Objective To observe expression of human antithrombin Ⅲ (hAT-Ⅲ) gene in vascular endotheliallike cells(VELCs) after transfected. Methods Human bone marrow mesenchymal stem cells(BMMSCs) were isolated, cultured and proliferated in vitro, and were differentiated into VELCs. Then, the VELCs were divided into experimental group cells and control group cells randomly. Plasmid DNA with hAT-Ⅲ gene was transfected into VELCs by liposome mediate. At last, the hAT-Ⅲ expression was determined by reverse transcriptpolymerase chain reaction(RT-PCR), immunohistochemical stain(IHCS), Westernblotting and chromogenic substrate assay at 72h and 96h respectively. In the control group, the plasmid DNA was replaced by TE buffer, and the other methods were the same as the experimental group. Results RT-PCR showed that the specific DNA fragment of hAT-Ⅲ could be amplifed in the experimental group cells, none in the control group. IHCS showed positive expression of hAT-Ⅲ in the experimental group cells, negative in the control group. Westernblotting showed that the specific band of hAT-Ⅲ could be detected in the experimental group cells culture fluid, none in the control group. Chromogenic substrate assay showed that the hAT-Ⅲ activity of the experimental group cells was 9.50%±1.52%, the control group was 1.83%±1.17%， there was statistically difference between two groups(t=7.910，Plt;0.01). Conclusion The hAT-Ⅲ gene could be transfected into VELCs and expressed successfully.
Objective To integrate the result of whole genome expression data and whole genome promoter CpG island methylation data, to screen the epigenetic modulated differentially expressed genes from transformed porcine bone marrow mesenchymal stem cells (BMSCs) after long-term cultivation. Methods Bone marrow from 6 landrace pigs, 3-month-old about 50 kg weight, was aspirated from the medullary cavity of the proximal tibia. The BMSCs were isolated, and purified by Ficoll density gradient centrifugation combined with adherent culture method. The transfor mation of BMSCs was tested by several methods including cell morphology observation, karyotype analysis, clone forming in soft agarose, serum requirement assay, and tumor forming in mice. The Agilent Pig 4x44k Gene Expression Microarray was used to investigate the differentially expressed mRNA. The methylated genes expression profile was performed using customized pig methylation chip. The gene expression and DNA methylation profiles were integrated to find out the epigenetic modulated differentially expressed genes, and to complete the bioinformatic analysis. Results BMSCs showed a change in appearance, from the initial spindle shape to a more flatted morphology then to small contact shape. After additional passages, BMSCs gradually acquired recovery of proliferating capacity and transformation properties such as anchorage-independent growth, chromosomal abnormality, and tumor formation in nude mice. The gene chip analysis demonstrated that 257 genes were upregulated and 315 genes were downregulated during long-term cultures as well as multiple signal pathways transduction involved, such as cell cycle, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton, pathways in cancer, and P53. The analysis from methylation chip of coding genes suggested epigenetic regulation was involved in BMSCs spontaneous transformation and play a important role on it; 962 genes were hypermethylated and 1219 genes were hypomethylated, which were involved in the biological process of cellular metabolic, structure, and tumor generation. The combined analysis of genes regulated by methylation in the transformation process of BMSCs found that the methylation changes of the 35 genes were contrary to the direction of expression change (correlation coefficient r=–0.686, P=0.000); in which the methylation level of 21 genes promoter regions were increased while the gene expression decreased, and the methylation level of the 14 genes promoter regions decreased and the gene expression increased. At the same time, KEGG enrichment analysis revealed multiple genes regulated by methylation, involved in stem cell differentiation and multiple cell signaling pathways. Among the 14 down-regulated genes, many of them have the role of regulating the interaction of tumor and immunization, and the change of the methylation status of the CDKN3 promoter region may be closely related to the cell oncology. Conclusion The results deepen our understanding of the crucial role of coding genes methylation modification in BMSCs transformation, and may provide new approach to establish safe criteria for BMSCs clinical applications and transformation prevention.
