ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.
Objective To explore the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and the combination of bFGF and EGF in the neural differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and the role of Wnt/β-catenin signaling pathway in this process. MethodsThe identified 4th-generation hBMSCs were divided into five groups according to different induction conditions, namely control group (group A), EGF induction group (group B), bFGF induction group (group C), EGF and bFGF combined induction group (group D), and EGF, bFGF, and Dickkopf-related protein 1 (DKK-1) combined induction group (group E). After 7 days of continuous induction, the cell morphology was observed by inverted fluorescence phase contrast microscopy, levels of genes that were related to neural cells [Nestin, neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)] and key components of the Wnt/β-catenin signaling pathway (β-catenin and Cyclin D1) were detected by RT-PCR, and the levels of proteins that were related to neural cells (Nestin and GFAP) as well as genes that were involved in Wnt/β-catenin signaling pathway [β-catenin, phosphorylation β-catenin (P-β-catenin), Cytoplasmic β-catenin, and Nuclear β-catenin] were explored by cellular immunofluorescence staining and Western blot. ResultsWhen compared to groups A and B, the typical neuro-like cell changes were observed in groups C-E, and most obviously in group D. RT-PCR showed that the relative expressions of Nestin, NSE, and MAP-2 genes in groups C-E, the relative expressions of GFAP gene in groups D and E, the relative expression of NSE gene in group B, the relative expressions of β-catenin gene in groups C and D, and the relative expressions of Cyclin D1 gene in groups B-D significantly increased when compared with group A (P<0.05). Compared with group E, the relative expressions of Nestin, NSE, MAP-2, GFAP, β-catenin, and CyclinD1 genes significantly increased in group D (P<0.05); compared with group C, the relative expression of Nestin gene in group D significantly decreased (P<0.05), while NSE, MAP-2, and GFAP genes significantly increased (P<0.05). The cellular immunofluorescence staining showed that the ratio of NSE- and GFAP-positive cells significantly increased in groups C-E than in group A, in group D than in groups C and E (P<0.05). Western blot assay showed that the relative expression of NSE protein was significantly higher in groups C and D than in group A and in group D than in groups C and E (P<0.05). In addition, the relative expression of GFAP protein was significantly higher in groups C-E than in group A and in group D than in group E (P<0.05). Besides, the relative expressions of β-catenin, Cytoplasmic β-catenin, Nuclear β-catenin, and the ratio of Nuclear β-catenin to Cytoplasmic β-catenin were significantly higher in groups C and D than in group A and in group D than in group E (P<0.05), whereas the relative expression of P-β-catenin protein was significantly lower in groups C and D than in group A and in group D than in group E (P<0.05). Conclusion Different from EGF, bFGF can induce neural differentiation of hBMSCs. In addition, EGF can enhance the hBMSCs neural differentiation of bFGF, while the Wnt/β-catenin signaling pathway may play a positive regulatory role in these processes.
ObjectiveTo investigate whether transplantation of islet cells combined with bone marrow mesenchymal stem cell (BMSCs) of the pancreatic subcapsular promoting revascularization of pancreatic islets in rats, so as to reduce the loss of islet cells after transplantation and improve the success rate of islet cell transplantation. MethodsThe model of diabetic rat was established. The BMSCs and islet cells were cultured and identified, then the simple islet cells, simple BMSCs, and combination of islet cells and BMSCs were injected into the pancreatic subcapsular of the islet cell group, BMSCs group, and combination group, respectively. In addition, the same amount of normal saline was injected into the same site as the control group. There were 10 rats in each group. The changes of blood glucose and serum insulin in different time point were detected in each group. The mRNA expressions of angiogenesis factors such as hypoxia inducible factor-1α (HIF-1α), stromal cell derived factor 1α (SDF1α), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor 2 (FGF2) were determined by real-time quantitative PCR. Results① The blood glucose levels of the islet cell group and combination group were lower than those of the control group and the BMSCs group within 15 d after surgery (P<0.05) and decreased to the normal level, which of the combination group could still maintain the normal level until on day 29 (P<0.05), but which of the islet cell group began to increase on day 15 after surgery and was similar to that in the BMSCs group (P>0.05). ② Compared with the control group and the BMSCs group, the insulin levels were higher in the islet cell group and combination group on day 1, 3, 7, 15, and 29 after surgery (P<0.05), especially in the combination group. ③ The expression levels of HIF-1α, SDF1α, VEGF, and FGF2 mRNAs in the combination group were higher than those the other three groups, and the differences were statistically significant (P<0.05). ConclusionsIslet cell transplantation of pancreatic subcapsular could decrease blood glucose level in diabetic rats. Hypoglycemic effect of single islet cell transplantation gradually weakens on day 15 d after surgery. After BMSCs combined with islet cells transplantation, the glycemic effect of rat is stable for a longer time. Expressions of angiogenesis factors of BMSCs combined with islet cells transplantation rat are high, which combined with pathological sections suggests that BMSCs could promote vascular recanalization of islet transplantation.
