ObjectiveTo research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. MethodsPeripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. ResultsGFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type Ι gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061±0.013 vs. 0.004±0.002, t=-7.700, P=0.031; 0.029±0.008 vs. 0.003±0.001, t=-5.741, P=0.020; 0.679±0.067 vs. 0.142±0.024, t=-12.998, P=0.000). ConclusionAd-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.
【Abstract】 Objective To review the recent progress of cell therapy in cl inical appl ications. Methods Therecent l iterature about cell therapy in cl inical appl ications was extensively reviewed. Results Based on the advances in cell biology, especially the rapid progress in stem cell biology, an increasing number of cl inical trials about cell therapy for management of various diseases, such as cardiovascular system diseases, neural system diseases, musculo-skeletal diseases, diabetes, stress urinary incontinence, and others, had been reported with encouraging results. All these showed that cell therapy had great potentials in cl inical appl ication. Conclusion Cell therapy provides a novel approach for the treatment of many human diseases. However, the mechanism remains to be fully elucidated.
Objective To investigate the effect of the serum from severe burn patients on the biology characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, so as to explore the feasibility of hUCMSCs transplantation for treating severe burn. Methods The 3rd passage of hUCMSCs were randomly divided into 3 groups: 10% fetal bovine serum group (group A), 10% normal serum group (group B), and 10% burn serum group (group C). At 24 hours, 72 hours, and 6 days after culture, the cell morphology and density were observed by inverted microscope; the cell proliferation was assessed by MTT; after 6 days of culture, the cell cycle by propidium iodide staining and flow cytometry, the apoptosis by acridine orange/ethidium bromide staining, and the cell senescence by β-galactosidase staining; the levels of tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), platelet-derived growth factor (PDGF), and insulin-like growth factor 1 (IGF-1) in serum were detected by a double-antibody sandwich ELISA kit. Results hUCMSCs were long spindle/polygon in 3 groups. The cell fusion of group C was obviously faster than that in group A and group B. The cell proliferation curves showed that the velocity and number of cell proliferation in group C were significantly higher than those in group A and group B at 2-6 days after culture (P lt; 0.05). The rates of proliferation period (S) of hUCMSCs were 9.21% ± 1.02%, 11.79% ± 1.87%, and 20.54% ± 2.03%, respectively in groups A, B, and C at 6 days, and group C was significantly higher than that of group A and group B (P lt; 0.05). The hUCMSCs showed normal morphology and structure in 3 groups, and no apoptosis cells was observed. The positive cells percentage of group C (2.6% ± 0.1%) was significantly lower than that of group A (4.8% ± 0.2%) and group B (3.8% ± 0.4%) (P lt; 0.05). The levels of TNF-α, IL-1, PDGF, and IGF-1 in group C were significantly higher than those in group B (P lt; 0.05). Conclusion The higher levels of cytokines in serum from the severe burn patients can significantly stimulate hUCMSCs proliferation, prevent cells apoptosis, and reduce cells senescence. Therefore, it is feasible to use hUCMSCs transplantation for treating severe burn patients.
