OBJECTIVETo study the effect of basic fibroblast growth factor (bFGF) on the gene expression of epidermal growth factor receptor (EGFR) by periodontal ligament cells (PDLC) in culture and discuss the effect of bFGF in cell differentiation and periodontal regeneration.

METHODSHuman PDLC were cultured in vitro and cell clone was obtained by the method of limiting dilution. PDLC were stimulated by bFGF, and then gene expression of EGFR was assessed by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe mRNA expression of EGFR was promoted by bFGF and the effect was dose-dependent.

CONCLUSIONThe promotion effect of the mRNA expression of EGFR in PDLC may be one of important factors which participate in the healing process of periodontitis and provide partly theoretical basis of cell differentiation and periodontal regeneration.

Cell Differentiation ; Cells, Cultured ; Fibroblast Growth Factor 2 ; Humans ; Periodontal Ligament ; RNA, Messenger ; Receptor, Epidermal Growth Factor ; Regeneration

OBJECTIVETo explore the effects of basic fibroblast growth factor (bFGF) on human bone marrow mesenchymal stem cell (hBMMSC) damaged by irradiation and its underlying mechanisms.

METHODShBMMSC was irradiated with 0, 6, 12 Gy X ray, then flow cytometry, cell counting kit-8 (CCK-8), Western blot and alizarin red staining were used to detect the effects of X ray on apoptosis, proliferation and osteogenic differentiation of hBMMSC; 0, 1, 5, 10, 20 ng/ml bFGF was added to hBMMSC irradiated with X ray for selecting the suitable bFGF reaction concentration; then the Western blot was used to detect the expression of PDGFRα so as to evaluate whether the expression of PDGFRα participated in bFGF-mediated recovery of hBMMSC proliferation and osteogenic differentiation after irradiation.

RESULTSThe proliferation and osteogenic differentiation of hBMMSC decreased remarkably after irradiation. bFGF promoted the recovery of proliferation and osteogenic differentiation of irradiated hBMMSC compared with untreated irradiated hBMMSC (P < 0.05); 5 ng/ml bFGF was identified as the optimal concentration. A significant difference in the number of apoptotic cells could be detected only between the 0 Gy group and 12 Gy group at the 24 h time point, while no differences were detected at later time points. Irradiated hBMMSC showed remarkable decrease of PDGFRα expression, while the PDGFRα expression increased after bFGF was added.

CONCLUSIONIrradiation dose not show significant effect on apoptosis of hBMMSC, but the bFGF displays a effect on repairing the irradiation damage of hBMMSC and promotes the recovery of hBMMSC proliferation and osteogenic differentiation. The damage of hBMMSC proliferation and osteogenic differentiation associates with downregulation of PDGFRα expression induced by irrediation. PDGFRα involves in repairing effect of bFGF on irradiation damage of hBMMSC.

Apoptosis ; Bone Marrow Cells ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Fibroblast Growth Factor 2 ; Humans ; Mesenchymal Stromal Cells ; Osteogenesis ; Receptor, Platelet-Derived Growth Factor alpha

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. METHODS: The 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. RESULTS: When 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.
Humans ; beta Catenin/metabolism* ; Bone Marrow Cells ; Cell Differentiation ; Cells, Cultured ; Epidermal Growth Factor/metabolism* ; Mesenchymal Stem Cells ; Wnt Signaling Pathway ; Neurons ; Fibroblast Growth Factor 2/metabolism*

BACKGROUND: Elevated serum levels of growth differentiation factor-15 (GDF-15) are associated with type 2 diabetes. Therefore, the effects of atorvastatin on metabolic parameters and GDF-15 levels in patients with type 2 diabetes and dyslipidemia were evaluated. METHODS: In this prospective randomized trial from February 2013 to March 2014, 50 consecutive type 2 diabetic patients with a low density lipoprotein cholesterol (LDL-C) levels > or =100 mg/dL were enrolled. The patients were divided into two groups based on the amount of atorvastatin prescribed, 10 mg/day (n=23) or 40 mg/day (n=27). The effect of atorvastatin on metabolic parameters, including lipid profiles and GDF-15 levels, at baseline and after 8 weeks of treatment were compared. RESULTS: The baseline metabolic parameters and GDF-15 levels were not significantly different between the two groups. After 8 weeks of treatment, the total cholesterol (TC) and LDL-C levels were significantly decreased in both groups. The mean changes in TC and LDL-C levels were more significant in the 40 mg atorvastatin group. The GDF-15 level was decreased in the 10 mg atorvastatin group, from 1,460.6+/-874.8 to 1,451.0+/-770.8 pg/mL, and was increased in the 40 mg atorvastatin group, from 1,271.6+/-801.0 to 1,341.4+/-855.2 pg/mL. However, the change in the GDF-15 level was not statistically significant in the 10 or 40 mg atorvastatin group (P=0.665 and P=0.745, respectively). CONCLUSION: The GDF-15 levels were not significantly changed after an 8-week treatment with atorvastatin in type 2 diabetic patients.
Cholesterol ; Cholesterol, LDL ; Diabetes Mellitus, Type 2* ; Dyslipidemias* ; Growth Differentiation Factor 15 ; Humans ; Prospective Studies ; Atorvastatin Calcium

