Abstract:Aim To observe the effect of Klotho gene modified bone marrow mesenchymal stem cells (BMSC) transplantation on myocardial fibrosis in rats with heart failure. Methods Isolation, culture and amplification of rat bone marrow mesenchymal stem cells. Klotho transfection to BMSC and RT-PCR detection of Klotho mRNA expression in BMSC. The rat model of chronic heart failure were randomly divided into normal group, saline group, EGFP-BMSC group and EGFP-Klotho-BMSC group, with 6 rats in each group. The cardiac function was detected by Doppler ultrasound after 28 days of cell transplantation. The survival and distribution of the cells were observed by fluorescence microscope. The pathological changes of myocardial tissue were observed by HE staining. Masson staining was used to detect the content of myocardial collagen deposition and the degree of myocardial fibrosis. Results Klotho combined with BMSC treatment can better inhibit myocardial fibrosis, reduce myocardial interstitial collagen deposition and improve cardiac function. Conclusion Klotho gene combined with bone marrow mesenchymal stem cells could attenuate cardiomyocytes hypertrophy and restrain collagens hyperplasy in the progression of myocardial fibrosis.

Abstract:Aim To investigate the effect of bone marrow mesenchymal stem cells（BMSC）transplantation on injured vessels endothelium repair and vascular restenosis after carotid balloon injury in rabbits. Methods 48 rabbits of carotid artery stenosis model were established and randomly divided into BMSC transplantation group （n24） and control group （n24）. BMSC were cultured in vitro and transfected with recombinant adenovirus-mediated enhanced green fluorescent protein gene. Balloon injured carotid artery of rabbits, meanwhile, BMSC（107/kg）were infused into rabbits injured artery by external carotid artery in BMSC transplantation group, and the same amount of PBS solution were infused into the control group. 1 week after transplantation, BMSC homing were detected by immunohistochemical techniques. 2 weeks after transplantation, the expression of platelet-endothelial cell adhesion molecule（CD31）, α-smooth muscle actin and proliferating cell nuclear antigen（PCNA）were detected by immunohistochemical staining. 4 weeks s after transplantation, the incidences of the vessels stenosis were detected by carotid artery arteriography, the neointimal area and the ratio of the intima/media area were examined by hematoxylin and eosin staining. Results BMSC with GFP homing were detected in injured vessels intima at 1 week after BMSC transplantation. CD31 continues to have expression in intima of BMSC transplantation group, while the control group did not express. The expression of PCNA in BMSC transplantation group were decreased significantly compared with control group（23.43%±2.80% vs 50.49%±3.60%，P<0.05）. The expression of SM α-actin in BMSC transplantation group were elevated significantly compared with control group（0.437± 0.049 vs 0.197±0.032， P<0.01）. The neointimal area（0.103±0.022 vs 0.214±0.024，P<0.01）and the ratio of the intima/media area（0.771±0.096 vs 1.646±0.223，P<0.01）were significantly decreased in BMSC transplantation group than control group at 4 week. The incidences of the vessels stenosis were decreased compared with control group（39.64%±2.30% vs 63.31%±2.82%，P<0.05）. Conclusion BMSC transplantation can promote repairing of endothelial and phenotypic transforming of vascular smooth muscle cells after carotid balloon injury in rabbits and inhibits neointima hyperplasia. BMSC transplantation can also reduce the restenosis of injured vessels.