Abstract: Objective To investigate the effect of autologous bone marrow mesenchymal stem cells (MSCs) transplantation on cardiac function and their proliferation and differentiation in the post-infarct myocardium in rabbits. Methods Twenty New Zealand rabbits were randomly divided into two groups, the autologous bone marrow mesenchymal stem cells group (MSCs group,n=10) and control group (n=10). Myocardial infarct model was set up by ligation of the left anterior descending (LAD), two weeks after establishment of the infarct model,either 400μl of cell suspension (total cells 1×106) labled by 1,1’-dioctadecyl3,3,3’,3’-tetramethyl indocarbocyanine perchlorate (Dil) or a comparable volume of L-DMEM medium were autologously transplanted into several different points of the periphery of the scar respectively. To evaluate the heart function, echocardiography were performed before modeling,two weeks after modeling, 2 and 4 weeks after the cells transplantation for asurements of left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD), tocalculate left ventricular eject fraction(LVEF) and left ventricular fractional shortening (LVFS). Meanwhile the myocardial contrast echocardiography (MCE) were performed for evaluating the blood perfusion of the post-infarct myocardium. Eight weeks after the transplantation, the animalswere undergoing euthanasia, specimens were acquired for pathology. Results Echocardiography indicated that:The LVEF and LVFS between two groups were fundamentally the same before modeling,two weeks after modeling respectively (0.72±0.08 vs. 0.71±0.04,0.56±0.11 vs. 0.55±0.09; 0.35±0.06 vs. 0.35±0.04, 0.24±0.08 vs. 0.23±0.03, Pgt;0.05), but those were improved significantly in group MSCs when compared with control group at two weeks and four weeks after the cells transplantation(0.71±0.05 vs. 0.60±0.05，0.72±0.07 vs. 0.62±0.08 and 0.34±0.03 vs. 0.29±0.01， 0.35±0.06 vs. 0.27±0.05 respectively,Plt;0.05). There were no differences in LVESD and LVEDD between two groups in any time points(Pgt;0.05). MCE showed the blood perfusion of the infarct myocardium were improved two and four weeks after the cell transplantation. Pathology indicated that Dil positive cells were survived in MSCs transplanted hearts, stained positively for αsarcomeric actin and desmin eight weeks after cell transplantation, HE slides indicated that the capillary density in all the cells transplanted hearts were much higher when compared with control group (38.6±7.6/mm2 vs. 21.4±3.9/mm2，Plt;0.05). ConclusionMSCs can differentiate into cardiomyocytes, improve myocardial perfusion and cardiac function when transplanted into ischemic myocardium.
Abstract: Objective To transplant the microencapsulated recombinanted Chinese hamster ovary (CHO ) cells into the infracted myocardium of rodent animals and investigate whether vascular endothelial growth factor (VEGF) secreted by the implanted CHO cells could augment angiogenesis and improve cardiac function. Methods The cDNA of VEGF was transferred into CHO cells with plasmid stable transfection. After microencapsulation, the cell growth in microcapsules and the VEGF level in the culture supernatant were evaluated. Two weeks after myocardial infarction, the microencapsulated CHO cells (MC-CHO group ) were implanted into the border of infracted myocardium, as well as similar amount of CHO cells (CHO group ) , blank microcapsule (MC group ) and non-serum culture medium (control group ) as controls, 12 rats per group. The cardiac function improvement was evaluated 3 weeks after transplantation, while the survival status of implanted CHO cells, in situ secretion of VEGF and capillary density were assayed by histology. Results CHO cells could grow and proliferate after microencapsulation. The secretion of VEGF was detectable in culture media supernatant, with the highest concentration of 3 852 pg/m l at day 8. As compared to the other three groups, the left ventricular dimension and cardiac function were significantly improved in MC-CHO group 3 weeks after transplantation. The capillary density of MC-CHO group were increased significantly than those of CHO group, MC group and control group (22. 3±3. 1 vs. 15. 6±2. 8, 11. 4±2. 5, 13. 2±2. 7 vessels per 0.13 mm2, P lt; 0.05). The implanted microcapsule maintained its original shape and protected theCHO cells in it. Conclusion M icroencapsulaed recombinanted CHO cells transplantation might be a promising app roach to augment angiogenesis and improve the cardiac function in infarction myocardium.
Objective To summarize the recent progress of cell-based approaches for promoting bone regeneration in distraction osteogenesis (DO). Methods Recent literature concerning enhancement of bone regeneration following DO using cell-based approaches was reviewed and analyzed. Results An overview of 4 different cell-based approaches was mainly provided: single cell injection, cell scaffold-based strategies/injectable tissue engineered bone, microtissue technology or cell aggregate technology, and stem cell gene therapy. Each has its advantages and disadvantages. Other methods are still in the experimental research except that compound injection of bone marrow mesechymal stem cells and platelet-rich plasma has been applied to clinical practice. Conclusion The cell-based approach is a promising strategy in the field of bone regenerative medicine. These approaches have bright future in promoting bone regeneration and reducing the treatment period in DO in the clinical application. However, well-designed preclinical studies are required to establish safe and effective guidelines for cell-based approaches to promoting bone regeneration during DO.
