in radiation group while those in AA model group were obviously higher compared to the other 2 groups. The number of adipose cells observed in the pathological slice of femoral bone in MSCs group and radiation group was no difference while that in AA model group was much higher. CONCLUSION: MSCs transfusion improves the hematopoiesis in mice with aplastic anemia by regulating the functions of immune cells.     

Transfection of calcitonin gene-related peptide mediated by lentivirus vector in vitro and its effects on cardiac stem cell viability

AIM: To investigate the effect of calcitonin gene-related peptide (CGRP)transfection into c-kit^pos cardiac stem cells (c-kit⁺CSCs) on the cell viability. METHODS: Under the sterile condition, the auricles of SD rats were taken out,and then c-kit⁺CSCs were collected through enzyme digestion and immunomagnetic bead separation (MACS). The cells were identified by flow cytometry. c-kit⁺CSCs were transfected with enhanced green fluorescent protein CGRP lentiviral vector (Lv-EGFP-CGRP) or enhanced green fluorescent protein lentiviral vector (Lv-EGFP). The cells were randomly divided into Lv-EGFP-CGRP-CSCs group, Lv-EGFP-CSCs group and CSCs group. The transfection was observed under the fluorescence microscope. The transfection efficiency was detected by flow cytometry. The CGRP protein secretion in the cell culture supernatants was detected by ELISA. The viability of c-kit⁺CSCs transfected with Lv-EGFP-CGRP or Lv-EGFP was measured by CCK-8 assay. RESULTS: c-kit⁺CSCs were isolated and cultured successfully. The expression positive rate of c-kit was 91.0% and the expression positive rates of CD45 and CD34 were 4.5% and 4.0%, respectively. After transfected with lentivirus for 48 h, the stable fluorescence in c-kit⁺CSCs was observed under fluorescence microscope. The transfection efficiency were 80% when MOI was 20. The level of CGRP was significantly increased in Lv-ECFP-CGRP-CSCs group compared with Lv-EGFP-CSCs group and CSCs group (P<0.05). Meanwhile, transfection with lentiviral vector in each group did not affect the viability of c-kit⁺CSCs. CONCLUSION: Transfection of Lv-EGFP-CGRP into c-kit⁺CSCs was successful. The secretion of CGRP was found in the transfected c-kit⁺CSCs and the viability was not changed after transfection. CGRP-modified c-kit⁺CSCs may play a role in treating myocardial infarction.     

Role of Oct3/4 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of Oct3/4 in inducing differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons in vitro. METHODS: Lentivirus (LV) vector containing Oct3/4 gene was constructed and transfected into rat bone marrow MSCs. The MSCs were divided into non-transfection group, transfection group (transfected with Oct3/4 -LV) and negative control group (transfected with FU-PCG-NC-LV). β-mercaptoethanol (β-ME) was used to induce differentiation of MSCs into neurons. Morphological changes and the fluorescence in transfected MSCs were observed under inverted fluorescence microscope. The expression of Oct3/4 and microtubulin-associated protein 2(MAP-2) at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of Oct3/4 and the neural cell specific markers neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP) were determined by immunocytochemical method. The viability of the MSCs was analyzed by MTT assay. RESULTS: The results of PCR confirmed that the Oct3/4 -LV was successfully constructed and the virus titer was 2×10¹¹ TU/L. The best transfection efficiency and survival rate appeared when multiply of infection(MOI) was 10 and at 48 h, and the fluorescence of MSCs was mostly displayed. The efficiency of transfection was up to 83.4%±2.2%. The shape of the MSCs was changed in transfection group, and the survival rate of the MSCs in transfection group was significant lower than that in other groups (P<0.05). MSCs were induced by β-ME to differentiate into neurons and the best efficiency of induction was observed in transfection group. The typical neuronal morphology was observed in transfection group after induction and the expression levels of NSE and MAP-2 were higher than those in other groups (P<0.05). Compared with other groups, the expression of Oct3/4 in transfection group was significantly increased (P<0.01). Furthermore, the expression of Oct3/4 was time-dependently decreased and there was significant difference between before induction and 5 h after induction (P<0.05). CONCLUSION: Oct3/4 may have an important role in regulating the differentiation of rat MSCs into neurons.     