We investigated an association between serum Growth Differentiation Factor 15 (GDF15) level and cardiovascular risk in patients with newly diagnosed type 2 diabetes mellitus (T2D). A total of 107 participants were screened for T2D and divided into a T2D group and a control group (without diabetes). We used the Framingham risk score (FRS) and the New Pooled Cohort Equation score to estimate the 10-year risk of atherosclerotic cardiovascular disease. Serum GDF15 levels were measured using an enzyme-linked immunosorbent assay. Correlation analyses were performed to evaluate the associations between GDF15 level and cardiovascular risk scores. The mean serum GDF15 level was elevated in the T2D group compared to the control group (P < 0.001). A positive correlation was evident between serum GDF15 level and age (r = 0.418, P = 0.001), the FRS (r = 0.457, P < 0.001), and the Pooled Cohort Equation score (r = 0.539, P < 0.001). After adjusting for age, LDL-C level, and body mass index (BMI), the serum GDF15 level was positively correlated with the FRS and the New Pooled Cohort Equation score. The serum GDF15 level is independently associated with cardiovascular risk scores of newly diagnosed T2D patients. This suggests that the level of GDF15 may be a useful predictive biomarker of cardiovascular risk in newly diagnosed T2D patients.
Body Mass Index ; Cardiovascular Diseases ; Cohort Studies ; Diabetes Mellitus, Type 2* ; Enzyme-Linked Immunosorbent Assay ; Growth Differentiation Factor 15* ; Humans

OBJECTIVE: To understand the differentiation of mesoderm-derived Flk1 cells on different locations of the early mouse embryonic development and to explore the potential of Flk1 cells to differentiate into mesenchymal lineage, like pericytes during vascular development. METHODS: Based on the Cre-LoxP system conditional knockout study strategy, the Flk1-Cre mice and ROSA26 reporter mice were used for lineage-tracing studies. The fate of the Flk1 progenitor cells was traced with the GFP population. The detection of mesoderm marker Flk1, hematopoietic cell-specific marker CD45, endothelial cell-specific markers CD31, CD144, and Emcn (endomucin), pericyte specific markers PDGFRβ and NG2, using the methods of immunohistochemistry, immunofluorescence, and flow cytometry should be combined to solve the concerned problems. RESULTS: Immunohistochemical staining of different fractions of E8.5-10.5 in the early embryogenesis of Flk1-Cre; ROSA26-EYFP mouse lineage showed that there were multiple populations in the Flk1 cell-derived GFP population surrounding hematopoietic sites, such as dorsal aortas, limb buds and yolk sac. In addition to hematopoietic cells, the CD31/Emcn typical endothelial cells distributed specifically along the blood vessel wall, there were many types of cell populations, such as mesenchymal-like cells. The immunofluorescence demonstrated that the cells of this group are neither hematopoietic, non-endothelial cells around the blood vessels, which are NG2+ pericytes. FACS analysis also confirmed that Flk1 cells contributed to pericytes. In addition, in different hematopoietic sites of the embryo, a small population of CD31+CD140B+ cell populations with a mesenchymal-like morphology was observed in the GFP population. CONCLUSION In the early stages of embryogenesis, mesoderm-derived Flk1 populations not only contribute to hematopoietic, endothelial, and muscle lineages, but also have a differentiation potential for mesenchymal lineage, like pericytes. The presumably observed CD31CD140B cell population may be a group of endothelial cells differentiated from Flk1 progenitor cells and undergoing an endothelium-to-mesenchymal transition, EndMT, gradually losing the endothelial surface-specific marker and also starting to express a pericyte surface-specific marker.
Animals ; Cell Differentiation ; Cell Lineage ; Mesoderm ; Mice ; Stem Cells ; Vascular Endothelial Growth Factor Receptor-2 ; Yolk Sac

Objective To determine whether the signaling activation of bone morphogenetic protein 2(BMP2)can induce myeloid-derived suppressor cells(MDSC)to secret transforming growth factor β(TGF-β),further enhancing the differentiation and infiltration of regulatory T lymphocytes(Treg)into tumor tissue. Methods The BMP2-induced mRNA and protein expression of TGF-β in MDSC was detected by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay(ELISA),respectively.The effect of BMP2-induced TGF-β secretion by MDSC on Treg differentiation was then determined by flow cytometry.Finally,we implanted the recombined human bone morphogenetic protein 2(rhBMP2)collagen gels into tumor-burdened mice to examine the role of BMP2 in Treg differentiation via MDSC-secreted TGF-β
Animals ; Bone Morphogenetic Protein 2 ; Cell Differentiation ; Mice ; Myeloid-Derived Suppressor Cells ; Neoplasms ; T-Lymphocytes, Regulatory ; Transforming Growth Factor beta

OBJECTIVETo screen specific small interfering RNA (siRNA) targeting mouse BMP9 gene and identify its function in BNLCL.2 fetal liver cells and C3H10 cells.

