Objective To study the effect of two cytokines, basic fibroblast growth factor(bFGF) and insulin-like growth factor-I(IGF-I), on cell proliferation in chondrocytes of adult rabbits. Methods The primary chondrocytes of adult rabbits were harvested and cultured with bFGF and IGF-I at different concentrations,respectively, as well as with the mixture of the two cytokines; the quantity of cultured chondrocytes was detected by MTT assay at the 24th, 48th and 72th hours; and the final fold increase of different groups was measured by cell count for the 3rd passage; and the proliferation index of the groups was recorded by flowing cytometer on the 14th day. Results ① The cultured chondrocytes with either bFGF, IGF-I or their mixture were significantly more than that of control group at the 24th, 48th and 72th hours (P＜0.01). ② After the 3rd passage, the final folds of proliferation were significantly higher in the groups with cytokinesthan in the control group (P＜0.01); and the final fold with the mixture ofcytokines was significantly higher than that of both IGF-I and bFGF (P＜0.01). ③ Theproliferation index was significantly higher in the groups with cytokines than in the control group (P＜0.01); the proliferation index with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P＜0.05); besides, proliferation index was higher when cytokine was applied twice than once (P＜0.05). Conclusion bFGF and IGF-I could promote chondrocytes proliferation of adult rabbits obviously and they are synergistic in cell proliferation.
ObjectiveTo investigate the angiogenesis mechanisms of vascular endothelial growth factor (VEGF) combined with basic fibroblast growth factor (bFGF) for arteriosclerosis obliterans of rat hind limb.
MethodsThe models of hind limb arteriosclerosis obliterans of 60 male SD rats were established and randomly divided into four groups:normal saline (NS) group, VEGF group, bFGF group, and VEGF+bFGF group. The saline 1 mL and 100μg/L rhVEGF 1 mL were respectively injected into the abdominal cavity on every other day in the NS group and VEGF group. The 100μg/L rhbFGF 1 mL was multiply injected into the hind limb medial vastus muscle in the bFGF group. The 100μg/L rhVEGF 1 mL and 100μg/L rhbFGF 1 mL were respectively injected into the abdominal cavity and the hind limb medial vastus muscle on every other day in the VEGF+bFGF group. The angiogenesis of rat hind limb arteriosclerosis obliterans was observed on day 30 by digital subtraction angiography (DSA). The VEGF and bFGF protein and mRNA expressions in the hind limb medial vastus muscle tissues were tested by the Western blot and RT-PCR methods respectively.
Results①The number of new collateral vessel in the VEGF+bFGF group was significantly more than that in the bFGF group (P < 0.05), VEGF group (P < 0.05), and NS group (P < 0.001), which in the VEGF group or bFGF group was significantly more than that in the NS group (P < 0.001), and which had no significant difference between the VEGF group and bFGF group (P > 0.05).②The protein and mRNA expressions of VEGF and bFGF in the VEGF+bFGF group were significantly higher than those in the bFGF group (P < 0.001), VEGF group (P < 0.001), and NS group (P < 0.001), which in the VEGF and bFGF were significantly higher than those in the NS group (P < 0.001), which had no significant difference between the VEGF group and bFGF group (P > 0.05).
ConclusionsVEGF and bFGF in combination could increase the expressions of VEGF and bFGF in the rat hind limb ischemia region tissue and promote vascular endothelial cell proliferation and differentiation, and capillary sprouting growth, make angiogenesis of ischemic area, it is provided a new clinical treatment of peripheral arterial disease.
OBJECTIVE To explore the effect of basic fibroblast growth factor (bFGF) combined with autogenous vein graft conduit on peripheral nerve regeneration. METHODS Fifty four New Zealand rabbits were divided into three groups. The main trunk of sciatic nerve of rabbit in one side was severed and bridged by autogenous vein. 0.2 ml bFGF solution (4,000 U/ml) was intravenously injected to the vein graft conduit as group A, the same amount of saline solution as group B, and no solution injection as group C. Microscopic examination, axon video analysis and nerve conduct velocity were performed at the 10th, 30th, and 100th day after operation. RESULTS The nerve fibers were grown into vein graft conduit in all groups at 30th after operation, they were more and regular in group A than that of group B and C, and the axon regeneration rate in group A was more than that of group B and C. CONCLUSION bFGF combined with autogenous vein graft conduit can markedly promote nerve regeneration.
