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 review the research progress in cell therapy and tissue engineering approach to regenerate salivary gland so as to provide a theoretical basis for the treatment of salivary hypofunction. Methods The recent literature on cell therapy and tissue engineering for the regeneration of salivary glands was reviewed and summarized. Results It is feasible to repair the salivary function by using various stem cells to repair damaged tissue, or by establishing salivary gland tissueex vivo for salivary gland function restoration and reconstruction. However, the mechanism of three dimensional culturing salivary organoids during organogenesis and function expressing and the potential influence of tissue specific extracellular matrix during this process should be further studied. Conclusion Basic research of cell therapy and salivary tissue engineering should be deeply developed, and a standardized culturing system should be establishedin vitro. In addition, it is of great significance to study thein vivo effects of salivary gland-specific cells, non salivary gland epithelial cells and transplanted gene-transfected stem cells.
【Abstract】 Objective To review the progress and cl inical appl ication of cellular therapy for stress urinaryincontinence (SUI). Methods The l iterature about cellular therapy of SUI was extensively reviewed. Results Becauseof having no or poor regeneration capacity, the cl inical application of chondrocytes and myoblasts were l imited. Based on the rapid progress in stem cell biology, an increasing number of animal experiments and cl inical trials about cellular therapy of SUI have been reported with encouraging results. All these show that cellular therapy has great potential in cl inical application. Stem cells are considered as ideal seeded-cells for treatment of SUI. Conclusion Cellular therapy, especially stem cells, provides a novel approach for treatment of SUI, but the mechanism needs further study.
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.
Retinitis pigmentosa (RP) is a genetic disorder of photoreceptor cell apoptosis and retinal pigment epithelium (RPE) cell atrophy caused by gene mutation. The clinical manifestations are night blindness, peripheral visual field loss and progressive vision loss. RPE cell apoptosis plays an important role in the progression of RP, and exogenous implantation of RPE cells as an alternative therapy has shown certain efficacy in animal experiments and clinical trials. With the diversification of cell sources, the update of surgical techniques and the continuous emergence of biological materials, more possibilities and hopes are provided for cell therapy. To further promote the development of this field in the future, it is still necessary to strengthen the cooperation between medicine, bioengineering and other disciplines in the future to jointly promote the innovation and development of therapeutic methods. It is believed that RPE cell transplantation therapy will show a brighter prospect in the future
ObjectiveTo study the external biocompatibility bewteen the mouse induced pluripotent stem cells (miPSCs) and poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBHHx). MethodsAfter we recovered and subcultured miPSCs, we divided them into two groups. There was one group cultured with material of PHBHHx films outside the body. We observed the adhesive pattern of miPSCs on film by fluorescence of 4, 6-diamidino-2-phenylindole (DAPI) staining. The cell vitality was detected by cell counting kit-8 (CCK-8). The morphology of miPSCs attached on the film was visualized under scanning electron microscope (SEM). We used the traditional petri dish to culture miPSCs and detect the cell activity by CCK-8. ResultsMiPSCs can adhere and proliferate on PHBHHx films. The result of cell vitality which detected by CCK-8 showed that there was a statistical difference in OD value between culturing on PHBHHx films and traditional cultivation (0.617±0.019 vs. 0.312±0.004, P < 0.05). ConclusionThere are adhesion and proliferation on the surface of cells patch made by miPSCs co-culturing with PHBHHx film. Compared with traditional culturing in the cell culture dish, culturing in PHBHHx films have great advantages in the process of adhesion and proliferation. PHBHHx can be used as one of the scaffold for stem cells treating various disease.
ObjectiveTo review the advances in utilizing paracrine effect of stem cells in knee osteoarthritis (OA) treatment.MethodsThe researches in applying stem cells derived conditioned medium, extracellular matrix, exosomes, and microvesicles in knee OA treatment and cartilage repair were reviewed and analyzed.ResultsThe satisfying outcomes of using different products of stem cells paracrine effect in knee OA condition as well as cartilage defect is revealed in studies in vitro and in vivo. The mechanism including suppressing the intraarticular inflammation, the apoptosis of chondrocytes, and the degradation of cartilage matrix, while enhancing the synthesis of cartilage matrix, the differentiation of in-situ stem cells into chondrocytes and the migration to the affected area. The effectiveness can be further improved supplemented with the tissue engineering methods or gene modification.ConclusionCompared with the traditional stem cell therapy, applying the products from paracrine effect of stem cells in knee OA treatment is more economical and safer, presenting great potential in clinical practice.