ObjectiveTo investigate whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
Objective To investigate effect of bone marrow mesenchymal stem cells (BMSCs) via portal vein injection on transforming growth factor-β receptor 1 (TGF-βR1) and TGF-βR2 in rats with acute liver failure (ALF). Methods Sixty male SD rats were randomly divided into a normal control group, ALF model group, and BMSCs treatment group, with 20 rats in each group. The rats of normal control group were directly sacrificed without other treatment. The ALF models were made in the rats of BMSCs treatment group and ALF model group, then were treated with BMSCs and equal volume of normal saline respectively. On day 7 after treatment, the 1-week survival situation of rats was observed, the pathological change was observed by HE staining, the apoptosis of liver cells was detected by TUNEL method, and the TGF-βR1 and TGF-βR2 proteins expressions were detected by Western blot method. Results ① The 1-week survival rate of the BMSCs treatment group was significantly higher than that of the ALF model group (P<0.05). ② In the ALF model group, the liver cells were diffuse necrosis, the lobular structure was indistinct, and a large number of bridging necrosis. In the BMSCs treatment group, the infiltrations of inflammatory cells were decreased, and the structure of hepatic lobules gradually recovered, and the normal hepatocytes were seen around it. ③ The apoptosis indexes of the BMSCs treatment group and the ALF model group were significantly higher than those in the normal control group (P<0.05), which in the BMSCs treatment group was significantly lower than that of the ALF model group (P<0.05). ④ The TGF-βR1 and TGF-βR2 proteins expressions in the liver tissues of the ALF model group were significantly higher than those of the normal control group (P<0.05), which of the BMSCs treatment group were significantly lower than those of the ALF model group (P<0.05). Conclusion BMSCs could inhibit apoptosis of hepatocytes in ALF. Its mechanism might be related to expressions of TGF-βR1 and TGF-βR1 proteins, but its specific regulatory pathway needs to be further studied.
Objective To compare the cl inical outcomes of the core decompression combined with autologous bone marrow mesenchymal stem cells (BMSCs) transplantation with the isolated core decompression for the treatment of earlyavascular necrosis of the femoral head (ANFH). Methods From May 2006 to October 2008, 8 patients (16 hips) with earlyANFH were treated. There were 7 males and 1 female with an average age of 35.7 years (range, 19-43 years). According to the system of the Association Research Circulation Osseous (ARCO): 4 hips were classified as stage II a, 2 as stage II b, 1 as stage II c, and 1 as stage III a in group A; 2 hips were classified as stage II a, 2 as stage II b, 3 as stage II c, and 1 as stage III a in group B. The average disease course was 1.1 years (range, 4 months to 2 years). The patients were randomly divided into 2 groups according to left or right side: group A, only the core decompression was used; group B, both the core decompression and autologous BMSCs transplantation were used. The Harris score and visual analogue scale (VAS) score were determined, imaging evaluation was carried out by X-rays and MRI pre- and post-operatively. The erythrocyte sedimentation rate, C-reactive protein, l iver function, renal function, and immunoglobul in were detected for safety evaluation. Results All incisions healed by first intention. Eight patients were followed up 12-42 months (23.5 months on average). The cl inical symptoms of pain and claudication were gradually improved. The Harris scores and VAS scores of all patients were increased significantly at 3, 6, and 12 months after operation (P lt; 0.05). There was no significant difference between groups A and B 3 and 6 months after operation (P gt; 0.05), but there was significant difference between groups A and B 12 months after operation (P lt; 0.05). The necrosis area of femoral head in groups A and B were 18.13% ± 2.59% and 13.25% ± 2.12%, respectively, showing significant difference (P lt; 0.05). In group A, femoral head collapsed 12 months after operation in 1 case of stage III. No compl ication of fever, local infectionoccurred. Conclusion The core decompression and the core decompression combined with BMSCs transplantation are both effective for the treatment of early ANFH. The core decompression combined with BMSCs transplantation is better than core decompression in the rel ief of pain and postponing head collapse.