Objective To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 μg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. Results All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group (P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group (P<0.05); the bFGF group showed an increase compared to model group (P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant (P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1β, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group (P<0.05). The above indexes in bFGF group significantly improved when compared with model group (P<0.05). Conclusion bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.
OBJECTIVE To determine the effect of basic fibroblast growth factor (bFGF) on the biological behaviour of ovarian epithelial neoplasm. METHODS Ten cases of normal ovarian tissues and eighty cases of ovarian epithelial tumor tissues were detected by immunohistochemical methods. Mias-2000 Picture Analysis System was used to study the relationship of bFGF expression intensity and microvessel count, FIGO stage, pathological grade and classification of ovarian epithelial neoplasm. RESULTS 1. Expression of bFGF was mainly in cytoplasm and nucleus in several cells of borderline and malignant tumor. 2. The expression intensity of bFGF was closely related to the malignant degree of ovarian epithelial neoplasm. The density of bFGF expression was (3.35 +/- 3.52)% in normal ovarian epithelium, (19.25 +/- 21.73)% in benign tumor, (33.78 +/- 10.86)% in borderline tumor and (48.18 +/- 12.93)% in malignant tumor. The results indicated that bFGF might play an important role in carcinogenesis of ovarian epithelial neoplasm. 3. The expression intensity of bFGF was increased with the FIGO stage of ovarian tumor. 4. The expression intensity of bFGF was increased accompanying with the decrease of differentiation degree in ovian neoplasm. 5. In borderline tumor, expression intensity of bFGF in serous cystadenoma was significantly higher than in mucinous cystadenoma, which indicated bFGF might be an important factor in canceration of ovarian epithelial neoplasm. CONCLUSION bFGF may play important roles in carcinogenesis, development, invasion and metastasis of ovarian epithelial neoplasm.
ObjectiveTo discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction.
MethodsThe hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining.
ResultsThe flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001).
ConclusionThe hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.
Objective The biological effects of fibroblast growth factor (FGF) may be different under different intensities and durations. To investigate the impact of sustained increasing FGF signal upon the development of epiphyseal plate. Methods Epiphyseal plates cultured in vitro were obtained from embryonic C57BL/6J mice, and were divided into control group (0.1% DMSO), basic FGF (bFGF) group (100 μg/L bFGF and 0.1% DMSO), and PD98059 group (100 μg/L bFGF and 50 μmol/L PD98059 with 0.1% DMSO). The total length (TL) and ossified tissue length (OSL) of the cultured bones weremeasured with Calcein staining 6 days after culture. The expressions of Indian hedgehog (Ihh), collagen type II (Col II), and Col X genes were detected by real-time fluorescent quantative PCR 7 days after culture. Results The embryonic bones cultured in vitro continued growth. At 6 days after culture, there was no significant difference in increased percentage of TL between bFGF group and control group (P gt; 0.05), the increased percentage of OSL in bFGF group was significantly less than that in control group (P lt; 0.05). There was no significant difference in the increased percentage of TL and OSL between PD98059 group and control group (P gt; 0.05), but they were significantly higher than those of bFGF group (P lt; 0.05). At 7 days after culture, the gene expressions of Ihh, Col II, and Col X in bFGF group significantly decreased when compared with those in control group (P lt; 0.05). There was no significant difference in the gene expressions of Col II and Col X between PD98059 group and control group (P gt; 0.05), but the gene expressions were significantly higher than those of bFGF group (P lt; 0.05); the expression of Ihh in PD98059 group was significantly higher than that in control group and bFGF group (P lt; 0.05). Conclusion Sustained increasing FGF signal may affect the Col II and Col X expressions by down-regulating Ihh, which may lead to the development retardation of epiphyseal plate cultured in vitro. The external signal regulated kinase pathway may play an important role in the process.
Objective To investigate the promotion effects of the collagen membrane incorporating bFGF impregnated microspheres on the wound healing of the pigskin losing its full-thickness layers. Methods The bFGF containing microspheres was added into the dry microspleres.The collagen membranes were prepared by incorporating bFGF-impregnated microspheres, and 6 York pig models of skin wounds with loss of their full-thickness layers were established for the ob servation of the effects on the wound healing. Results The healing time and the 28day healing rate were 27.30±1.14 days and 98.12%±1.97%, respectively.The healing rate was significantly higher and the healing time was significantl y shorter in the experimental group than in the control group (Plt;0.05). The histological examination showed that the proliferation condition of the epidermiswasalso much better in the experimental group. Conclusion Incorporation of bFGF-impregnated microspheres into the collagen membrane is a promising method of pro moting the healing of the wound with a loss of the fullthickness skin.