Effect of lentiviral vector of microRNA-9-1 on differentiation of mouse bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of microRNA-9 in inducing bone marrow mesenchymal stem cell(MSCs) differentiation into neurons.METHODS: The lentiviral vector of microRNA-9-1(microRNA-9-1-LV) was constructed and transfected into mouse MSCs. The cells were divided into non-transfected group, transfected group(transfected with microRNA-9-1-LV) and negative control group(transfected with FU-RNAi-NC-LV). MSCs were treated with β-mercaptoethanol(β-ME) as an inducer for triggering the cells to differentiate into neurons. The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope. The mRNA expression of microtublin-associated protein 2(MAP-2) was detected by RT-PCR. The expression of neuron-specific markers,neuron-specific enolase(NSE), MAP-2 and glial fibrillary acidic protein(GFAP), were measured by immunocytochemical method. The viability of MSCs was determined by MTT method. RESULTS: The results of PCR confirmed successful construction of mouse microRNA-9-1-LV. The virus titer was 1×10¹² TU/L(TU, transduction unit). The best transfection efficiency(up to 91.3%±4.2%) and survival rate appeared when multiply of infection(MOI)was 20 and on 4th day. β-ME induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group. The expression levels of NSE and MAP-2 in transfected cells were higher than those in the cells of other group(P<0.05).CONCLUSION: MicroRNA-9-1-LV has high transfection efficiency in mouse MSCs. Higher differentiation rate from mouse MSCs to neurons is induced by β-ME after the cells are transfected with microRNA-9-1-LV.     

Role of caveolin-1 in differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the role of caveolin-1 in bone marrow mesenchymal stem cells (MSCs) differentiation into neurons. METHODS: The MSCs used in the experiment were divided into non-transfected group, transfected group (transfected with Rn-caveolin-1 siRNA), positive control group (transfected with Rn-MAPK-1 control siRNA) and negative control group (transfected with negative control siRNA). The MSCs were induced by β-ME to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The mRNA expression of caveolin-1 and MAPK-1 was detected by RT-PCR. NSE, NF-M and GFAP, the neural cell specific markers, were detected by immunocytochemistry staining. The survival ratio of MSCs was detected by MTT method. RESULTS: The fluorescence of MSCs was mostly displayed at 72th h after transfection and the efficiency of transfection was up to 81.5%±2.8%. Meanwhile, the mRNA expression of caveolin-1 in transfected MSCs was decreased (P<0.05). No significant difference of the survival MSCs ratio between transfected group and other groups was observed by MTT method. β-ME induced MSCs into neural cells. The differentiation efficiency of MSCs transfected with Rn-caveolin-1 siRNA was the highest and the expression of NSE and NF-M was increased significantly compared to the other groups (P<0.01). The expression of caveolin-1 was increased persistently with time and the highest expression was observed 6 d after induction. Furthermore, there was significant difference between before induction and 6 d after induction. CONCLUSION: Lipid raft labeled with careolin as marker protein has important role in regulating MSCs differentiation into neurons.     

Ectopic hTERT gene expression in human bone marrow mesenchymal stem cells

AIM: To investigate the effect of transfection of hTERT gene into human mesenchymal stem cells (MSCs) on their telomerase activity and life-span.METHODS: Human MSCs were transfected with a pEGFP-hTERT plasmid by liposome-mediated transfection. Then the hTERT mRNA expression in MSCs was detected by RT-PCR. The activity of telomerase in transfected MSCs was detected by PCR and ELISA. The telomerase-positive MSCs was cultured in vitro and induced into neuron-like cells with EGF and bFGF. Neuron-specific markers (NF-M, MAP₂) were detected by RT-PCR.RESULTS: hTERT fragment was identified in the hTERT-transfeced cells but not in the untransfected human bone marrow MSCs. The untransfected human MSCs remained telomerase-negative but the hTERT-transfected cells showed robust telomerase activity. The telomerase-negative MSCs entered a nondividing state and senesced after about 20 to 25 passages. In test group, however, telomerase-positive MSCs to date had undergone 35 passages. RT-PCR analysis showed that telomerase-positive MSCs expressed neuron-specific markers, such as NF-M or MAP₂ after induced with EGF and bFGF in vitro. CONCLUSION: Ectopic expression of the hTERT gene in human MSCs reconstitutes telomerase activity. The transfection of hTERT gene into human MSCs extends their replicative life span and maintains their multipotent differentiation capacity.     