ObjectiveTo prepare dopamine modified and cartilage derived morphogenetic protein 1 (CDMP1) laden polycaprolactone-hydroxyapatite (PCL-HA) composite scaffolds by three-dimensional (3D) printing and evaluate the effect of 3D scaffolds on in vitro chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsA dimensional porous PCL-HA scaffold was fabricated by 3D printing. Dopamine was used to modify the surface of PCL-HA and then CDMP-1 was loaded into scaffolds. The surface microstructure was observed by scanning electron microscope (SEM) and porosity and water static contact angle were also detected. The cytological experiment in vitro were randomly divided into 3 groups: group A (PCL-HA scaffolds), group B (dopamine modified PCL-HA scaffolds), and group C (dopamine modified and CDMP-1 laden PCL-HA scaffolds). The hBMSCs were seeded into three scaffolds, in chondrogenic culture conditions, the cell adhesive rate, the cell proliferation (MTT assay), and cell activity (Live-Dead staining) were analyzed; and the gene expressions of collagen type Ⅱ and Aggrecan were detected by real-time fluorescent quantitative PCR.ResultsThe scaffolds in 3 groups were all showed a cross-linked and pore interconnected with pore size of 400–500 μm, porosity of 56%, and fiber orientation of 0°/90°. For dopamine modification, the scaffolds in groups B and C were dark brown while in group A was white. Similarly, water static contact angle was from 76° of group A to 0° of groups B and C. After cultured for 24 hours, the cell adhesion rate of groups A, B, and C was 34.3%±3.5%, 48.3%±1.5%, and 57.4%±2.5% respectively, showing significant differences between groups (P<0.05). Live/Dead staining showed good cell activity of cells in 3 groups. MTT test showed that hBMSCs proliferated well in 3 groups and the absorbance (A) value was increased with time. The A value in group C was significantly higher than that in groups B and A, and in group B than in group A after cultured for 4, 7, 14, and 21 days, all showing significant differences (P<0.05). The mRNA relative expression of collagen type Ⅱ and Aggrecan increased gradually with time in 3 groups. The mRNA relative expression of collagen type Ⅱafter cultured for 7, 14, and 21 days, and the mRNA relative expression of Aggrecan after cultured for 14 and 21 days in group C were significantly higher than those in groups A and B, and in group B than in group A, all showing significant differences (P<0.05).ConclusionCo-culture of dopamine modified and CDMP1 laden PCL-HA scaffolds and hBMSCs in vitro can promote hBMSCs’ adhesion, proliferation, and chondrogenic differentiation.
The regulation of epigenetics on bone marrow mesenchymal stem cells (BMSCs) has been a research hot spot in medical area. This paper mainly summarizes the progress of the regulation of DNA methylation, histone acetylation, small interfering RNA (siRNA) induced gene silence and microRNA (miRNA) on BMSCs. Our analysis shows that the regulation of epigenetics on BMSCs plays a significant role in the repair of bone tissue, nervous tissue and cardiac muscle.
