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ObjectiveThis study aimed to investigate the effects of eugenol on inhibiting the inflammatory activation of human umbilical cord mesenchymal stem cells （HUC-MSCs） and the pro-inflammatory phenotype of hepatic stellate cells （HSCs） in liver fibrosis， and to explore their underlying mechanisms. MethodsHUC-MSCs were cultured and identified in vitro， and the toxicity of eugenol to HUC-MSCs was evaluated by MTT method. The effect of eugenol on the migration ability of HUC-MSCs was investigated by in vitro scratch test. The expression of α-SMA， COL1A1， Smad2/3 and p-Smad2/3 of LX-2 cells activated by TGF-β1 treated with EU-MSCs-CM and MSCs-CM were detected by WB assay. EU-MSCs-CM and MSCs-CM treated THP-1 macrophages stimulated with Lipopolysaccharide （LPS） were analyzed for the expression of surface markers CD11b， CD86， and CD206 by flow cytometry. Additionally， the expression of pro-inflammatory genes TNF-α， IL-1β， and IL-6 in THP-1 macrophages was detected by qPCR. ResultsThe results of MTT method showed that the viability of the cells remained above 90% after 24 h and 48 h treatment at 0， 7.5， 15 μg/mL. In vitro scratches showed that eugenol treatment enhanced HUC-MSCs migration. WB results showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment significantly inhibited the expression of α-SMA， COL1A1， Smad2/3， and p-Smad2/3 of activated HSCs. Flow cytometry showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment had a more significant inhibitory effect on CD86， a M1-type polarization marker in THP-1 macrophages. The results of qPCR experiment showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment more significantly inhibited the expressions of TNF-α， IL-1β and IL-6 of THP-1 macrophage proinflammatory genes. ConclusionsEugenol enhances the inhibitory effect of HUC-MSCs on inflammatory activation of HSCs， possibly by regulating TGF-β1/Smads signaling pathway. It also enhances the inhibitory effect of HUC-MSCs on the pro-inflammatory phenotype of macrophages. Proinflammatory macrophages can promote inflammatory activation of HSCs.
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Objective To investigate the role of human umbilical cord mesenchymal stem cell-derived extracellular vesicle (hUC-MSC-EV) in the regeneration of fibrotic liver. Methods C57BL/6 mice were randomly divided into the 70% normal liver resection group (Oil+PHx group), 70% liver fibrosis resection group (CCl4+PHx group) and 70% liver fibrosis resection+mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment group (CCl4+PHx+MSC-EV group), with 8 mice in each group. LX-2 cell lines were assigned into the phosphate buffer solution (PBS) group, transforming growth factor (TGF)-β group and TGF-β+MSC-EV group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) in mice after partial liver resection were detected in each group. The expression levels of liver fibrosis and proliferation-related parameters were analyzed in each group. The messenger RNA (mRNA) expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in LX-2 cells were detected in each group, and their effects on HGF expression in mouse liver were observed. Results Compared with the Oil+PHx group, the serum levels of AST, ALT and LDH were up-regulated, and the degree of fibrosis was more severe, the positive area of Sirius red and α-smooth muscle actin (α-SMA) staining was larger, and the expression level of α-SMA protein was up-regulated in the CCl4+PHx group. Compared with the CCl4+PHx group, the serum levels of AST, ALT and LDH were decreased, the degree of fibrosis was slighter, the positive area of Sirius red and α-SMA staining was decreased, and the expression level of α-SMA protein was down-regulated in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the Oil+PHx group, the protein expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) were lower in the CCl4+PHx group. Compared with the CCl4+PHx group, the protein expression levels of Ki67 and PCNA were increased in the CCl4+PHx+MSC-EV group, and the differences were statistically significant (all P < 0.05). Compared with the PBS group, the expression level of CollagenⅠ mRNA in LX-2 cells was increased, the expression level of α-SMA protein was up-regulated and the expression level of HGF protein was decreased in the TGF-β group. Compared with the TGF-β group, the expression level of CollagenⅠ mRNA in LX-2 cells was decreased, the expression levels of HGF mRNA and protein were increased, and the expression level of α-SMA protein was decreased in the TGF-β+MSC-EV group, the differences were statistically significant (all P < 0.05). The expression level of HGF protein in the CCl4+PHx group was lower than that in the Oil+PHx group, whereas the difference was not statistically significant (P > 0.05). The expression level of HGF protein in the CCl4+PHx+MSC-EV group was higher than that in the CCl4+PHx group, and the difference was statistically significant (P < 0.05). Conclusions The regenerative capacity of fibrotic liver is weaker than that of normal liver. hUC-MSC-EV may alleviate liver fibrosis and improve liver regeneration by promoting HGF secretion from actived hepatic stellate cells and effectively enhancing the regenerative capacity of fibrotic liver.
