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Objective To investigate the effect of glutamine in combination with umbilical cord blood mesenchymal stem cells ( MSCs) transplantation on intestinal ischemia-reperfusion injury in rats.Methods Umbilical cord blood mesenchymal stem cells were isolated, and were labeled with CM-DiI fluorescent dye.Eighty Sprague-Dawley rats were randomly divided into normal control group, ischemia reperfusion injury group, glutamine group, MSCs transplantation group and combined group with 15 rats in each group.The control group received saline enema.The injury group was treated with TNBS ( ethanol dilution) enema.The glutamine group at 1 h after TNBS received intravenous injection of 0.45 g/kg glutamine.The rats of MSCs transplantation group had tail vein injection of 1 ×1010/L umbilical cord blood mesenchymal stem cell suspension, and the combined group received intravenous injection of glutamine 0.45 g/kg and 1 ×1010/L umbilical cord blood mesenchymal stem cell suspension.ELISA was used to detect the midgut fatty acid binding protein (iFABP), interleukin 6 (IL-6), and superoxide dismutase (SOD) content in the rat serum.The water content of intestinal tissue was detected at 1 h and 3 h after reperfusion in each group.The expressions of NF-kB, Bcl-2 and caspase-3 mRNA and proteins in the rat intestinal epithelial cells after treated with glutamine in combination with MSCs were detected by RT-PCR and Western blot assays.Results The fluorescent tracer method revealed that the transplanted MSCs cells were distributed in the intestinal mucosal lymphoid tissues and glandular epithelial cells, indicating that MSCs might be involved in the repair process of intestinal ischemia-reperfusion injury.The content of serum IFABP and IL-6 in the injured group was significantly higher than that in the control group, while significantly reduced in the glutamine group, MSCs transplantation group and combined group, with the most obvious in the combined group.The content of SOD in the injury group was significantly lower than that in the control group, and significantly increased than that in the glutamine group, MSCs transplantation group, with the most striking in the combined group ( P0.05).Compared with the control group, the caspase-3 and NF-kB mRNA and protein expressions in the intestinal mucosal epithelial cells of the injury group were significantly increased, and the expressions of Bcl-2 mRNA and protein were significantly reduced ( P 0.05), but there was a significant difference between these two groups and the combined group (P<0.05).Conclusions After treated with glutamine and MSCs transplantation, the degree of intestinal ischemia reperfusion injury is obviously reduced in rats.It may be mediated through inhibiting the expression of caspase-3 and NF-kB and promoting the expression of Bcl-2.
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BACKGROUND:Bone marrow mesenchymal stem cel s can repair intestinal ischemia-reperfusion injuny by interfering inflammatory reactions after intestinal ischemia-reperfusion to protect intestinal barrier functions. In recent years, umbilical cord blood mesenchymal stem cel s are gradual y used as a substitute source of bone marrow mesenchymal stem cel s. OBJECTIVE:To investigate the effects of umbilical cord blood mesenchymal stem cel s on acute intestinal ischemia-reperfusion injury. METHODS:Umbilical cord blood mesenchymal stem cel s were induced, isolated in vitro and tracked by CM-DiI fluorescent labeling. Sixty-three Sprague-Dawley rats were equivalently randomized into three groups:control group received normal saline enema, intestinal ischemia-reperfusion injury group with ethanol diluted trinitro-benzene-sulfonic acid and transplantation group administrated with 1×1010/L umbilical cord blood mesenchymal stem cel suspension via the tail vein at 1 hour after trinitro-benzene-sulfonic acid modeling. At 3 days after transplantation, colon tissues were removed in each group to observe pathological changes of the intestinal tract by hematoxylin-eosin staining. Besides, expression of leptin mRNA in the colon tissues and cyclooxygenase-2 in the mucosa were detected by RT-PCR and immunohistochemistry method, respectively. RESULTS AND CONCLUSION:Transplanted umbilical cord blood mesenchymal stem cel s distributed in the intestinal lymphoid tissue and among glandular epithelial cel s, suggesting that these stem cel s might be involved in the process of intestinal ischemia-reperfusion injury repair. Compared with the control group, intestinal injury in the injury group was significantly aggravated, and most intestinal epithelial cel s shed;and the transplantation group appeared to have significantly reduced intestinal damage and significantly less cel shedding. Expression of leptin mRNA was significantly higher in the injury group than the transplantation group fol owed by the control group, and there were significant differences among the three groups (P<0.05). Additional y, expression of cyclooxygenase-2 in the injury group was significantly higher than that in the control group (P<0.05);compared with the injury group, expression of cyclooxygenase-2 was significantly lower in the transplantation group (P<0.05). To conclude, leptin and cyclooxygenase-2 may be involved in acute intestinal ischemia-reperfusion injury, and umbilical cord blood mesenchymal stem cel transplantation significantly lessens intestinal ischemia-reperfusion injury, which provides an experimental basis for human treating acute intestinal ischemic injury.