ObjectiveTo investigate the anti-apoptotic ability of synovium-derived mesenchymal stem cells (SMSCs) by comparing the apoptosis induced by tumor necrosis factor α (TNF-α) between SMSCs and bone marrow mesenchymal stem cells (BMSCs). MethodSMSCs and BMSCs were isolated with tissue adhering and density gradient centrifugation respectively, and cells at passages 3-5 were used in further experiments. After immunophenotype identification and differentiation induction, cells were divided into 4 groups. In the experimental groups, apoptosis of SMSCs and BMSCs were induced by 20 ng/mL TNF-α and 10 μg/mL cycloheximide, and cells were cultured in normal culture medium in the control groups. Cellular morphology were observed by inverted phase contrast microscope. After apoptosis induction for 24 hours, cell viability was determined by cell counting kit 8 assay and apoptotic index was detected by flow cytometer. Moreover, the level of Cleaved Caspase-8, 3 were determined by Western blot. ResultsBoth SMSCs and BMSCs accorded with the definition criteria of MSCs according to results of immunophenotype identification and differentiation induction. After apoptosis induction, cells became shrinking and partially floated and cellular morphologies became worse than those in the control groups. After apoptosis induction for 24 hours, cell viabilities of SMSCs and BMSCs in the control groups were both 100%, and no apoptotic cells were observed. However, cell viabilities of SMSCs and BMSCs in the experimental groups were 60.13%±8.63% and 46.55%±10.54% respectively, which were both significantly lower than those in the control groups (P<0.05) , and cell viability in the SMSCs experimental group was significantly higher than that in the BMSCs experimental group (t=3.152, P=0.006) . The apoptotic index was 36.54%±8.63% in the SMSCs experimental group and was 53.77%±11.52% in the BMSCs experimental group, both were significantly higher than the control groups (1.12%±0.24% and 1.35%±0.31%) (P<0.05) . What's more, it was significantly lower in SMSCs experimental group than that in BMSCs experimental group (t=3.785, P=0.001) . Moreover, no expression of Cleaved Caspase-8, 3 was detected in the control groups. But the levels of Cleaved Caspase-8, 3 were significantly enhanced in the experimental groups and they were lower in SMSCs than in BMSCs (t=13.870, P=0.000; t=7.309, P=0.000) . ConclusionsTNF-α induced apoptosis is lower in SMSCs than in BMSCs, which means that SMSCs may have stronger anti-apoptosis ability than BMSCs.
ObjectiveTo explore the effect and mechanisms of bone marrow mesenchymal stem cells (BMSCs) on healing quality of acetic acid-induced gastric ulcer. MethodsForty-eight clean grade male Wistar rats were used to establish the model of gastric ulcer with acetic acid and were randomly divided into 3 groups after 3 days of modeling, 16 rats each group. After the abdominal cavity was open and stomach was pulled out, no treatment was given in group A, 150 μL phosphate buffered saline (PBS) and 150 μL BMSCs at passage 4+PBS (1×108 cells/100 μL) were injected into the gastric wall surrounding the ulcer at 5 different points in groups B and C respectively. After 10 days, the ulcer area was measured, the mucosal thickness and the number of dilated glands were tested in the regenerative mucosa by histological method. And the expression of vascular endothelial growth factor (VEGF) was detected at ulcerative margin by immunohistochemical method. ResultsThe ulcer area in group C was significantly smaller than that of groups A and B (P<0.01), but no significant difference was found between groups A and B (P>0.05). HE staining showed that group C had thicker regenerative gastric mucosa, less dilated glands, and more regular mucosal structure than groups A and B, showing significant differences in regenerative gastric mucosa thickness and dilated glands number (P<0.01), but no significant difference between groups A and B (P>0.05). Immunohistochemical staining showed that the positive expression of VEGF in the ulcer margin mucosa of group C was significantly higher than that of groups A and B. The integral absorbance (IA) value of VEGF expression in group C was significantly higher than that in groups A and B (P<0.01), but no significant difference between groups A and B (P>0.05). ConclusionBMSCs can accelerate ulcer healing by the secretion of VEGF, and improve the quality of ulcer healing.