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AIM: To evaluate the efficacy of transplantation of human umbilical cord mesenchymal stem cells(hUCMSCs)in the treatment of corneal alkali burn in rabbits, and study the infiltration of polymorphonuclear neutrophils(PMNs)and the changes of vascular endothelial growth factor(VEGF)expression.METHODS: Corneal alkali burn models were established in right eyes of 75 healthy Japanese white rabbits, which were divided into three groups(group A, B and C), with 25 rabbits in each group. Group A was treated with amniotic membrane combined with hUCMSCs on the day after corneal alkali burn. Group B was treated with amniotic membrane only. Group C did not give any treatment after corneal alkali burn. At 3, 7, 14, 21 and 28d after corneal alkali burn, the corneal recovery was observed by slit lamp and photographed, the growth of corneal neovascularization(CNV)was scored, and corneal tissue was separated to make pathological sections. PMNs infiltration was observed by hematoxylin-eosin(HE)staining, and the expression of VEGF was determined by immunohistochemical staining.RESULTS: The growth of CNV in group A was much slower than that in group B at 14d after alkali burn. The CNV growth score around lesions of group A was significantly lower than that of group B(P<0.05). The quantity of PMNs increased on the 3d with the stromal layer of cornea infiltrated, relatively decreased on the 7d, shown a peak on the 14d, and then decreased gradually. Early infiltration after alkali burn was in the corneal stroma of the lesion area, and the extent of infiltration was equal to the ulcer area at later stage. The cell densities of corneal PMNs in group A and group B were significantly lower than those in group C at all time points after alkali burns(P<0.05), and those in group A were significantly lower than group B at 14 and 21d(P<0.05). The expression levels of corneal VEGF in all groups after alkali burn reached peak at 7～14d and decreased significantly at 28d, and the expression levels of VEGF in group A and group B at all time points after alkali burn were significantly lower than those in group C(P<0.05), and group A was significantly lower than that in group B at 7, 14 and 21d(P<0.05).CONCLUSION: The transplantation of hUCMSCs after alkali burn cornea can reduce the formation of CNV and inhibit corneal revascularization after alkali burn. The corneal pathological lesions and vascularization are closely related to PMNs and VEGF.
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Objective: To observe the effects of exosomes derived from human umbilical cord mesenchymal stem cells on the proliferation and invasion of pancreatic cancer cells, and to analyze the contents of exosomes and explore the mechanisms affecting pancreatic cancer cells. Methods: Exosomes extracted from human umbilical cord mesenchymal stem cells were added to pancreatic cancer cells BxPC3, Panc-1 and mouse models of pancreatic cancer, respectively. The proliferative activity and invasion abilities of BxPC3 and Panc-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The expressions of miRNAs in exosomes were detected by high-throughput sequencing. GO and KEGG were used to analyze the related functions and the main metabolic pathways of target genes with high expressions of miRNAs. Results: The results of CCK-8 cell proliferation assay showed that the absorbance of BxPC3 and Panc-1 cells in the hucMSCs-exo group was significantly higher than that in the control group [(4.68±0.09) vs. (3.68±0.01), P<0.05; (5.20±0.20) vs. (3.45±0.17), P<0.05]. Transwell test results showed that the number of invasion cells of BxPC3 and Panc-1 in hucMSCs-exo group was significantly higher than that in the control group (129.40±6.02) vs. (89.40±4.39), P<0.05; (134.40±7.02) vs. (97.00±6.08), P<0.05. In vivo experimental results showed that the tumor volume and weight in the exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) group were significantly greater than that in the control group [(884.57±59.70) mm(3) vs. (695.09±57.81) mm(3), P<0.05; (0.94±0.21) g vs. (0.60±0.13) g, P<0.05]. High-throughput sequencing results showed that miR-148a-3p, miR-100-5p, miR-143-3p, miR-21-5p and miR-92a-3p were highly expressed. GO and KEGG analysis showed that the target genes of these miRNAs were mainly involved in the regulation of glucosaldehylation, and the main metabolic pathways were ascorbic acid and aldehyde acid metabolism, which were closely related to the development of pancreatic cancer. Conclusion: Exosomes derived from human umbilical cord mesenchymal stem cells can promote the growth of pancreatic cancer cells and the mechanism is related to miRNAs that are highly expressed in exosomes.
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Camundongos , Animais , Humanos , MicroRNAs/metabolismo , Exossomos/genética , Sincalida/metabolismo , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/genética , Células-Tronco Mesenquimais/metabolismo , Cordão UmbilicalRESUMO
Objective:To investigate the effect of human umbilical cord mesenchymal stem cells (hUC-MSC) on the apoptosis of retinal ganglion cells (RGCs) in diabetic retinopathy (DR) model rats and on the regulation of p38 mitogen-activated protein kinase (p38MAPK) pathway.Methods:Forty-five SPF male 8-week old SD rats were selected.The DR rat model was established by intraperitoneal injection of streptozotocin (STZ) combined with a high-sugar and high-fat diet.The fasting blood glucose (FBG) and body weight of the rats were measured every week during the high-sugar and high-fat diet, and fundus angiography was used to observe the circulation and leakage of retinal blood vessels.Forty rats with successful modeling were randomly divided into model group and hUC-MSC injection group according to the random number table method, with 20 rats in each group.Another 20 normal rats fed routinely were served as control group, and intraperitoneally injected with the same amount of citric acid buffer.The hUC-MSC injection group was injected intravitreously with hUC-MSC, and the control group and model group were injected intravitreously with the same amount of phosphate buffer saline (PBS). Fluoro gold (FG) retrograde tracer labeling RGCs was used to observe the number of survived RGCs.Hematoxylin-eosin staining was used to observe the pathological changes of retina.TUNEL method was used to observe the apoptosis of RGCs.Western blot was used to detect B cell lymphoma /leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax), p38MAPK and phosphorylated (p-) p38MAPK protein expression in retinal tissues.The