Seeding cells play an important role in the peripheral nerve damage repair. Seeding cells studied consequently in peripheral nerve are Schwann cells, bone marrow mesenchymal stem cells and neural stem cells. Schwann cells, the first seeding cells, are various unique glial cells in the peripheral nervous system, which can form the myelin sheath for insulation and package of the neuron projecting axons in the peripheral nervous system so that the conduction velocity of the nerve signal was accelerated. It can be proved that Schwann cells played an important role in the maintenance of peripheral nerve function and in the regeneration process after peripheral nerve injury. The second, bone marrow mesenchymal stem cells are the various mesenchymal stem cells mainly exist in the systemic connective tissues and organs. These functional stem cells are often studied at present, and it has been found that they have exuberant proliferation and differentiation potentials. Neural stem cells, mentioned the third in sequence, are the kind of pluripotent cells with multi-directional differentiation, which could conduct the self-renewal function, and generate and differentiate neurons, astrocytes and oligodendrocytes through asymmetric cell division. These three types of seed cells are discussed in this paper.
ObjectiveTo investigate the effect of granulocyte colony-stimulating factor (G-CSF) mobilizing the bone marrow mesenchymal stem cells (BMSCs) homing to the spinal cord injury sites in rats, and to evaluate the feasibility of G-CSF mobilizing the BMSCs home to the injured spinal cord. MethodsTwenty-four healthy adult female Sprague Dawley rats were injected with 1 mL green fluorescence protein labeled BMSCs (GFP-BMSCs, 1×106 cells/mL) into tail vein at 12 hours before operation. They were randomly divided into sham operation group (group A), sham operation+G-CSF group (group B), spinal cord injury group (group C), and spinal cord injury+G-CSF group (group D), with 6 rats in each group. In groups C and D, spinal cord injury model was established by T10 level spinal cord hemisection. In groups A and B, only laminectomy was performed without injury to the spinal cord. Groups B and D were injected with G-CSF (10 μg/kg·d) at 1 hour after operation for 3 consecutive days, and groups A and C were injected with the same amount of saline. The Basso-Beattie-Bresnahan (BBB) score was used to estimate the neurological function of rats and the expressions of tumor necrosis factor α (TNF-α) and stromal-derived factor 1 (SDF-1) were detected by ELISA method at 1, 3, 7, 14, 21, and 28 days after operation. The spinal cord samples of rats were sacrificed at 28 days after operation for immunohistochemical staining to observe the expression of cytokines, including SDF-1, brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and TNF-α, and immunofluorescence staining to observe GFP-BMSCs positive cells, double-stained fluorescent yellow GFP/neuronal nuclear antigen (NeuN) positive neurons, and GFP/glial fibrillary acidic protein (GFAP) positive neurons. The number of glial cells and apoptosis were detected by TUNEL method. ResultsThe BBB score of groups A and B had no significant change at each time point after operation. At 1 day after operation, the BBB score of groups C and D decreased to the lowest level, and then gradually increased. The BBB score of group D was significantly higher than that of group C at all time points except 1 day after operation (P<0.05). At 3, 7, 14, 21, 28 days after operation, the levels of TNF-α and SDF-1 in groups C and D were significantly higher than those in groups A and B (P<0.05), but the levels of TNF-α in group D were significantly lower than those in group C at each time point, and the levels of SDF-1 were significantly higher than those in group C (P<0.05). Immunohistochemical staining showed that the expressions of SDF-1, BDNF, VEGF, and TNF-α in groups C and D were significantly higher than those in groups A and B (P<0.05); the expressions of SDF-1, BDNF, and VEGF in group D were significantly higher than those in group C, and the expression of TNF-α was significantly lower than that in group C (P<0.05). Immunofluorescence staining showed that the number of GFP-BMSCs, GFP/NeuN, and GFP/GFAP positive cells in groups C and D were significantly higher than those in groups A and B, and in group D than in group C (P<0.05). TUNEL assay showed that the number of apoptotic cells in groups C and D was significantly lower than that in groups A and B, and in group D than in group C (P<0.05). ConclusionG-CSF can mobilize BMSCs to the spinal cord injury site and promote repair effect by down-regulating TNF-α to promote the anti-apoptosis function and up-regulating SDF-1, BDNF, VEGF to promote BMSCs migration.