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BACKGROUND:Stem cells expansion technology in vitro is affected by the microenvironment of the culture system. So, to find an effective method is particularly important to promote celladherent and growth. OBJECTIVE:To compare the effects of different culture media on cellexpansion. METHODS:Human umbilical cord mesenchymal stem cells at a density of 5.0×104 cells/cm2 were inoculated into Corning? polystyrene culture dishes coated with or without poly-L-ornithine and Corning? cellBIND culture dishes. celladhesion and proliferation were observed, and expressions of celladhesion proteins and cellmarkers were detected. RESULTS AND CONCLUSION:celladhesion was promoted when cells were cultured in Corning? cellBIND? Surface medium coated with poly-L-ornithine for 24 hours, and the cultured cells grew at the logarithmic phase. The cellproliferation was also enhanced, and the cells expressed celladhesion protein but not the cellmarkers of CD73, CD90, CD105. Corning? cellBIND? Surface medium was superior to Corning? polystyrene culture medium in the improvement of celladhesion and proliferation. Additional y, both of these two media showed no influence on cellphenotype. These findings indicate that Corning? cellBIND? Surface medium can promote celladhesion and proliferation, but shows no effects on cyclin and cellphenotype. This medium coated with poly-L-ornithine can further accelerate celladhesion and proliferation, and stably express cellphenotype of human umbilical cord mesenchymal stem cells.
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BACKGROUND:Stem celltumorigenicity is a practical issue concerning stability in the clinical application of stem cells. Therefore, it is particularly important to clear whether stem cells have tumorigenic ability or not. Nude mice occupy an increasingly important position in oncology, immunology, and safety evaluation of drugs and biological products. OBJECTIVE:To observe the tumorigenicity of neural stem cells and mesenchymal stem cells in Balb/c nude immunodeficient mice. METHODS:Balb/c nude mice were randomly divided into control group, negative group, positive group, neural stem cellgroup and mesenchymal stem cellgroup. HepG-2 cells, RPE cells, passage 4 neural stem cells and mesenchymal stem cells were injected subcutaneously into nude mice from different groups, respectively. After 12 weeks of injection, anatomical observation was performed to detect the tumor formation in the transplantation site. Meanwhile, soft agar colony formation assay was applied to investigate neural stem celland mesenchymal stem cellclone in vitro. RESULTS AND CONCLUSION:After 12 weeks of injection, the tumorigenicity study results showed that no tumor developed in the transplantation site in the control group, negative group, neural stem cellgroup and mesenchymal stem cellgroup. Histopathologic examinations also showed no abnormality in these groups. Soft agar colony formation assay showed in soft agar resistance medium, neural stem cells and mesenchymal stem cells did not exhibit clone ability. These findings indicate that neural stem cells and mesenchymal stem cells undergoing short-term passages have no tumorigenic growth.
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Objective To study the effect of dexmedetomidine(DEX)intrathecal administration on formation of chronic neuropathic pain induced by chronic constriction injury(CCI)in rats. Methods One hundred and twenty healthy adult male Sprague-Dawley(SD)rats were divided randomly into four groups(each n=30):sham group, model group,30μg/kg and 60μg/kg DEX intrathecal injection group(D30 group,D60 group). The CCI model was installed by left sciatic nerve ligaturing,sham surgery was done by exposing the sciatic nerve without ligation,and 30 μg/kg,60 μg/kg DEX(each,10 μl)and 10 μl normal saline were given intrathecally in D30,D60 and model groups respectively,all kinds of injection being once a day for 7 days. Hind paw mechanical contraction reflex threshold(MWT),heat-shrinkable reflex latency(TWL)and the score of motor dysfunction of hind extremity in rats were recorded on 1 day before ligation and 1,3,7 days after operation,and before ligation and 1,7,14 and 21 days after operation,the contents of tumor necrosis factor-α(TNF-α),interleukin(IL-1βand IL-6)in intumescentia lumbalis were measured by enzyme linked immunosorbent assay (ELISA). Results Compared with before operation,there were no significant differences in values of MWT,TWL,the motor function evaluation,TNF-α, IL-1β,and IL-6 at each time points(all P>0.05);however,with the prolongation of time,MWT and TWL were decreased,and the motor function evaluation,TNF-α,IL-1β,and IL-6 were increased in all the other groups. Compared with those of the sham group,MWT and TWL were declined,and the motor function evaluation,TNF-α, IL-1β,and IL-6 were elevated in model group. Compared with those of model group,30μg/kg and 60μg/kg DEX could significantly raise MWT(g)and TWL(s),obviously improve motor function and remarkably decrease the contents of TNF-α(pg/mg),IL-1β(pg/mg)and IL-6(pg/mg)in the spinal cord of CCI rats from 1 day after operation. And the changes in 60 μg/kg DEX group were more significant than those in 30 μg/kg DEX intrathecal injection group〔postoperative 1 day MWT:39.3±1.3 vs. 20.3±2.2,TWL:10.9±0.4 vs. 8.2±1.1,motor function score:2.00±0.00 vs. 2.00±0.75,TNF-α:33±7 vs. 125±18,IL-1β:108±12 vs. 203±34,IL-6:156±39 vs. 405±75,all P<0.05〕. Conclusions The DEX intrathecal administration has certain degree of dose-dependent therapeutic effect on hyperalgesia in CCI rat models. The mechanism is related to the amelioration of inflammatory reaction at the lumbar segment of spinal cord.