| Objective:Isolation, cultivation of the mesenchymal stem cells in C57BL/c mice in vitro, the well proliferated cells were selected in this trial.Methods:Bone marrow mononuclear cells were isolated with density gradient centrifugation and adherence screening method. BMSCs were obtained by removing the non-adherent cells and observed with light microscope. Then the BMSCs were purified and expanded through passaging in time. The surface antigens of cells in the third passage were tested with flow cytometry. ALP dye and oil red O staining were used to identify the potentials in differentiating into osteoblasts and adipocytes.Results:The density of the third and following passages of mice BMSCs was idendical. Cells appeared in spindle shape, as the fibroblast cells. The growth curves were similar through the third to the twelfth passage. Growth speed in the following passages became faster than the primary cells.Flow cytometry show that cells in the third passage strongly express the stem cells surface antigen CD29(98.10%), but little haematopoietic stem cells specific antigen, such as CD45(10.50%) and CD34(15.6%). So the cells obtained in this trial were not haematopoietic stem cells.After appropriate induction, ALP and oil red O staining were both positive in the cells.Conclusions:With the methods of density gradient centrifugation and adherence screening, we can obtain high purified mice MMSCs. The isolated BMSCs express the mesenchymal stem cells specific surface antigens. Also, these cells have pluripotent differentiating ability, such as osteoblasts or adipocytes, and are suitable for cell transplantation. Objective:Packaging and transfecting TGF-?1 gene to BMSCs via lentiviral vector system.Methods:The TGF-?1 gene was recombined to a HIV-1 lentiviral vector with eGFP by infusion technique. The combined plasmid was identified by polymerase chain reaction(PCR), enzyme digestion and sequencing and transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles. The lentiviral liter was detected by real-time fluorescence quantitative PCR(RT Q-PCR) and was transducted to mouse BMSCs by optimum MOI. The expression of TGF-?1 protein and infection rate was observed with fluorescent microscope. We adopted hemiquantitative RT-PCR to analyze the expression of TGF-?1 mRNA.Results:In-Fusion technology was adopted to construct the TGF-?1 gene recombinant lentiviral vector, which was identified by the polymerase chain reaction(PCR), enzyme digestion and sequencing. The transfected 293T cells were identified to express correctly. The optimum MOI of BMSCs was 10 with the 1×108TU/ml lentiviral titer and BMSCs infected by TGF-?1 gene recombinant lentiviral vector can express TGF-?1 efficiently.Conclusions:The TGF-?1 & eGFP fusion gene recombinant lentiviral vector, which was constructed by In-Fusion technology and transfected to BMSCs, can express TGF-?1 efficiently. Objective:To investigate the effects and mechanisms of TGF-?1 gene modified BMSCs implantation on myocardial repairing in a mice model of acute myocardial infarction.Methods:AMI was induced in mices by permanent ligation of the left anterior descending coronary artery. A total of 50 mices were randomly divided into five groups:Sham group, the study animals undergone sham operation; PBS group, the animals were transplanted with PBS,n=10; BMSCs groups,the animals were transplanted with BMSCs (without gene or Lentiviral vector); LV-NullBMSCs groups,the animals were transplanted with EGFP gene modified BMSCs,n=10; TGF-?1BMSCs group:the animals were transplanted with TGF-?1 gene modified BMSCs, n=10; All the animals received exogenous transplantation of BMSCs 7 days after myocardial infarction(concentration of 1×106BMSCs/100ul,amount of 25ul, injected at multiple points); Two weeks after AMI, echocardiography were performed to evaluated the cardiac function, subsequently, the cardiac sample were obtained. Hematoxylin Eosin staining was used to observe the myocardial pathological changes after BMSCs transplantation. Middle transver section was stained with Masson'Trichrome to measure the infarct size and collagen volume with the application of Image-pro plus 5.0 analysis software. Immuno-histochemistry was applied to evaluate the positive expression of?-SMA, Vimentin, collagen?and?, TIMP-1,MMP-9,and IL-6. Also, laser scanning confocal microscope was used to observe the survival and differentiation of transplanted BMSCs.Results:Compared with Sham group, the myocardial tissue pathological changes were significantly more lighter in all MSCs implantation groups, importantly, the myocardial tissue pathological changes in the TGF-?1 BMSCs group were significantly more lighter; The survival number of transplanted cells was significantly larger in TGF-?1BMSCs group than in other groups, as evidenced by the number of DAPI-positive markers BMSCs in the TGF-?1BMSCs group was significantly more than other groups(P<0.05). There was no significant difference in infarct size between all the four MSCs implantation groups. Compared with the PBS group, the thicknes of the infact-zone was significantly thicker in these four MSCs implantation group. Of note, the increase in wall thicknes of the infact-zone in the TGF-?1BMSCs group was more significant than that in BMSCs group and LV-NullBMSCs group (P<0.01). A greater improvement in cardiac function was obtained in the TGF-?1BMSCs group than that in the BMSCs group, LV-NullBMSCs group(P<0.05). The expression of?-SMA, Vimentin, collagen 1 and collagen III,TIMP-1 were all up-regulated in the infareted area, whereas MMP-9, IL-6 levels were decreased(P<0.05). Interestingly, we found BMSCs could decrease the synthesis of collagen I and collagen III in non-infareted area. Laser scanning confocal microscope demonstrated that the transplanted BMSCs could differentiate into myofibroblast. Of note,the number of myofibroblast was significantly larger in the TGF-?1 BMSCs group than that in the BMSCs and LV-NullBMSCs group(P<0.01).Conclusion:Our findings suggest that (1) implantation of BMSCs could increase the wall thickness of the infarct area, contribute to the myocardial infarction repairing and healing, therefore, improving the cardiac function. (2) TGF-?1 gene modified BMSCs could remarkably promote the differentiation of BMSCs into myofibroblast in the infracted area; (3) TGF-?1 gene modified BMSCs could up-regulate the expression of TIMP-1, collagen I, III and attenuate the the expression of MMP-9, IL-6, accelerating the repairment and protecting myocardial expansion; Implantation of TGF-?1 gene modified BMSCs to improve the repair of infracted myocardium was feasible and efficacious;... |
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