ObjectiveTo explore the possible active mechanism of the basic fibroblast growth factor (bFGF) long circulation l iposome (LCL) (bFGF+LCL) on spinal cord traction injury in rats at the level of proteomics.
MethodsTwenty Sprague Dawly rats were randomly divided into groups A and B, 10 rats in each group. The models of spinal cord traction injury was established at T12-L3 spines. The rats were not treated in group A, and the rats were treated with bFGF+LCL (20μg/ kg) in group B. At 3 weeks after operation, the rats were sacrificed for harvesting T13-L2 spinal tissue specimens. The protein was extracted and quantified in the spinal tissue firstly. The proteins from spinal tissue were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. The different expression profiling was established in each group, and the differentially expressed protein was determined by comparing the level of each spot with gel imaging software and manually. The proteins were identified by nano ultra-high performance liquid chromatography-electrospray tandem mass spectrometry (NanoUPLC-ESI-MS/MS), and the proteins were classified.
ResultsThe differentially expressed protein spots were found in 2 groups. Compared with group A, 4 spots were up-regulated and 6 were down-regulated in group B. NanoUPLC-ESI-MS/MS results showed that 18 significant proteins were identified in 26 differentially expressed proteins, including 4 apoptosis-related proteins, 3 nerve signal transduction related proteins, 7 proteins involved in metabolism, 1 unknown function protein, and 3 unnamed proteins.
ConclusionThe differentially expressed proteins are found in spinal cord traction injury of rats treated with bFGF+LCL. bFGF+LCL can affect the proteins expression in rats with spinal cord traction injury. The possible active mechanism is that it has protective and repair effects on injured spinal cord by nerve signal transduction, and regulation of nerve cells apoptosis and metabolism.
Objective To evaluate the effect of the local del ivery of basic fibroblast growth factor 2 (bFGF-2) on the osseointegration around titanium implant of diabetic rats. Methods The bFGF-2-loaded poly (lactic-co-glycol ic acid) microspheres were prepared by water/oil/water (W/O/W) double-emulsion solvent evaporation method. Thirty-five male SPF level Sprague Dawley rats, weighing 220-250 g and aged 9 weeks, were selected as experimental animals. Ten rats were fedwith the routine diet as normal control group. The other 25 rats were made the diabetic animal model by giving high fat-sugar diet and a low dose streptozotocin (30 mg/ kg) intravenously; 20 rats were made the diabetic animal model successfully. Then 20 rats were randomly divided into diabetic control group (n=10) and bFGF-2 intervention group (n=10). A hole was drilled in the right tibia bone of all rats, and the titanium implant treated by micro-arc oxidation surface was planted into the hole. Simultaneously, the previously prepared microspheres and blood were mixed and were loaded on the surface of the implant before it was implanted into the rats of the bFGF-2 intervention group. At 4 and 8 weeks, the tibia containing implants was harvested, embedded with resin and made undecalcified tissue sl ices to compare the osseointegration. Results At 4 weeks, the implants of the normal control group were surrounded by new lamellar bone with continuity; whereas the tissue around the implants of the diabetic control group contained l ittle woven bone and some fibrous tissue; and obvious new formed bone with continuity was observed in bFGF-2 intervention group. At 8 weeks, the results of 3 groups were similar to those at 4 weeks. At 4 weeks, the percentage of bone-implant contact (BIC) in diabetic control group was significantly less than those in normal control group (P lt; 0.05) and in bFGF-2 intervention group (P lt; 0.05); the BIC in bFGF-2 intervention group was less than in normal control group, but showing no significant difference (P gt; 0.05). After 8 weeks, the BIC in normal control group and in bFGF-2 intervention group were significantly greater than that in diabetic control group (P lt; 0.05), but there was no significant difference between bFGF-2 intervention group and normal control group (P gt; 0.05). Conclusion Local del ivery of bFGF-2 around titanium implants may improve the osseointegration in diabetic rats.