METHODSFour pairs of double-stranded DNA fragments for silencing mouse BMP9 were annealed in vitro and cloned into pSOS-BMP9 vector with BMP9 gene to construct pSOS-simBMP9 plasmid. The 4 pSOS-simBMP9 plasmids were separately transfected in HEK293 cells via Lipofectamine, and the gene silencing efficiency was assessed by GFP detection. BNLCL.2 fetal liver cells were infected with the constructed adenovirus simBMP9s, and their BMP9 expression was detected with RT-PCR and Western blotting. C3H10 cells were co-infected with Ad-simBMP9 and Ad-BMP9, and the inhibitory effect on BMP9-induced osteoblasts was evaluated by alkaline phosphatase (ALP) activity.

RESULTSGFP expression in the two simBMP9 groups was significantly decreased in HEK293 cells, and the endogenous expression of BMP9 was reduced by 50%-70% by adenovirus-mediated simBMP9 in the fetal liver cells. ALP activity in C3H10 cells was significantly higher in BMP9 group than in the control group (P<0.01), while the activity of the two Ad-simBMP9-infected groups was significantly lower than that in Ad-BMP9-infected group (P<0.01).

CONCLUSIONTwo specific siRNA targeting mouse BMP9 gene have been obtained, which can effectively inhibit both endogenous and exogenous expressions of BMP9 to facilitate the study of the mechanisms of BMP9 in liver cell differentiation.

Animals ; Cell Differentiation ; Cell Line ; Fetus ; Growth Differentiation Factor 2 ; biosynthesis ; genetics ; Hepatocytes ; metabolism ; Humans ; Mice ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVEWe ascertained the effect of bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) by a series of experiments: Proliferation and differentiation of bone marrow stromal cells (BMSCs) in vitro, ectopic and in situ bone formation and loaded porous calcium phosphate cement (CPC) on the repair of bone defects around dental implants.

METHODSBMSCs from Beagle dogs were cultured in vitro with basic culture medium containing BMP-2, bFGF, and BMP-2+bFGF. Proliferation and differentiation of BMSCs were quantified using methyl thiazolyl tetrazolium (MTT) and alkaline phosphatase (ALP) test. The CPC seeded with BMSCs and BMP-2, bFGF, combined BMP-2 with bFGF were implanted subcutaneously into nude rats in ectopic bone formation, and were implanted into critical-sized bone defects of Beagle dogs in the in situ bone formation. The bone formation was detected by histology examination and quantified using an image analysis system. Polychrome sequential fluorescent labels and fluorescence histological examinations of undecalcified sections were performed post-operatively.

RESULTSIt was determined that BMP-2+bFGF promoted BMSCs statistically significant proliferation and differentiation compared to either BMP-2 or bFGF in vitro. The CPC with BMP-2+bFGF group yielded more bone than those with either BMP-2 or bFGF in ectopic bone formation test. The percentages of newly ectopic formed bone were higher in the BMP-2+bFGF group (48.79% +/- 11.31%) than those in other groups (BMP-2 group, 30.71% +/- 10.85%; bFGF group, 27.33% +/- 9.67%; and the control group, 10.65% +/- 6.05%). Undecalcified showed that new bone was actively formed in the BMP-2+bFGF group after 12 weeks in the in situ bone formation test. The bone mineralization apposition rate (MAR) was better in the BMP-2+bFGF group than in other groups (P<0.01).

CONCLUSIONBMP-2 combined with bFGF are more effective than one alone in promoting the formation of new bone.

Animals ; Bone Marrow Cells ; Bone Morphogenetic Protein 2 ; Bone and Bones ; Calcium Phosphates ; Cell Differentiation ; Cells, Cultured ; Dogs ; Fibroblast Growth Factor 2 ; Mesenchymal Stromal Cells ; Rats

The objective was to explore the feasibility of differentiation of human umbilical cord blood mononuclear cells into endothelial cells induced by cytokines in vitro and to study the possibility of using cord blood stem cells in ischemic diseases therapy. The cells were isolated from umbilical cord blood by using lymphocyte separation solution, and committedly differentiated by using VEGF, bFGF and IGF-I in a liquid culture system. The results showed that the combination of cytokines produced a large number of caudated adherent cells and flow cytometric analysis revealed endothelial marker vWF expressed in about 80% cells, and the endothelial -specific Weibel-Palade body was detected in the cytoplasm by electronic microscope. It is concluded that human umbilical cord blood mononuclear cells may be induced to differentiate into endothelial cells induced by VEGF, bFGF and IGF-I. Human umbilical cord blood MNC may be an ideal source of adult stem cells for the treatment of the ischemic disease.
Cell Differentiation ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; ultrastructure ; Fetal Blood ; cytology ; Fibroblast Growth Factor 2 ; pharmacology ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Leukocytes, Mononuclear ; cytology ; Microscopy, Electron ; Vascular Endothelial Growth Factor A ; pharmacology