Objective Construction of viable tissue engineered bone is one of the most important research fields in the cl inical appl ication of bone tissue engineering, to investigate the function of nerve factors in bone tissue engineering by celldetection in vitro and construction of neurotization tissue engineered bone in vivo. Methods Fifty-four healthy New Zealandwhite rabbits, male or female, weighing 2-3 kg, were involved in this study. Bone marrow mesenchymal stem cells (BMSCs) from the bone marrow of white rabbits were cultured. The second passage of BMSCs were treated with sensory nerve or motor nerve homogenates, using the LG-DMEM complete medium as control. The prol iferation and osteogenic differentiation of the cells were observed and tested by the MTT assay, alkal ine phosphatase (ALP) stain, and collagen type I immunocytochemistry identification. The osteogenic induced BMSCs were inoculated in β tricalcium phosphate (β-TCP) biomaterial scaffold and cultured for 72 hours, then the β-TCP loaded with seed cells was implanted in the rabbit femur with 15 mm bone and periosteum defects. Fifty-four New Zealand white rabbits were randomly divided into three groups (n=18): sensory nerve bundle (group A) or motor nerve bundle (group B) were transplanted into the side groove of β-TCP scaffold, group C was used as a control without nerve bundle transplantation. X-ray detection was performed at the 4th, 8th, and 12th weeks after operation.
Objective To investigate the influence of different transplantating times on the survival and immigration of the bone marrow mesenchymal stem cells (BMSCs) in injured spinal cord by subarachnoid administration, and to evaluate the most optimal subarachnoid administration times for BMSCs. Methods Eight adult male rats (weighing 120 g) were used to isolate BMSCs that were cultured, purified and labeled with Hoechst 33342 in vitro. Another 75 adult Wistar rats (weighing 220 g) were made the spinal cord injury (SCI) models at T9,10 level according to the improved Allen’s method and were randomly divided into 5 groups (groups A, B, C, D, and E, n=15). The labeled BMSCs at 1 × 107/mL 0.1 mL were injected into subarachnoid space of the rats via a catheters under the subarachnoid space in groups A (one time at 1 week), B ( two times at 1 and 3 weeks), C (3 times at 1, 3, and 5 weeks) and D (5 times at 1, 3, 5, 7, and 9 weeks) and 0.2 mL phosphate-buffered sal ine (PBS) was injected in group E (5 times at 1, 3, 5, 7, and 9 weeks) as blank control. The neurological functions were evaluated using the Basso-Beattie-Bresnahan (BBB) scale 1, 3, 5, 7, 9, and 12 weeks after transplantation. The migration, survival, differentiation, and histomorphological changes of BMSCs were observed by HE, immunohistochemistry, and fluorescence microscopy. Results At 3 weeks after injury, there were significant differences in the BBB scores between group E and groups A, B, C, D (P lt; 0.01), and between groups A, B and groups C, D (P lt; 0.01). At 7, 9, and 12 weeks, the BBB scores were significantly higher in groups C and D than in groups A and B (P lt; 0.01), and in group B than in group A (P lt; 0.01). There were no significant differences in the BBB scores between groups C and D (P gt; 0.05). The fluorescence microscopy showed that the transplanted BMSCs survived and grew in the injured region at 3 weeks after injury and as time went on, the transplanted cells gradually decreased in group A; in groups B, C, and D, BMSCs count reached the peak values at 5 and 7 weeks and then gradually decreased. At 12 weeks, the survival BMSCs were significantly more in groups C and D than in groups A and B (P lt; 0.01). HE staining showed that the formation of cavity was observed in each group at 3 weeks after injury and the area of cavity gradually decreased in groups A, B, C, and D. At 12 weeks, the area of cavity was the miximal in groups C and D, moderate in groups A and B, and the maximal in group E. The immunohistochemistry staining indicated that the expression of NF-200 was more intense in groups C and D than in groups A and B. The expression of NF-200-positive fibers was more intense in group C. Conclusion Multiple administration of BMSCs promotes the restoration of injured spinal cord and improves neurological functions, and three times for BMSCs transplantation is best