Effects of rhTGF-β1 on proliferation and osteogenic differentiation of MSCs in SD rats

AIM：To investigate the effects of recombinant human transforming growth factor β1 (rhTGF-β1) on the ability of proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), as well as its effects on the expression of bone morphogenetic protein 2 (BMP-2), Smad4 and core binding factor α1 (Cbfa1). METHODS：SD rat MSCs were isolated and purified by the differential time adherent method. MTT assay was used to confirm the optimal concentration of rhTGF-β1 for the proliferation of MSCs. The optimal concentration for differentiation of MSCs into osteoblast was also determined by observing the activity and positive staining of alkaline phosphatase. According to the different induction conditions, MSCs were divided into 4 groups:control group, classic group, rhTGF-β1 group, and rhTGF-β1+classic group. Alkaline phosphatase, type I collagen, bone Gla protein and calcium nodes were detected to evaluate the osteogenic differentiation. BMP-2 was detected by ELISA and the mRNA expression of Smad4 and Cbfa1 was analyzed by real-time quantitative PCR. RESULTS：The optimal concentrations of rhTGF-β1 for the proliferation of MSCs and for the osteogenic differentiation of MSCs were 10 and 5 μg/L, respectively. The MSCs in classical group and rhTGF-β1 group were promoted to osteogenic differentiation, and the mRNA expression of BMP-2, Smad4 and Cbfa1 was increased. rhTGF-β1 induced osteogenic differentiation of MSCs in the early and middle terms. However, in rhTGF-β1+classic group, the osteogenic differentiation of MSCs was more obvious in the late term. CONCLUSION:The induction conditions of classical group, rhTGF-β1 group and rhTGF-β1+ classical group promote the differentiation of MSCs by increasing BMP-2 secretion and starting the TGF-β superfamily/Smads signaling pathway to regulate the differentiation of MSCs.     

Effect of Zfp521 expression on differentiation of rat bone marrow mesenchymal stem cells into neurons

AIM: To investigate the effect of zinc finger protein 521 (Zfp521) on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neurons. METHODS: Rat MSCs were cultured by conventional method in vitro and divided into non-transfection group, transfection group (transfected with Rn-Zfp521-siRNA) and negative control group (transfected with negative control siRNA). MSCs were induced by β-mercaptoethanol (β-ME) to differentiate into neurons. The fluorescence expressed by transfected MSCs was observed under inverted fluorescence microscope. The expression of Zfp521 was detected after transfection by RT-PCR. Immunohistochemistry, RT-PCR and Western blotting were used to detect the expression levels of neuron-specific enolase (NSE),microtubule-associated protein 2(MAP-2) and Zfp521 after induction. RESULTS: The fluorescence of MSCs was mostly displayed 72 h after transfection and the efficiency of transfection was up to 84.1%±2.3%. Meanwhile, the mRNA expression of Zfp521 was decreased (P<0.05). MSCs were induced by β-ME to differentiate into neurons. The differentiation efficiency of MSCs transfected with Rn-Zfp521-siRNA was the highest and the expression of NSE and MAP-2 was significantly increased compared with other groups (P<0.05). Zfp521 was detected in all groups, and the expression level of Zfp521 was significantly decreased after induction (P<0.01). CONCLUSION: Zfp521 may be down-regulated during the differentiation. The inhibition of Zfp521 promotes the neural differentiation of MSCs. Zfp521 may play an important role in regulating MSCs differentiation into neurons.     