use and care of the rats complied with the ARVO statement.The study protocol was approved by an Animal Ethics Committee of Zhengzhou central Hospital Affiliated to Zhengzhou University (NO.2980316).Results:The FBG of control rats was maintained at a normal level, and the body weight gradually increased over time, and was gradually decreased as the course of disease prolonged.The retinal blood vessels ran normally without fluorescein leakage in the control group.In the modeling group, the FBG was maintained at a high level, and the body weight increased slowly and gradually decreased with the prolongation of the disease course since the second week after modeling.The distal retinal vessels were found twisted with large area of capillary fluorescein leakage in the modeling group.The density of RGCs in the control group, model group and hUC-MSC injection group were (2 136.10±215.17), (849.40±167.82), (1 549.20±183.26) cells/mm 2, with significant overall differences ( F=115.218, P<0.01). The density of RGCs in the model group and the hUC-MSC injection group were significantly lower than that of the control group, and the density of RGCs in the hUC-MSC injection group was significantly higher than that of the model group, and the differences were statistically significant (all at P<0.05). The retina in the control group was with clear structure, distinct layers, and a large number of complete RGCs.The number of RGCs in the model group was significantly reduced with nuclear pyknosis, thinned and atrophied RGC layer.The retinal structure was relatively complete, and there were more RGCs in the hUC-MSC injection group than the model group.The apoptosis rates of RGCs in the control group, model group and hUC-MSC injection group were (2.16±1.11)%, (43.47±2.26)%, (20.75±2.18)%, with significant overall difference ( F=445.159, P<0.01). The apoptosis rates of RGCs in the model group and hUC-MSC injection group were significantly higher than that of the control group, and the apoptosis rate of RGCs in the hUC-MSC injection group was lower than that of the model group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of Bax, Bcl-2 and p-p38MAPK proteins in the retina tissues among the three groups ( F=30.982, 12.526, 73.158, all at P<0.01). The relative expression of Bax and p-p38MAPK protein were significantly higher, and the relative expression of Bcl-2 protein was significantly lower in the hUC-MSC injection group and the hUC-MSC injection group than those of the control group, and the differences were statistically significant (all at P<0.05). The relative expression of Bax and p-p38MAPK protein was significantly lower, and the relative expression of Bcl-2 protein was significantly higher in the hUC-MSC injection group than those in the model group, and the differences were statistically significant (all at P<0.05). There was no significant difference in the relative expression of p38MAPK protein among the three groups ( F=1.182, P=0.322). Conclusions:Intravitreal injection of hUC-MSC can inhibit the apoptosis of RGCs in DR model rats and protect the retinal structure of rats, which may play an anti-apoptotic effect by inhibiting the p38MAPK signaling pathway.
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Objective:To investigate the protective effect of human umbilical cord mesenchymal stem cell conditioned medium (HucMSC-cm) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) and relevant mechanism of action.Methods:Forty 6-week-old male C57BL/6 mice were selected and randomized (random number) into the sham group, LPS group, LPS + HucMSC-cm (LPS+cm) group, and LPS+HucMSC-cm+Compound C (LPS+cm+cc) group, with 10 mice in each group. Mice were intratracheally injected with LPS (5 mg/kg) to establish ALI model, and intratracheally injected with hucMSC-CM (50 μL) 4 h after LPS treatment. Mice in the LPS+cm+cc group were intraperitoneally treated with Compound C (15 mg/kg) prior to LPS treatment. Neutrophils in peripheral blood were counted with the automated hematology analyzer 72 h after LPS administration. After that, mice were sacrificed, and the lung tissue pathology was observed using hematoxylin eosin (HE) staining. Besides, the expressions of IL-6, ICAM-1, VCAM-1 and P-AMP-activated protein kinase (P-AMPK) in the lung tissues were analyzed by Western blot and immunohistochemical assay. In vitro, human lung microvascular endothelial cells (HuLEC-5a) were cultured and divided into three groups: control group, LPS group (10 μg/ mL), and LPS + HucMSC-cm group. After 24 h of treatment, the expressions of p-AMPK and AMPK were detected by Western blot, and the expressions of IL-6 and IL-8 were detected by real-time fluorescence quantitative PCR. Oneway analysis of variance was used to compare the mean values of normally distributed measurement data between groups. Comparisons between two groups were performed using the Tukey’s multiple comparison test. Results:Compared with the sham group, the LPS group showed lungs with congestion and swelling, thickened pulmonary septum, and inflammatory cell infiltration. Moreover, in the LPS group, the protein expressions of IL-6 ( P=0.003), ICAM-1 ( P<0.001) and VCAM-1 ( P=0.001) were increased significantly, while the expression of p-AMPK was decreased ( P=0.013), accompanied by an increase in the proportion of neutrophils in peripheral blood ( P<0.001). Compared with the LPS group, the LPS+HucMSC-cm group demonstrated eased congestion, edema and pathological injury of lung tissue, reversed protein expressions of IL-6 ( P=0.003), ICAM-1 ( P=0.002), VCAM-1 ( P=0.006) and P-AMPK ( P=0.002), as well as decreased proportion of neutrophils in peripheral blood ( P<0.005). Compared with the LPS+HucMSC-cm group, the LPS+cm+cc group exhibited more severe lung histopathological injury, significantly increased protein expressions of IL-6, ICAM-1 and VCAM-1 in lung tissues, as well as decreased expression of P-AMPK protein. The results of immunohistochemistry were consistent with those of protein. In vitro experiment, after LPS treatment, the mRNA expressions of IL-6 ( P<0.001) and IL-8 ( P=0.027) were increased and p-AMPK protein expression ( P=0.005) was decreased as compared with the control group. In comparison with the LPS group, the LPS+HucMSC-cm group showed decreased mRNA expression levels of IL-6 ( P=0.003) and IL-8 ( P=0.002), but increased protein level of p-AMPK ( P=0.003). Conclusions:HucMSC-cm has a protective effect against LPS-induced acute lung injury, which is mainly attributed to the inhibited expression of adhesion molecules and inflammatory factors under the activation of AMPK.