Objective To investigate tissue engineered spinal cord which was constructed of bone marrow mesenchymal stem cells (BMSCs) seeded on the chitosan-alginate scaffolds bridging the both stumps of hemi-transection spinal cord injury (SCI) in rats to repair the acute SCI. Methods BMSCs were separated and cultured from adult male SD rat. Chitosan-alginate scaffold was produced via freeze drying, of which the structure was observed by scanning electron microscope (SEM) and the toxicity was determined through leaching l iquor test. Tissue engineered spinal cord was constructed by seeding second passage BMSCs on the chitosan-alginate scaffolds (1 × 106/mL) in vitro and its biocompatibil ity was observed under SEM at 1, 3, and 5 days. Moreover, 40 adult female SD rats were made SCI models by hemi-transecting at T9 level, and were randomly divided into 4 groups (each group, n=10). Tissue engineered spinal cord or chitosan-alginate scaffolds or BMSCs were implanted in groups A, B, and C, respectively. Group D was blank control whose spinal dura mater was sutured directly. After 1, 2, 4, and 6 weeks of surgery, the functional recovery of the hindl imbs was evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating score. Other indexes were tested by wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retrograde tracing, HE staining and immunofluorescence staining after 6 weeks of surgery. Results Chitosan-alginate scaffold showed three-dimensional porous sponge structure under SEM. The cells adhered to and grew on the surface of scaffold, arranging in a directional manner after 3 days of co-culture. The cytotoxicity of chitosan-alginate scaffold was in grade 0-1. At 2, 4, and 6 weeks after operation, the BBB score was higher in group A than in other groups and was lower in group D than in other groups; showing significant differences (P lt; 0.05). At 4 and 6 weeks, the BBB score was higher in group B than in group C (P lt; 0.05). After 6 weeks of operation, WGA-HRP retrograde tracing indicated that there was no regenerated nerve fiber through the both stumps of SCI in each group. HE and immunofluorescence staining revealed that host spinal cord and tissue engineering spinal cord l inked much compactly, no scar tissue grew, and a large number of neurofilament 200 (NF-200) positive fibers and neuron specitic enolase (NSE) positive cells were detected in the lesioned area in group A. In group B, a small quantity of scar tissue intruded into non-degradative chitosan-alginate scaffold at the lesion area edge, and a few of NSE flourescence or NF-200 flourescence was observed at the junctional zone. The both stumps of SCI in group C or group D were filled with a large number of scar tissue, and NSE positive cells or NF-200 positive cells were not detected. Otherwise, there were obviously porosis at the SCI of group D. Conclusion The tissue engineered spinal cord constructed by multi-channel chitosan-alginate bioscaffolds and BMSCs would repair the acute SCI of rat. It would be widely appl ied as the matrix material in the future.
ObjectiveTo investigate the effects of bone marrow mesenchymal stem cells (BMSCs) transplantation for treating spinal cord injury (SCI) in rat and the cytokine expression changes in the local injury tissues. MethodsBMSCs were separated from Sprague Dawley (SD) rat and cultured with the whole bone marrow culture method. rAd-EGFP was used to transfect the 5th generation BMSCs for green fluorescent protein (GFP) label. Twelve SD rats were randomly divided into experimental group (n=6) and control group (n=6). After the T10 SCI model was established with Allen's impact device in 2 groups, 1×106 GFP-labeled BMSCs and PBS were administered by subarachnoid injection in situ in experimental group and control group, respectively. Basso-Beattie-Bresnahan (BBB) score was used to detect the motor function at immediat, 1, 2, 3, 4, and 5 weeks after SCI. At 5 weeks, the spinal cord tissues were harvested for the histological and immunofluorescent staining examinations to measure the expressions of neural marker molecules, including Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific nuclear protein (NeuN). Cytokine was analyzed with antibody array. ResultsAt 5 weeks, 2 rats died of urinary tract infection in 2 groups respectively, the other rats survived to the end of experiment. BBB score of experimental group was significantly higher than that of control group at 1, 2, 3, 4, and 5 weeks (P < 0.05). At 5 weeks, histological results showed that there were many cells with regular arrangement in the experimental group; there were less cells with irregular arrangement in the control group. Compared with the control group, Nestin and NeuN expressions significantly increased (P < 0.05), and GFAP expression significantly decreased (P < 0.05) in the experimental group. Leptin and ciliary neurotrophic factor levels were higher in the experimental group than the control group, but granulocyte-macrophage colony-stimulating factor, tumor necrosis factorα, interleukin 1β, and tissue inhibitor of metalloproteinases 1 levels were lower in the experimental group than the control group. ConclusionBMSCs transplantation can improve survival and regeneration of nerve cells and enhances the recovery of nerve function by regulating secretion of cytokines from grafted BMSCs.