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells

AIM: To investigate the effects of calcitonin gene-related peptide (CGRP) on myocardin expression and phenotypic switch in vascular smooth muscle cells (VSMCs). METHODS: VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ (AngⅡ), AngⅡ + CGRP or AngⅡ + CGRP + CGRP_8-37. The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot. RESULTS: The expression of myocardin in cultured VSMCs showed downregulation along with time expansion. The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05). However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05). Furthermore, at 48 h in cultured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group. After treatment with CGRP, the levels of myocardin and α-SMA become higher (P<0.05) but OPN was lower (P<0.05) in CGRP group than those in AngⅡ group. CGRP_8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP_8-37 group compared with CGRP group. CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein, which may be accomplished by the combination of CGRP and its receptor.     

Differentiation of bone marrow mesenchymal stem cells in dilated cardiomyopathy

AIM: To investigate the feasibility of differentiation of bone marrow mesenchymal stem cells (MSCs) into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy (DCM). METHODS: 20 rabbits were randomly divided into two groups. MSCs were isolated from bone marrow and cultured. Adriamycin was applied to the two groups to create rabbit DCM models. At 3 weeks after the creation of DCM models, the experiment group animals received intramyocardial injection of autologous MSCs. 4 weeks after transplantation, the implanted sites were examined to identify the labelled cells and to investigate its differentiation through immunofluorescence. RESULTS: At 4 weeks after the MSCs transplantation, the implanted cells were found in the experiment group and some differentiated into cardiomyocytes and vascular endothelial cells, which was not founded in the control group. CONCLUSION: Autologous bone marrow mesenchymal stem cells can differentiate into cardiomyocytes and vascular endothelial cells in dilated cardiomyopathy.     

Effect of livin-modified BM-MSCs transplantation on cardiac function following acute myocardial infarction in a rat model

AIM: To study the effect of livin gene-modified bone marrow mesenchymal stem cells(BM-MSCs) transplantation on the cardiac function following acute myocardial infarction in a rat model and the expression of livin, caspase-3, caspase-7 and caspase-9 in the livin gene-modified BM-MSCs. METHODS: The MSCs were obtained by the whole bone marrow culture method, and the apoptosis of the MSCs after infection with adenovirus vector carrying enhanced green fluorescent protein(EGFP) gene and livin recombinant vector(rAd-livin) were detected by flow cytometry. The expression of livin, caspase-3, caspase-7 and caspase-9 was detected by Western blot. After permanent left anterior descending artery occlusion, the rats were randomized to receive intramyocardial injection of DMEM without cells(vehicle group), or containing MSCs(MSCs group), MSCs(EGFP)(rAd-control/MSCs group) or MSCs(livin)(rAd-livin/MSCs group). Left ventricular systolic pressure(LVSP), left ventricular end-diastolic pressure(LVEDP), the maximum increased rate of left ventricular pressure(-dp/dt_max) and the maximum decline rate of left ventricular pressure(+dp/dt_max) were recorded for evaluating the cardiac functions. RESULTS: The apoptosis of rAd-livin/MSCs was significantly decreased as compared with MSCs and rAd-control/MSCs(P<0.05). Meanwhile, the expression of caspase-3, caspase-7 and caspase-9 was significantly downregulated as compared with the other 2 groups(P<0.05). The cardiac function in rAd-livin/MSCs group was significantly improved as compared with DMEM group, and those in the other 2 groups got the similar results, but the function in rAd-livin/MSCs group was better improved. Meanwhile, the number of surviving cells in rAd-livin/MSCs group was significantly improved as compared with the other 2 groups. CONCLUSION: The apoptosis of MSCs is decreased after rAd-livin transfection, and the expression of caspase-3, caspase-7 and caspase-9 is also significantly downregulated while the expression of livin is significantly upregulated. Transplantation of livin-modified BM-MSCs by lentiviral vector results in better prognosis for treating myocardial infarction by enhancing cell survival.     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Biological characteristics of chronic myelogenous leukemia bone marrow derived mesenchymal stem cells