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.
Objective To investigate the effects of chondroitinase ABC (ChABC) combined with bone marrow mesenchymal stem cells (BMSCs) in repair spinal cord injury of rats. Methods Primary BMSCs were isolated and cultured from the femur and tibia of neonatal Sprague Dawley (SD) rats. The spinal cord injury model was established in 24 adult SD male rats (weighing, 200-230 g), which were randomly divided into control group (group A), BMSCs transplantation group (group B), ChABC injection group (group C), and ChABC and BMSCs transplantation group (group D), 6 rats in each group. At 7 and 14 days after injury, Basso-Beattie-Bresnahan (BBB) score criteria was used to evaluate the hindlimb motor function; at 14 days after injury, the injured spinal cord tissue was perfused and stained by HE for further calculation of the injury area. Immunofluorescence staining were used for observing the expressions of glial fibrillary acidic protein (GFAP)/chondroitin sulfate proteoglycan (CSPG) and GFAP/growth associated protein 43 (GAP43). Results At 7 days after injury, three joints movement of the hindlimbs were recovered in all groups, and no significant difference in the BBB score was found among 4 groups (P gt; 0.05). At 14 days after injury, no load drag was observed in 3 joints of the hindlimbs in groups A, B, and C, but weight-bearing plantar or occasional dorsalis pedis weight-bearing walking was observed in group D with no plantar walking. The BBB score of group D was significantly higher than that of the other 3 groups (P lt; 0.05). HE staining showed that the cavity formed in the damage zone, and there were a large number of macrophages in the cavity and its surrounding, which was wrapped by scar tissue. The damage area of group D was significantly smaller than that of the other 3 groups (P lt; 0.05). At 14 days after injury, the GFAP/CSPG double immunofluorescence staining showed that the astroglial scar damage zone in group D was significantly reduced, and no cavity formation was found. And the fluorescence intensity in groups C and D was significantly lower than that in group B (P lt; 0.05). The GFAP/GAP43 double immunofluorescence staining showed that GAP43-positive fibers passed through the damage zone in group D and the fluorescence intensity in group D was significantly higher than those in groups B and C (P lt; 0.05). Conclusion Inhibition of astrocytes secreting CSPG by ChABC combined with BMSCs transplantation in early injury may promote the regeneration of nerve fibers, and repair spinal cord injury in rats.
Objective To explore repair role of allogeneic bone marrow mesenchymal stem cells （BM-MSCs） transplantation on treating hepatic ischemia reperfusion injury （HIRI） in rats. Methods Ten rats were executed to get BM-MSCs， then BM-MSCs were cultured in vitro and dyed by 4，6-diamidino-2-phenylindole （DAPI）. Models of 70% hepatic ischemia reperfusion injury were eatablished. Thirty two rats were randomly divided into sham operation group （Sham group）， ischemia reperfusion group （I/R group）， Vitamin C group （VC group）， and BM-MSCs group. Serum samples were analyzed for ALT and AST， and hepatic tissue were for superoxide dismutase （SOD） and malondialdehyde （MDA）. Liver sections were stain with hematoxylin and eosin （HE） for histological analysis， TUNEL staining was applied to detect hepatic apoptosis. Serum and tissues were both collected at 24 h after reperfusion. Results The isolated BM-MSCs maintained vigorous growth in vitro. Specific markers for MSCs antigens CD29 and CD44 were detected by flow cytometry， but antigens CD34 and CD45 were not be detected. Models of HIRI were stable， and BM-MSCs were detected around the periportal area by DAPI staining. Compared with I/R group， levels of ALT， AST， MDA， and AI in the VC group and BM-MSCs group decreased at 24 h after reperfusion （P＜0.05）， meanwhile SOD level increased （P＜0.05）. Compared with VC group， levels of ALT， AST， MDA， and AI in the BM-MSC group decreased at 24 h after reperfusion （P＜0.05）， meanwhile SOD level increased （P＜0.05）. Conclusion BM-MSCs could protect HIRI by alleviating oxidative stress and inhibiting cellular apoptosis.