ObjectiveTo investigate the bone regeneration potential of cell-tissue engineered bone constructed by human bone marrow mesenchymal stem cells (hBMSCs) expressing the transduced human bone morphogenetic protein 2 (hBMP-2) gene stably. MethodsThe full-length hBMP-2 gene was cloned from human muscle tissues by RT-PCR and connected into a vector to consturct a eukaryotic expression system. And then the gene expression system was transduced to hBMSCs with lipidosome. hBMSCs were transfected by hBMP-2 gene (experimental group) and by empty plasmid (negative control group), untransfected hBMP-2 served as blank control group. RT-PCR, dot-ELISA, immunohistochemical analysis and ALP activity were performed to compare and evaluate the situation of hBMP-2 expression and secretion after transfection. hBMSCs transfected by hBMP-2 gene were seeded on hydroxyapatite (HA) and incubated for 4 days to construct the hBMP-2 gene modified tissue engineered bone, and then the tissue engineered bone was observed by the inverted phase contrast microscope and scanning electron microscope. Then the hBMP-2 gene modified tissue engineered bone (group A, n=3), empty plasmid transfected hBMSCs seeded on HA (group B, n=3), hBMSCs suspension transfected by hBMP-2 gene (group C, n=3), and hBMP-2 plasmids and lipidosome (group D, n=3) were implanted into bilateral back muscles of nude mice. The osteogenic activity was detected by HE staining and alcian blue staining after 4 weeks. ResultsAt 48 hours and 3 weeks after transfection, RT-PCR and dot-ELISA results indicated that the transfected hBMSCs could express and secrete active and exogenous hBMP-2 stably. The immunohistochemical staining was positive, and the ALP activity in the transfected hBMSCs was significantly higher than that in two control groups (P < 0.05). The transfected hBMSCs had a good attaching and growing on the three-demension suface of HA under inverted phase contrast microscope and scanning electron microscope. In vivo study indicated that a lot of new bone formation was obviously found at 4 out of 6 sides of back muscles in group A. Some new bone formation at both sides of back muscles was observed in 1 of 3 mice in group B. No new bone formation was found in group C. A few new bone formation was observed at one side of back muscles in group D. ConclusionThe tissue engineered bone constructed by hBMP-2 gene modified hBMSCs and HA is able to express and secrete active hBMP2 stably and can promote new bone formation effectively in muscles of nude mice.
ObjectiveTo investigate the effect of LOC103693069 on hypoxic apoptosis of bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs from 1-week-old Sprague Dawley rat bone marrow were isolated, cultured, and passaged by the whole bone marrow adherent culture method. After identification of adipogenic, chondrogenic, and osteogenic differentiation, the 3rd generation cells were treated with hypoxia under 5%O2, 1%O2, and anaerobic conditions. After 48 hours, the cell viability, apoptosis, and apoptosis-related proteins [hypoxia inducible factor 1α (HIF-1α), Caspase-3, B cell lymphoma/leukemia 2 (Bcl-2)] expressions were detected, and normal BMSCs were used as controls. Based on the research results, the concentration group with the most obvious apoptosis was selected and used for subsequent experiments. After 48 hours of hypoxia treatment, BMSCs were taken and analyzed by gene chip and real-time fluorescence quantitative PCR (qRT-PCR) to screen the most significantly down-regulated gene and construct their high-expression, low-expression, and negative control lentiviruses; BMSCs were transfected with the different lentiviruses, respectively. After qRT-PCR detection confirmed that the transfection was successful, the BMSCs were treated with hypoxia for 48 hours to observe the cell viability and the expressions of apoptosis-related proteins. ResultsAfter cell viability, apoptosis, and apoptosis-related proteins were detected, cell apoptosis was the most significant under anaerobic conditions after 48 hours. The above indicators were significantly different from other groups (P<0.05), and this group was used for treatment conditions for subsequent experiments. Gene chip analysis showed that after 48 hours of hypoxia treatment, AC125847.1, LOC102547753, AABR07017208.2, and LOC103693069 were significantly down-regulated in BMSCs, and the expressions of LOC103693069 was the most significant down-regulation detected by qRT-PCR (P<0.05). It was selected to construct lentivirus and transfect BMSCs. Afterwards, qRT-PCR detection showed the successful transfection into the cells. After hypoxia treatment, the apoptosis rate and the expressions of apoptosis-related proteins of BMSCs overexpressed by the gene were significantly reduced (P<0.05). Conclusion LOC103693069 can relieve the hypoxic apoptosis of BMSCs.