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Impaired immunomodulatory capacity and oxidative stress are the key factors limiting the effectiveness of mesenchymal stem cell transplantation therapy. The present study was aimed to investigate the effects of jujuboside A (JuA) on the protective effect and immunomodulatory capacity of human umbilical cord mesenchymal stem cells (hUC-MSCs). Hydrogen peroxide was used to establish an oxidative damage model of hUC-MSCs, while PBMCs isolated from rats were used to evaluate the effect of JuA pre-treatment on the immunomodulatory capacity of hUC-MSCs. Furthermore, Hoechst 33258 staining, lactate dehydrogenase test, measurement of malondialdehyde, Western blot, high-performance liquid chromatography; and flow cytometry were performed. Our results indicated that JuA (25 μmol·L-1) promoted the proliferation of hUC-MSCs, but did not affect the differentiating capability of these cells. JuA pre-treatment inhibited apoptosis, prevented oxidative damage, and up-regulated the protein expression of nuclear factor-erythroid factor 2-related factor 2 and heme oxygenase 1 in hUC-MSCs in which oxidative stress was induced with H2O2. In addition, JuA pre-treatment enhanced the inhibitory effect of hUC-MSCs against abnormally activated PBMCs, which was related to stimulation of the expression and activity of indoleamine 2,3-dioxygenase. In conclusion, our results demonstrate that JuA pre-treatment can enhance the survival and immunomodulatory ability through pathways related to oxidative stress, providing a new option for the improvement of hUC-MSCs in the clinical setting.
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Animais , Humanos , Ratos , Diferenciação Celular , Peróxido de Hidrogênio/metabolismo , Células-Tronco Mesenquimais , Estresse Oxidativo , Saponinas , Cordão Umbilical/metabolismoRESUMO
BACKGROUND: Stem cell transplantation has a significant neuroprotective effect on neurological diseases. Current transplantation methods such as arteriovenous transplantation and brain stereotactic transplantation are not suitable for clinical application in preterm infants. OBJECTIVE: To explore the feasibility of nasal transplantation of human umbilical cord mesenchymal stem cells and human neural stem cells for the treatment of white matter injury in premature rat infants. METHODS: Human umbilical cord mesenchymal stem cells were prepared from human umbilical cord tissue, and human neural stem cells were prepared from human embryonic brain tissue. In vitro migration of two kinds of cells was assessed by Transwell method. Forty 3-day-old Sprague-Dawley rats were randomly divided into sham operation group, model control group, human umbilical cord mesenchymal stem cell transplantation group and human neural stem cell transplantation group, with 10 rats in each group. Rats in all groups except the sham operation group were treated with right common carotid artery ligation and hypoxia for 90 minutes to establish a rat model of white matter injury in the preterm infant. Totally 1×106 cells were delivered intranasally in the transplantation group at 3 days after injury. Each nostril was infused with 5×105, and each nostril was infused once. On day 7 after injury, MBP immunofluorescence staining was used to detect the expression of myelin basic protein in the white matter of the brain to identify the damage of the white matter injury model. At 24 hours after transplantation, human umbilical cord mesenchymal stem cell migration was detected by anti-HuNu immunohistochemical method and human neural stem cell migration was detected by CM-Dil fluorescent labeling method. RESULTS AND CONCLUSION: (1) On day 7 after modeling, compared with the normal side, the positive area of MBP decreased in cingulate band, corpus callosum and external capsule of the affected side in the model of brain white matter injury in preterm infants (P < 0.05), indicating a successful modeling. (2) In vitro experiments showed that the migration rate of human neural stem cells was the same as that of human umbilical cord mesenchymal stem cells. (3) At 24 hours after the nasal transplantation, human umbilical cord mesenchymal stem cells migrated to the cortex, corpus callosum and hippocampus on the normal side and the damaged side, and human neural stem cells migrated to the damaged cortex, corpus callosum and hippocampus, and human umbilical cord mesenchymal stem cells migrated more than human neural stem cells. (4) Overall, these findings indicate that 24 hours after the nasal transplantation, human umbilical cord mesenchymal stem cells could survive and migrate to the normal side and the injury side, and human neural stem cells could survive and migrate to the injury side; and the migration of human umbilical cord mesenchymal stem cells was more extensive than that of human neural stem cells.
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BACKGROUND: Transplanting islet-like cells induced by human umbilical cord mesenchymal stem cells (hUC-MSCs) into type 1 diabetic mice can reduce blood glucose level and improve the symptoms of diabetes mellitus. However, there are few reports on intraperitoneal transplantation. OBJECTIVE: To study the therapeutic effect of transplantation of islet-like cells induced by hUC-MSCs in different ways for the treatment of type 1 diabetic mice. METHODS: The hUC-MSCs were isolated and cultured by tissue explants adherent method and differentiated into islet-like cells. The 3 of 15 male C57BL/6J mice were used as normal group, and the remaining mice were taken to prepare a mouse model of type 1 diabetes using intraperitoneal injection of streptozotocin. After successful modeling, nine model mice were randomly divided into diabetes group, tail vein-islet-like cells group, and abdomen-islet-like cells group, with three mice in each group. After 10 days of modeling, the normal group and diabetic group were not treated. The tail vein-islet-like cells group was injected with 5×105 cells/0.4 mL islet-like cells via the tail vein and the abdomen-islet-like cells group was intraperitoneally injected with 5×105 cells/0.4 mL islet-like cells. During the treatment, the blood glucose and insulin levels were measured twice a week; glucose tolerance test was performed at 28 days after cell transplantation; and fasting insulin level was detected at 42 days after cell transplantation. RESULTS AND CONCLUSION: (1) Compared with the diabetic group, in the tail vein-islet-like cells group, the blood glucose level began to decrease on the 10th day after transplantation and maintained until the 31st day, and the insulin level and glucose tolerance significantly improved (P < 0.05). However, there was no significant improvement in blood glucose level, insulin level and glucose tolerance in the abdomen-islet-like cells group. (2) To conclude, transplantation of hUC-MSCs induced islet-like cells for the treatment of type 1 diabetic mice via tail vein is an ideal transplantation method, and the effect of intraperitoneal injection is unsatisfactory.
