Objective To construct a mammalian expression vector ofbasic fibroblast growth factor (bFGF) and to investigate the expression of bFGFin vitro and in vivo. Methods A mammalian expression vector pcDNA3.1/myc-His(-)C-bFGF was constructed with gene cloning technique. The mammalian expression system was prepared and purified. The expression of bFGF cDNAin cultured transfected cells was examined by RT-PCR and cell immunohistochemistry. The recombinant plasmids, pcDNA3.1/myc-His(-)C-bFGF and pCD2-VEGF121, were transferred into rabbit cervical muscle by direct injection of plasmid following electric pulses in vivo. The transferred gene expression and the biological effect were measured by use of histochemistry and immunohistochemistry analysis. Results The eukaryon expression system pcDNA3.1/myc-His(-)C-bFGF could express the target protein bFGF in vitro. The recombinant plasmids, pcDNA3.1/myc-His(-)C-bFGF and pCD2-VEGF121 were transferred into muscles flap in vivo successfully. The active proteins bFGF and VEGF121were expressed at high levels. Blood vessels increased significantly in the muscles, and blood circulation was improved by local angiogenesis. Conclusion Theeukaryon expression vector of bFGF is constructed and can be expressed successfully in vitro and in vivo. That is a primary preparation for the research on tissue transplantation and tissue engineering with bFGF gene therapy.
OBJECTIVE To study the early protective effects of basic fibroblast growth factor(bFGF) on the experimental acute spinal cord injury. METHODS Thirty-four SD rats were randomly divided into three groups, and were subjected to contusion of thoracolumbar spinal cord. A thin plastic tube was placed in subarachnoid space below the injury level for perfusion. The bFGF-treated rats were received 20 microliters bFGF(containing bFGF 100 U) at once, 30 min, 1, 2, 3, 4, 6, 12, 24 and 48 hours after injury, and an equal volume of normal saline was given to the control group at the same time. The injured spinal cord was detected by morphological observation and biochemical index after injury. RESULTS The degree of ionic disorder in bFGF-treated rats was significantly ameliorated and the contents of H2O were also markedly decreased. The morphological finding showed that the damages of gray and white matter in bFGF-treated rats were slighter than those of saline-treated rats. CONCLUSION bFGF has some protective effects on the secondary lesion of early spinal cord injury in rats.
Porpose To investigate the optimal concentration of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on DNA synthesis and their synergism indensity arrested human retinal pigment epithelial (RPE) cells. Methods Growth factor effects in cultured human RPE of the 6th generation were assessed by [3 H]-thymidine incorporation and radioautography. Results EGF and bFGF were potent stimulators when used alone,and their optimal concentrations were 10ng/ml in DMEM and 1ng/ml in 2% serum DMEM.When used in combination (10ng/ml EGF and 10ng/ml bFGF),they caused a significant enhancement of [3 H]-thymidine incorporation about 2.96 times. Conclusion EGF and bFGF were potent stimulators in RPE cells,and demonstrated synergism in their action. (Chin J Ocul Fundus Dis,1998,14:98-100)
Objective To evaluate the ability of inductive osteogenesis of allgraft demineralized bone containing basic fibroblast growth factor (bFGF/ALB) in repairing bone defect. Methods Thirty-two New Zealand white rabbits were randomly divided into four groups (groups A,B,C and D, n=8). A segmental bone defect of15 mm inlength was made on the bilateral radius respectively and the defects filled with ALB/bFGF in group A, with ALB in group B, with bFGF in group C and without any materials in group D serving as blank control. At 2, 4, 6 and 8 weeks after operation, all restored bones were evaluated by roentgenography, histological observation and Ca2+detection of osteotylus. Results The X-ray films showed that groups A and B had a little shadow of bone formation at 2 weeks, while groups C and D had transparent shadow; that group A had denser shadow and new bone formation at 4 weeks and 6 weeks, groups B and C had a little increase of shadow and group D had little shadow at fractured ends; and that group A had formation of bone bridge at 8 weeks, the new formed bone in fractured ends of group B closed with each other, the gap still existed in group C, and the defects filled with the soft tissue in group D. The Ca2+content of group A was higher than that of groups B, C and D at 4 weeks (Plt;0.05) and 8 weeks (Plt;0.01). The histological observaton showed that the degree of bone restoration of group A was superior to that of groups B, C and D. Conclusion bFGF/ALB is a good material to improve bone restoration.
OBJECTIVE: To study the stimulating effects of basic fibroblast growth factor(bFGF) on fibroblast function and its ability to expression of c-fos gene. Furthermore, to explore the possible network action between bFGF and oncogene in modulating wound healing. METHODS: Cultured rat fibroblasts were divided into bFGF stimulating group and control group. Fibroblasts in bFGF stimulating group were treated with bFGF in a dosage of 40 ng/culture hole, while the control fibroblasts were treated with the same vehicle without bFGF. The morphology, cell vitality and their ability to express c-fos gene in the fibroblasts in both groups were studied with MTT and immunohistochemical methods. RESULTS: All fibroblasts in bFGF treated groups were enlarged and showed increased vitality with MTT method. C-fos gene expression in bFGF stimulating group was increased, especially in nucleus when compared with those in control group. CONCLUSION: The results show that the function and the ability to express c-fos gene in bFGF treated fibroblasts are enhanced. Combined with our previous studies, it may make a conclusion that there is a network regulation mechanism between growth factors and some oncogenes.