AIM: To study the biology characteristics of mesenthymal stem cells (MSCs) derived from chronic myelogenous leukemia(CML) and normal adult bone marrow.METHODS: Mononuclear cells from chronic myelogenous leukemia (n=19) and normal adult (n=8) bone marrow were obtained, cultured in expanded medium with low serum concentration.Cell morphology, cell cycle, immunophenotype and in vitro differentiation capacity were investigated.The differentiations of osteocytes and adipocytes were detected by von Kossa staining and Oil-red O staining.The chimeric oncogene BCR/ABL and Ph chromosome, two hall marks of CML, were detected in CML derived MSCs, normal adult MSCs, CML derived hematopoietic cells and K562 cells.RESULTS: CML and normal adult derived MSCs showed similar characteristics in cell morphology, phenotype and growth pattern.A typital fibrablast like morphology was observed.Under suitable conditions, CML and normal adult MSCs had the similar ability to differentiate into adipocytes and osteoblasts in vitro.Moreover, CML and normal adult MSCs did not express BCR/ABL gene products and Ph chromosome was not observed.CONCLUSIONS: We isolated and cultured a population of cells with characteristics of multipotent stem cells from CML bone marrow.There were similar biologic characteristics and differentiation ability between normal adult and CML bone marrow-derived MSCs.     

Adrenomedullin gene transfection enhances the therapeutic effects of transplantation of bone marrow mesenchymal stem cells on cardiac function and ventricle remodeling in acute myocardial infarction rats

AIM: To investigate adrenomedullin gene transfection enhances the therapeutic effects of homogeneous transplantation of bone marrow mesenchymal stem cells (MSCs) on cardiac function and ventricle remodeling in acute myocardial infarction rats. METHODS: MSCs were isolated and expanded using the preplating method. The infection efficiency of adenovirus vector to MSCs was tested by X-gal staining. Ad-ADM expression in MSCs and its secretion in culture medium were measured by ELISA. The left anterior descending branch of rats was ligated to establish a myocardial infarction model. The MSCs were labeled by DAPI, and were directly implanted into the acute infarct site via focal injection. Four weeks later, cardiac function was evaluated using physiological recorder. Hearts were harvested and sliced to be analyzed by immunohistochemistry (factor Ⅷ and ADM) and the DAPI-labeled cells were identified. Sirius red staining was used to identify interstitial collagen on slides. Analysis of collagen type I and III was performed using a polarized filter on sections stained for collagen with Sirius red, and the ratio of collagen type I and III were detected. RESULTS: With X-gal staining, MSCs were effectively transfected by adenovirus in vitro. The transfection efficiency showed the dose-effect relationship with multiplicities of infection (MOI). When MOI was 150, the infection efficiency was 95.4%. The expression of ADM was traced in culture medium and expressed in the time-dependent manner. A maximum production of ADM was observed at 7 d after infection ［(26.53±1.42) ng/L vs (1.34±0.08) ng/L, P<0.05］, and ADM secretion reduced to normal level at 15 d ［(2.20±1.44) ng/L vs (1.52±0.33) ng/L, P>0.05］. DAPI-labeled MSCs transplantation was found in the hearts of the recipients. Immunohistochemical studies demonstrated that intense immunostaining for ADM was higher in Ad-ADM plus MSCs group, compared to other groups. Compared with control, MSCs transplantation significantly increased capillary density in infarct area (P<0.01). A combination of Ad-ADM trensfection and MSCs transplantation demonstrated a further increase in capillary density compared with Ad-ADM or MSCs alone. MSCs transplantation decreased the ratio of collagen type I and III, obviously improved the left ventricular functions. Furthermore the combination treatment resulted in further decrease in the ratio of collagen type I and III, and significantly improved the left ventricular functions. CONCLUSION: Ad-ADM transfection enhances the angiogenic potency of MSCs transplantation and decreases the ratio of collagen type I and III through increasing ADM expression in infarct area, thus contributes to reverse the ventricular remodeling and improves the cardiac function.