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Objective:To establish a three-level clinical grade human umbilical cord mesenchymal stem cells (hUC-MSCs) bank, including seed cell bank (SCB), master cell bank (MCB) and working cell bank (WCB), and provide hUC-MSCs with controllable quality for clinical research and application.Methods:247 human umbilical cord tissues were isolated, cultured, amplified, subcultured and frozen in GMP laboratory, and the biological characteristics, safety and stability of hUC-MSCs were tested in accordance with the requirements of relevant quality management control specifications.Results:247 strains of hUC-MSCs were isolated and prepared. The prepared hUC-MSCs have good purity and homogeneity without tumorigenicity, show good differentiation ability in biological efficacy, and have strong immunosuppressive effect in the process of co-culture with immune cells. These cells have passed the quality check of National Institute for Food and Drug Control. In this study, a three-level hUC-MSCs bank was established, and it was included into the National Stem Cell Translational Resource Center.Conclusions:A three-level clinical hUC-MSCs bank was successfully established and preliminarily applied to clinical research, which effectively promoted the standardized development of clinical stem cell resource bank and the clinical transformation and application of stem cells in China.
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Objective To explore the mechanism of human umbilical cord mesenchymal stem cell (HUC-MSC) alleviating ischemia-reperfusion injury (IRI) of liver cells through mitochondrial transfer. Methods Normal human liver cell line L02 was divided into the blank control group, oxygen-glucose deprivation (OGD) group, experimental control group, and L02 and HUC-MSC co-culture group (L02+HUC-MSC group). L02+HUC-MSC group was further divided into 10:1 co-culture subgroup (group A), 4:1 co-culture subgroup (group B), 2:1 co-culture subgroup (group C), 1:1co-culture subgroup (group D) and 1:2 co-culture subgroup (group E) according to different co-culture ratio of L02 and HUC-MSC. The apoptosis rate and relative reactive oxygen species (ROS) level of L02 cells were detected by flow cytometry. The MitoTracker positive rate of L02 cells was detected by flow cytometry. The mitochondrial transfer from HUC-MSC to L02 cells was observed by laser confocal microscope. Results The apoptosis rate and relative ROS level of L02 cells in the OGD group were significantly higher than those in the blank control group (both P < 0.05). Compared with the OGD group, the apoptosis rates of L02 cells in group B, C, D and E were significantly decreased (all P < 0.05), and the relative ROS level of L02 cells in group E was significantly declined (P < 0.05). The MitoTracker positive rate of L02 cells did not significantly differ between group A and experimental control group (P>0.05), whereas the MitoTracker positive rates of L02 cells in group B, C, D and E were significantly higher than that in the experimental control group in a concentration-dependent manner (all P < 0.05). Under the laser confocal microscope, mitochondrial transfer fromHUC-MSC to L02 cells could be observed through tunneling nanotube (TNT). Conclusions HUC-MSC may alleviate cell apoptosis and reduce ROS level of liver cells after IRI via direct mitochondrial transfer between cells.
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Objective:To investigate the long-term effect of the implantation of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) for type 1 diabetes mellitus.Methods:Fifteen patients with type 1 diabetes mellitus were treated with HUC-MSCs from September 2009 to December 2011 at Department of Endocrinology and Metabolism, the Second People′s Hospital. Patients were followed-up for 10 years and the parameters were collected including fasting blood glucose, HbA 1C, mean amplitude of glycemic excursions (MAGE), fasting C-peptide, daily insulin doses and glutamic acid decarboxylase antibody (GADA). Results:Among 15 patients, 1 patient (6.67%) was found with breast cancer. All patients with type 1 diabetes mellitus decreased daily insulin doses due to frequent hypoglycemia one week later. Six months later, 4 patients (26.67%) stopped insulin injection. While among the 4 patients, 1 patient (6.67%) had not yet used insulin until today and GADA was negative, the other 3 patients (20.00%) restarted insulin within 3-5 years after implantation with significantly less daily insulin doses [(18.00±1.00)U vs (29.00±1.73)U, P<0.01]. The remaining 11 patients (73.33%) with type 1 diabetes mellitus who did not stop insulin also had significantly lower daily insulin doses [(18.09±0.83)U vs (29.64±0.89)U, P<0.01]. The level of MAGE was signicantly decreased compared to those of pre-implantation [(6.14±0.25)mmol/L vs (9.72±0.32)mmol/L, P <0.01], while fasting C-peptide level was significantly improved[(0.91±0.03)nmol/L vs (0.11±0.01)nmol/L, P <0.01]. There were no significant differences in fasting blood glucose and HbA 1C before and after implantation. Conclusions:The implantation of HUC-MSCs for the treatment of type 1 diabetes mellitus can restore the function of islet β cells, decrease daily insulin doses and reduce blood glucose fluctuations in the long term. Although precise mechanisms are unknown, this therapy is expected to be an effective strategy for treatment of type 1 diabetes mellitus.