Abstract:Aim　To　investigate　the　regulating　effects　of　P38MAPK　signaling　pathway　on　the　differentiation　of　the　bone　marrow-derived　stem　cells（BMSC）　induced　by　BMP-2　toward　cardiomyocyte-like　cells　and　to　determine　the　possible　mechanisms. Methods　BMSC　were　separated　and　cultured　in　vitro.　The　third　passage　cells　were　divided　into　3　groups:　control　group,　BMP-2　inducer　group,BMP-2+P38MAPK　blocker　SB203580　group.　The　morphological　observation　was　performed　by　the　inverted　phase　contrast　microscope,　the　immunohistochemical　technique　was　used　for　detecting　the　expression　of　cardiac　specific　troponin（cTnT）　and　connexin　43（Cx43）,Western　blot　was　used　for　examining　of　the　changes　in　p-P38MAPK/P38MAPK　levels　in　BMSC　after　induced　with　the　BMP-2. Results　Expression　of　p-P38MAPK　of　BMSC　could　be　observed　at　15　min　after　induction　with　BMP-2,　increased　obviously　at　30　min,increased　increasingly　from　60　min.　Compared　with　inducer　group,the　expression　of　p-P38MAPK　of　blocker　group　decreased　obviously（P<0.05）.　The　expression　of　cardiac　specific　troponin（cTnT）　and　Cx43　were　negative　in　control　group,　and　positive　in　inducer　group　and　blocker　group.　Compared　with　inducer　group,　Cx43　positive　cells　increased　in　blocker　group（P<0.05）. Conclusion　BMP-2　could　induce　differentiation　of　BMSC　toward　cardiomyocyte-like　cells,and　the　P38MAPK　sinaling　pathway　plays　a　negative　regulating　role　during　induction.

Abstract:Aim To investigate whether bone marrow mesenchymal stem cells （MSC） reduce the apoptosis of human umbilical vein endothelial cells （HUVEC） induced by oxidized low density lipoprotein （ox-LDL）,and related mechanisms. Methods The HUVEC were divided into three groups: HUVEC were cultured in normol medium HUVEC were cultured with 100 mg/L ox-LDL for 24 h and HUVEC were cocultured with MSC with 100 mg/L ox-LDL for 24 h. Then cell apoptosis of different groups were mesured by flow cytometry. The content of VEGF,as well as TNF-α,in supernatant medium were determined by ELISA,and Real-time PCR was used to detect Bcl-2 and Bax mRNA expression.Results After 24 h stimulated by ox-LDL,the HUVEC apoptosis rate was increased,and the content of VEGF,TNF-α were significantly increased,while Bcl-2 mRNA expression was downregulated and Bax mRNA upregulated. The MSC cocultured with HUVEC could increase the VEGF levels,reduce TNF-α levels,as well as increase Bcl-2 mRNA expression but reduce the expression of Bax mRNA,HUVEC apoptosis was significantly decreased. Conclusions MSC can attenuate HUVEC apoptosis induced by ox-LDL,possibly through the increase of VEGF,reduction of TNF-α,and upregulation of Bcl-2 mRNA and downregulation of Bax mRNA,which provides a theoretical basis for MSC as a treatment for endothelial injury disease such as atherosclerosis.

Abstract:Aim To study the effect of transfection of adenovirus-mediated integrin-linked kinase(ILK) on rat bone marrow mesenchymal stem cell(BMSC) paracrine action.Methods BMSC were isolated and cultured in vitro by adherent culture.The recombinant adenoviral vector was constructed containing both human wild-type ILK cDNA and humanized recombinant green fluorescent protein(hrGFP).Corresponding virus with null content(adeno-null) was used as a control.BMSC were infected by adeno-ILK(MSC-ILK group) or adeno-null(MSC group) in serum-free medium.The infection efficiency of adeno-ILK was tested by GFP expression using flow cytometry,thus the best multiplicity of infection(MOI) was identified.The mRNA was extracted from BMSC and the gene expression of vascular endothelial growth factor(VEGF),fibroblast growth factor-2(FGF-2),and insulin-like growth factor-1(IGF-1) was examined by Real-time PCR.Results The gene expression of VEGF,FGF-2,and IGF-1 in the MSC-ILK group was 8.2±0.4,2.6±0.2 and 2.4±0.2 times(P<0.05 or P<0.01) higher than that in the MSC group respectively.Conclusion BMSC transfected by ILK gene could express higher VEGF,FGF-2,and IGF-1.