OBJECTIVE: To investigate the influence of basic fibroblast growth factor (bFGF) on adhesion characteristics of osteoblasts, aimed at the important problem in bone tissue engineering of how to promote the adherence of osteoblasts to extracellular matrix materials. METHODS: 5 ng/ml, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml bFGF were used to induce bone marrow stromal-derived osteoblasts of rabbit for 24 hours before incubation, and the common culture medium as the control. The attached cells were calculated with stereology method at 0.5 hour, 1st hour, 2nd hour, 4th hour, 8th hour after seeding. RESULTS: The number of attached cells was significant higher in the experimental group when induced by 10 ng/ml bFGF than that in the control group (P lt; 0.01); the number did not increase with the increase of bFGF concentration and there was no significant difference between the experimental group induced by 100 ng/ml bFGF and control group, and the number was even obviously lower in the experimental group when induced by 200 ng/ml than the control group (P lt; 0.01). CONCLUSION: bFGF can influence the adhesion characteristics of osteoblasts, 10 ng/ml bFGF can promote the adherence of osteoblasts to matrix materials, but 200 ng/ml bFGF may inhibit cell adhesion.
Objective To observe the effects of basic fibroblast growth factor (bFGF) on the expression of heat shock protein 70 (HSP70) in ratrs retina after iscbemia/reperfusion injury.Methods The rat model of experimental retinal ischemia/reperfusion injury was made by increasing the intraocular pressure. Tweenty-four Wistar rats were divided into normal (3 rats) and operation group (21 rats) randomly. The latter group was subdivided into group 0 hour, 4, 8, 12, 24, 48 and 72 hours after reperfusion, in which the left eyes of the rats were in the ischemia/reperfusion groups and the right ones were in the treatment groups (bFGF 2 t~g intracameral injection). The expression of HSP70 was observed by strept avidin-biotin complex (SABC) immunohistochemistry. Results No HSP70 positive cells were found in normal group; a few of HSP70 positive cells were found 0 hour after reperfusion [20.8±4. 5) cells/mm2], and increased gradually until reached the peak 24 hours later [(111.2±4.4) cells/mm2] and then decreased gradually. Few HSP70 positive cells were found 72 hours after reperfusion. The amount of HSP70 positive cells increased in treatment group at all time courses, and the peak time was earlier and longer than that in ischemia group. HSP70 positive cells distributed extensively in retinal ganglion cell layer and inner nucleous layer. The difference of the amount of HSP70 positive cells between the two groups was significant (Plt;0.05) 8, 12, 24, 48 and 72 hours after reperfusion.Conclusion bFGF can enhance the expression of HSP70 in rat’s retina after retinal ischemia/reperfusion injury.(Chin J Ocul Fundus Dis，2004，20：37-39)
Objective To select the appropriate media to culture the epidermal stem cells in vitro,and to observe the biological characteristics of the epidermal stem cells. Methods The epidermal stem cells were cultured in five different media, including FAD, FAD+1 ng/ml bFGF, FAD+5 ng/ml bFGF, FAD+10 ng/ml bFGF and K-SFM, and the same fetous fibroblasts were used as the nutrient cells. The proliferation ability was investigated by cell growth curve and MTT detection. Then the biological characteristics of epidermal stem cells were observed through phasecontrast microscope, cell growth curve, BrdU detection and FBM analysis. Results The epidermal stem cells grew best in FAD with bFGF and nutrient cells. And the epidermal stem cells retained proliferative capacity, and formed larger and more expandable clones in vitro. And 80.2% of the cells show a G0/G1 cycle, and the cells had long cell proliferation cycle. Conclusion The above results demonstrate that the media with bFGF and the use of nutrient layer were appropriate to culture epidermal stem cell in vitro. And the epidermal stem cells have a slow cell cycle, characteristics of immaturity.
OBJECTIVE: To explore the localization and expression characteristics of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in rat skins at different development stages (embryonic, newborn, adult). METHODS: Skins from embryonic, newborn and adult rats were taken, and detected by immunohistochemical technique. RESULTS: Positive immunohistochemical signal of EGF could be found in skins from embryonic, newborn and adult rats, mainly in the cytoplasm of the epidermal cells, fibroblasts, hair follicle epithelial cells, and endothelial cells. With the increase in age, the expression amount of EGF was increased. The positive signal of bFGF could be found in skins of newborn and adult rats, while the signal of bFGF in skin of embryonic rats was negative. CONCLUSION: The results indicate that endogenous EGF plays important role in epidermal development in embryonic stage and wound healing in adult after injury. The negative expression of bFGF in skin of embryonic rat indicate that the absence of bFGF may be one of the reasons for the non-scar healing in embryonic stage.
OBJECTIVE: To further explore the effects of fibroblast growth factor on soft tissue repair. METHODS: Based on the data from our experiments and clinical trial and data from other reports, a further reconsideration about fibroblast growth factor and soft tissue repair was demonstrated, including embryonic development, histology, animal experiments, clinical trial and prospect. RESULTS: Amounts of basic and clinical data showed that fibroblast growth factor was needed in embryonic development. Exogenous fibroblast growth factor could accelerate wound healing. CONCLUSION: Fibroblast growth factor is a bioactive protein which can obviously promote wound healing, it has broad prospects of clinical application.