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At present, surgical and endoscopic interventions are mainly employed to treat ischemic-type biliary lesion (ITBL). Due to the disadvantages of single therapeutic strategy, high difficulty and expensive medical cost, it is urgent to identify a novel treatment option. Mesenchymal stem cell (MSC) has become potential seed cell for tissue and organ repair in regenerative medicine due to its high self-renewal capability, multi-directional differentiation potential, low immunogenicity and immunoregulatory effects, etc. Recent studies have demonstrated that MSC transplantation into ITBL animal models may not only home to the injured area, but also promote the repair of injured biliary tract tissues through anti-apoptotic and pro-angiogenic effect, which indicates that MSC transplantation is expected to become a new strategy for the treatment of ITBL. In this article, the biological characteristics of MSC, the mechanism and clinical application of MSC transplantation for ITBL were reviewed.
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【Objective】 To investigate the effect of Polymerized human cord hemoglobin (PolyCHb) on the sensitivity of hepatocellular carcinoma grafts to lumvalatinib in nude mice. 【Methods】 Hep3B hepatoma cells were subcutaneously transplanted in 18 nude mice to establish tumor graft model. Mice were randomly divided into 3 groups: control group (the saline 90 mg·kg-1·d-1), monotherapy group (Lenvatinib10 mg·kg-1·d-1), and sensitized group (Lenvatinib mg·kg-1·d-1, polyCHB 600 mg/kg twice a week) for 28 days. The tumor volume was measured regularly and the growth curve was drawn. On day 29, the nude mice were sacrificed, the tumor was stripped and weighed, and the pathomorphological differences of each group were evaluated by HE section staining. The expression levels of hypoxia-inducing factor (HIF-1α), CD34, VEGF, CD44, MMP-9, and Glut-1 in tumor tissues of each group were determined by immunohistochemistry. The content of reactive oxygen species (ROS) in tumor tissues of each group was determined by dihydroethyl ingot method. 【Results】 The tumor growth rate and tumor volume in the sensitized group decreased significantly compared with the control group and the solo drug group. On day 29, the tumor volumes of the control group, the monotherapy group and the sensitization group were (2 076.46±350.25)mm3, (1 035.96±84.16)mm3 and (892.66±104.46)mm3, respectively. Tumor weight was (1.61±0.52)g, (0.45±0.10)g, and (0.34±0.13)g, respectively. Immunohistochemical score of HIF-1α was 75±23 vs 45±18 vs 18±11, VEGF was 52±8 vs 67±16 vs 35±4, CD34 was 40±7 vs 50±13 vs 28±7, CD44 was 37±15 vs 30±7 vs 15±3, Glut-1 was 74±41 vs 51±30 vs 14±18, MMP-9 was 51±7 vs 62±20 vs 33±3, respectively(P<0.05). The malignant degree of the sensitized group was decreased by HE section staining, which was significantly lower than that of the solo drug group and the control. The ROS content in the sensitized group was higher than that in the solo drug group and the control. 【Conclusion】 PolyCHb can reduce the expression of HIF-1α and its downstream pathway related molecules by increasing oxygenation of hepatocellular carcinoma tissues in nude mice, delay tumor growth and reduce tumor volume in a certain period, thus increase the therapeutic effect of lenvalatinib on hepatocellular carcinoma grafts in tumor bearing nude mice models.
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Objective To study the effect of human umbilical cord mesenchymal stem cells (hUCMSCs) tail vein transplantation on depressive behavior in chronic unpredictable mild stress (CUMS) model mice. Methods The third generation hUCMSCs were abtained; Sixty C57BL/6 male mice were randomly divided into the normal model group (+ normal saline), the cell group (+hUCMSCs) and the fluoxetine(flu) group (+flu), with 15 mice in each group. The depression model of CUMS mice with a 6 week duration was constructed; From the third week, hUCMSCs and normal saline were transplanted into the mice by tail vein injection, and the mice were gavaged with flu daily from the fifth week; Weight changes in each group were recorded weekly; The depression model and therapeutic effect of mice were evaluated by sucrose preference test, tail suspension test, forced swimming test and open field test; The cell changes of CAI and CA3 region in hippocampus were observed by HE staining; Transmission electron microscopy was used to detect the changes of synapses in CA3 region of hippocampus; Real-time PCR was used to detect mRNA expressions of synaphysin (SYP) and postsynaptic density protein 95 (PSD-95), which are key proteins of synaptic plasticity. Results hUCMSCs improved weight decrease in depressed mice; behavioral experiments observed that the model group mice showed depressive behavior, while the cell group and the drug group mice depression were improved; HE staining showed that, compared with the model group, cells in CA 1 and CA3 regions of the hippocampus of mice in the cell group and the group were arranged orderly and the number of cells increased; Transmission electron microscopy showed that compared with the model group, the number of synapses in the CA3 region of the hippocampus was more, the synaptic gap was narrower, and the density of synaptic spines was higher in the cell group and the drug group; More mRNA expressions of SYP and PSD-95 were detected by Real-time PCR in the cell group and the drug group than in the model group. Conclusion hUCMSCs transplatation showes antidepressant effects, which are associated with improved synaptic plasticity in the hippocampus.