Objective To review and summarize the latest development of the therapy for the Duchenne muscular dystrophy (DMD). Methods Therecentlypublished articles related to the therapies for DMD were extensively reviewed and briefly summarized. Results The therapeutic approaches for DMD included the gene therapy, the cell therapy, and the pharmacological therapy. The gene therapy and the cell therapy were focused on the treatment for the cause of DMD by the delivery of the missing gene, the modification of the mutated gene, and the transfer of the normal cells including the stem cells, while the pharmacological therapy dealt with the downstream events caused by the dystrophin gene defect, slowed down the pathologic progress of DMD, and improved the DMD patient’s life quality and life span, by medication and other factor treatments. Conclusion There is still no cure for DMD because of various difficulties in replacing or repairing thedefected gene and of the multifaceted nature of the severe symptoms. Therefore,it is imperative for us to find out a more effective treatment that can solve these problems.
Objective To review the advance in the experimental studies and evaluate the potential therapeutic application of the mesenchymal stem cells(MSCs). Methods The related articles published in China and theother countries during the recent years were extensively reviewed and analyzed. Results The MSCs were widely used in the cell-transplantation therapy and the tissue engineering because of their pluripotency of differentiation into various kinds of cells. They were also frequently used in the gene therapy because they could stably express the transfected objective genes. Because of their immunomodulatory function, the MSCs could also be used in the immunotherapy. Conclusion The MSCs are the stem cells, which have characteristics of renewing themselves, having multipotency, and being easy to undergo amplification in vitro.The MSCs are ideal target cells for the cell therapy, tissue engineering, gene therapy, and immunotherapy.
Objective To evaluate the transfection efficiency and expression level of hepatocyte growth factor (HGF) by transfecting a recombinant adenovirus carrying HGF gene (Ad-HGF) into bone marrow mesenchymal stem cells (BMSCs) and to explore the effect of the expression supernatant on BMSCs in vitro so as to lay a foundation for the manufacture of gene medicine which expresses efficient cell factors. Methods Rat BMSCs were isolated using Percoll density gradient method and cultured according to the adherent property of BMSCs. The expression of c-Met was detected by immunohistochemical examination. BMSCs were infected with a recombinant adenovirus carrying green fluorescent protein gene (Ad-GFP) at multipl icity of infection (MOI, 0, 25, 50, 100, and 200 pfu/cell). To select an optimal MOI, the transfection efficiency and the degree of cell damage were assayed by flow cytometry and MTT, respectively, at 48 hours after transfecting. The expression of HGF in BMSCs transfected with optimal MOI Ad-HGF was measured with ELISA assay. MTT method was used to evaluate the prol iferation effect of HGF expression supernatant on BMSCs. Results Immunohistochemical staining showed that BMSCs expressed c-Met receptor for HGF. At 48 hours after transfecting with different MOI Ad-GFP (0, 25, 50, 100, and 200 pfu/cell), the transfection efficiencies were 0.34% ± 0.04%, 40.72% ± 0.81%, 61.72% ± 1.04%, 85.33% ± 0.83%, and 17.91% ± 0.63%, respectively; and the highest transfection efficiency was observed at 100 pfu/cell MOI. The cell damage was obviously observed when MOI was 200 pfu/cell. The expression of HGF in BMSCs reached the highest level after being transfected with 100 pfu/cell MOI Ad-HGF for 48 hours. The expression product could stimulate the prol iferation of BMSCs. The prol iferation of BMSCs gradually rose with the increase of HGF protein, and reached the highest level at 10% (320 pg). Conclusion BMSCs can be transfected efficiently with Ad-HGF and express HGF protein, which stimulates the prol iferation of BMSCs. It suggests that BMSCs is an ideal repair cells with gene vector.
ObjectiveTo review the recent research progress of skeletal myoblasts for cardiac repair. MethodsThe related literature about skeletal myoblasts for cardiac repair was reviewed, analyzed, and summarized. ResultsThe results of animal experiments and clinical studies have shown that skeletal myoblasts been transplanted into the regional myocardial infarction area in different ways can improve cardiac function. But there are some challenges such as high loss rate of skeletal myoblasts and resulting in ventricular arrhythmias. ConclusionFurther studies can improve the safety and effectiveness of skeletal myoblasts for cardiac repair in the future.