Abstract:Aim To investigate the effect and possible mechanism of bone marrow mesenchymal stem cells(BMSC)transplation on reparing injured vessels endothelium after carotid atherosclerosis stenosis angioplasty in rabbits.Methods Carotid atherosclemsis stenosis model of 48 rabbits had been successfully built up and randomly divided into BMSC transplantation group(n=24) and control group(n=24).BMSC were obtained by density gradient centrifugation and adherent cultured.Surface markers of BMSC were detected by flow cytometry,and BMSC pre-labled by DAPI.Balloon injured carotid artery of rabbits,meanwhile,BMSC(107/kg)were infused into injured artery of BMSC transplantation group rabbits by external carotid artery,and control group infused the same amount of PBS solution.The peripheral blood was collected and the vascular endothelial growth factor(VEGF) levels was detected by enzyme linked immunosorbent assay(ELISA) at preoperative and 3,7,14,28 days of BMSC transplantation.7 days after BMSC transplantation,DAPI labeled BMSC were detected by immunofluorescence microscopy.14 days after BMSC transplantation,the immunohistochemical staining was used to analyse platelet-endothelial cell adhesion molecule(CD31)expression in the injured vessels.28 days after BMSC transplantation,the neointimal area,the ratio of the intima/media area and vascular restenosis were analysed in the vascular tissue by hematoxylin and eosin staining.Results The expression of VEGF in BMSC transplantation group were elevated significantly compared with control group at 3,7,14,28 days after BMSC transplantation.7 days after BMSC transplantation,DAPI labeled BMSC were detected in injured vessels intima.14 days after BMSC transplantation,CD31 continues to express in intima of BMSC transplantation group,while the control group did not express CD31.The neointimal area(0.092 ± 0.009 vs 0.189 ± 0.007,P<0.01),the ratio of the intima/media area(0.698 ± 1.570 vs 1.630 ± 0.122,P<0.01) and the luminal stenosis ratio(41.70% ± 3.70% vs 61.28% ± 1.57%,P<0.01) were significantly decreased in BMSC transplantation group compared with control group at 28 days.Conclusion BMSC transplantation can promote repairing of endothelial after atherosclerotic stenosis carotid artery by balloon injury and reduce the restenosis of injured vessels.

Abstract:Aim To investigate the effect of 5-azacytidine on the differentiation of rat bone marrow mesenchymal stem cells(BMSC) into cardiomyocyte-like cells,and explore the role of fluid shear stress. Methods BMSC were isolated from rats marrow mononuclear cells by attaching growth.The 4 th generation of BMSC were exposed to 3,5,10,15 and 20 μmol/L 5-azacytidine for 12,24 and 48 hours respectively.10 μmol/L for 24 hours as best induction concentration was established based on α-actin expression of cardiomyocyte-like cells after 3 weeks by immunofluorescence.The modles of BMSC were divided into four groups: no fluid shear stress group,load 5 dyn/cm2 fluid shear stress group,load 15 dyn/cm2 fluid shear stress group and load 25 dyn/cm2 fluid shear stress group.After 24 hours,the morphology changes of BMSC was observed by inverted microscopy.RT-PCR was used to examine cTnI mRNA expression after 4 weeks later. Results BMSC gradually became spindle with the passage.5-Azacytidine induced BMSC increases cell bodies and extended long thin processes,branching occurs at the protruding end,some of the processes connecting adjacent cells to form a network,showed morphological transformation to the direction of the characteristics of myocardial cells.α-actin was expressed by inducation after 3 weeks.Through fluid shear stress mechanical stimulation,cTnI expression increased,positive bands was more significant than no fluid shear stress stimulation cells.It was the most obvious in 15 dyn/cm2 fluid shear stress group.However,the change of 25 dyn/cm2 fluid shear stress group was not increased at a positive correlation. Conclusions 5-Azacytidine can induce the differentiation of BMSC into cardiomyocyte-like cells.5-Azacytidine combined with shear stress not only induce the differentiation of BMSC into cardiomyocyte-like cells,but also cause cell differentiation,which seems to have a better effect than only 5-azacytidine.