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BACKGROUND: Vascular injury is a common complication after balloon dilatation. The development of umbilical cord mesenchymal stem cells (UC-MSCs) provides a new method for treating vascular injury. OBJECTIVE: To investigate the mechanism underlying the repair of damaged blood vessels by human UC-MSCs (hUC-MSCs) transfected with interleukin-8RA/B (IL-8RA/B) adenovirus. METHODS: hUC-MSCs and human umbilical vein endothelial cells (hUVECs) were collected and transfected with adenovirus vectors containing human IL-8RA and/or IL-8RB cDNAs and green fluorescent protein. A rat model of carotid artery injury was established. Sprague-Dawley rats were randomly divided into four groups: IL-8RA/B-hUCMSCs group, Il-8ra/B-hUVECs group, Null-hUCMSCs group, and control group, followed by injection of 0.5×106 corresponding cells (500 μL) and same volume of normal saline via the tail vein respectively at 1, 3, and 5 hours post-surgery. After 30 minutes of injection, the carotid artery was taken and the expression of green fluorescent protein was observed. After 24 hours, the serum levels of inflammatory and anti-inflammatory factors were measured by ELISA; and the infiltration of neutrophil cells and mononuclear macrophages was observed by immunohistochemistry. After 14 days, Evans blue staining was used to observe vascular endothelialization and fibrosis. After 28 days, the neointimal hyperplasia was observed by hematoxylin-eosin staining. RESULTS AND CONCLUSION: (1) After 30 minutes of IL-8RA/B-hUC-MSCs infusion, the expression of green fluorescent protein was observed in the injured vascular intima, and the fluorescence expression was higher than that of the other three groups. (2) After 24 hours of IL-8RA/B-hUC-MSCs infusion, the expression of inflammatory factors in the serum was significantly lower than that of the other three groups, while the expression of anti-inflammatory factor interleukin-10 was higher than that of the other three groups (P < 0.05). In addition, inflammatory cell infiltration in the IL-8RA/B-hUC-MSCs group decreased significantly. (3) hUC-MSCs overexpressing interleukin-8 receptor promoted re-endothelialization of injured vessels and reduced vascular fibrosis after 14 days of infusion. (4) IL-8RA/B-hUC-MSCs reduced vascular neointimal hyperplasia after 28 days of infusion. (5) Interleukin-8 receptor enhances the targeted homing ability of hUC-MSCs, allowing MSCs to migrate to the site of vascular injury, inhibit inflammation, reduce neointimal hyperplasia, and promote vascular repair.
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BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) have been drawing a great attention due to their potential therapeutic effect in a variety of diseases, including immune-mediated diseases. Further characterization of the immunomodulatory properties and action pathways of hUC-MSCs is necessary to ensure their safety and effectiveness in clinical application. OBJECTIVE: To investigate the immunomodulatory properties of hUC-MSCs. METHODS: HUC-MSCs were directly co-cultured with CFSE-labeled peripheral blood mononuclear cells (PBMCs) at the ratio of 1:5, 1:10, and 1:20, or indirectly co-cultured with CFSE-labeled PBMCs at the ratio of 1:5 via the Transwell co-culture system. Phytohemagglutinin- stimulated PBMC proliferation and the percentages of Th1, Th17 and Treg subgroups in the CD4+ T cells were determined by flow cytometry. The levels of tumor necrosis factor α and interferon γ were determined by ELISA. RESULTS AND CONCLUSION: After direct co-culture, hUC-MSCs significantly inhibited the phytohemagglutinin-stimulated PBMCs proliferation in a dose-dependent manner, whereas the inhibitory effect disappeared in the Transwell co-culture system. A significant decrease of Th1, Th17 cells and an increase of Treg cells were detected in the PBMCs co-cultured with hUC-MSCs compared to the PBMCs cultured alone. Furthermore, hUC-MSCs co-culture significantly reduced tumor necrosis factor α and interferon γ levels in the PBMCs. These findings indicate that cell-to-cell contact is essential for hUC-MSCs to inhibit the proliferation, differentiation and inflammatory factor secretion of immune cells.
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BACKGROUND: TDP43 may be a negative regulator of MAPK signaling pathway under hypoxic-ischemic conditions. However, its effect on JNK and p38 MAPK signaling pathways in osteoarthritis remains unclear.OBJECTIVE: To investigate the expression of chondrocyte lesion-related gene RACK1 in wild type TDP43 involved in osteoarthritis, and to analyze its stress effect. METHODS: Human umbilical cord mesenchymal stem cells were transfected by TDP43 lentivirus, and the ability to differentiate into chondrocytes in vitro was analyzed. Umbilical cord mesenchymal stem cells transfected by TDP43 lentivirus, empty vector and without transfection were co-cultured with chondrocytes for 12 days. The chondrocyte proliferation was detected at 0, 3, 6, 9 and 12 days of co-culture. The chondrocyte apoptosis rate was detected by flow cytometry at 3 days of co-culture. The expression levels of TDP43, RACK1, p38, JNK, AP-1 and cl-xl in chondrocytes were detected by qRT-PCR at 3 days of co-culture. RESULTS AND CONCLUSION: (1) After TDP43 lentivirus transfection, human umbilical cord mesenchymal stem cells could differentiate into chondrocytes. (2) The morphology of chondrocytes co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells showed significant change, and the cells became large with abundant branches. Chondrocytes co-cultured with empty vector transfected- or non-transfected umbilical cord mesenchymal stem cells were spindle-shaped in appearance and showed adherent growth with no morphological changes. (3) After co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells, the apoptosis of chondrocytes was promoted, and the cell proliferation was inhibited (P < 0.05). (4) The expression levels of TDP43, RACK1, p38, JNK, AP-1 and Bcl-xl in the chondrocytes co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells were significantly higher than those in the chondrocytes co-cultured with non-transfected- and empty vector-transfected-umbilical cord mesenchymal stem cells. (5) To conclude, high expression of TDP43 in chondrocytes can activate the expression of RACK1, and further regulate chondrocyte proliferation and apoptosis.
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BACKGROUND: Mesenchymal stem cells have been widely applied in autoimmune diseases and graft-versus-host diseases because of their immunomodulatory capabilities. However, mesenchymal stem cells have plasticity in immunomodulation, which leads to heterogeneity and instability when used in vivo. OBJECTIVE: To investigate the polarization of human umbilical cord derived mesenchymal stem cells to an immunosuppressive phenotype (MSC2) in the inflammatory microenvironment induced by interferon-γ and lipopolysaccharide. METHODS: Human umbilical cord-derived mesenchymal stem cells were isolated and cultured in vitro, and then were identified by morphological characteristics, surface markers, adipogenesis and osteoinduction activity. Human umbilical cord-derived mesenchymal stem cells were treated with interferon-γ (10 μg/L), lipopolysaccharide (100 μg/L), or their combination for 24 hours, respectively, and were then co-cultured with phytohemagglutinin pre-treated peripheral blood mononuclear cells for 5 days under direct or Transwell indirect contact. The percentages of regulatory T cells and T helper 1 cells were analyzed by flow cytometry at different times. The mRNA expression levels of Toll-like receptors 2, 3 and 4 were detected by real-time fluorescence quantitative PCR. RESULTS AND CONCLUSION: (1) Human umbilical cord derived mesenchymal stem cells exhibited spindle-shaped or fibroblast-like morphology, highly expressed CD73, CD90 and CD105, and lacked expression of CD34, CD45 and HLA-DR. (2) Under direct or indirect co-culture, human umbilical cord-derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide could promote the generation of regulatory T cells, which was superior to the interferon-γ, lipopolysaccharid, un-treated and control groups (P < 0.05). (3) The percentage of T helper 1 cells gradually decreased over time. (4) Under indirect co-culture, human umbilical cord derived mesenchymal stem cells pre-treated with interferon-γ and lipopolysaccharide were polarized into immunosuppressive MSC2 phenotype at an earlier period and highly expressed Toll-like receptor 3 (P < 0.05). (5) In conclusion, the combination of interferon-γ (10 μg/L) and lipopolysaccharide (100 μg/L) results in the high-efficient polarization of mesenchymal stem cells toward the MSC2 phenotype under indirect co-culture, and the immunosuppressive capability of MSC2 is independent of intercellular contact, which provides clinical evidence for the MSC2-derived exosome therapy in the future.
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BACKGROUND: A large number of studies mainly concern the proliferation effect of mesenchymal stem cells on hematopoietic stem cells in vitro and that bone marrow mesenchymal stem cell transplantation can reduce the death of hematopoietic cells caused by irradiation, increase the survival of bone marrow cells and repair hematopoiesis, while few of them investigate the repair of human umbilical cord blood mesenchymal stem cells transplantation on bone marrow hematopoiesis injury. OBJECTIVE: To explore the repair of hematopoietic microenvironment of bone marrow by human umbilical cord blood mesenchymal stem cells. METHODS: Male BALB/c mice were randomly divided into three groups. The mice in experimental group and control group were irradiated with total dose of 6-Gy X-ray to establish a mouse model of bone marrow hematopoietic injury. The normal group contained untreated normal mice. In the experimental group, CM-DiL labeled human umbilical cord blood mesenchymal stem cells were injected into the tail vein of each mouse at 5×106 (0.2 mL). The control group and the normal group received normal saline 0.2 mL through the tail vein. The peripheral blood hematology and bone marrow hematopoietic microenvironment repair were observed at 1, 5, 7, 14 and 21 days after cell transplantation. RESULTS AND CONCLUSION: Peripheral blood condition: At 1, 5 and 7 days after transplantation, the leucocyte, platelet, erythrocyte count and hemoglobin concentration in the experimental group and control group decreased progressively compared with the normal group. The most obvious decrease occurred on day 7. The trilineage recovered on day 14 after transplantation, basically returned to normal on day 21 after transplantation. Compared with the experimental group, the decrease of the trilineage in the control group was more obvious. The recovery was obvious faster in the experimental group than in the control group on day 14 after transplantation. Bone marrow smears: Bone marrow smears showed that the hematopoietic function was inhibited in the experimental group and the control group at 1, 5, 7, and 14 days after transplantation, especially on day 7. Bone marrow proliferation recovered on day 14 after transplantation. It was better in the experimental group than in the control group. On day 21 after transplantation, the hematopoietic function of bone marrow of mice in the experimental group and the control group recovered, and there was no difference between the experimental group and the control group compared with the normal group. Bone marrow pathological section: Bone marrow pathological sections showed that at 1, 5, 7, and 14 days after transplantation, the hematopoietic function of bone marrow in the experimental group and the control group was inhibited. On day 14 after transplantation, the bone marrow hematopoietic function of the experimental group and the control group began to recover, but the bone marrow proliferation of the experimental group was better than that of the control group. On day 21 after transplantation, there was no difference in the bone marrow proliferation between the experimental and the control groups and the normal group. The results suggested that human umbilical cord blood mesenchymal stem cells can promote the recovery of hematopoietic function of bone marrow.