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 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 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.
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 investigate the effects of basic fibroblast growth factor (bFGF) on the promoter activities of human alpha 1(I) procollagen gene and the interaction between bFGF and transforming growth factor-beta 1 (TGF-beta 1). METHODS: Fibroblasts of the hypertrophic scar and normal skin from a 3-year-old patient were primarily cultured and subcultured in vitro. Both of the fibroblasts were transient transfected with phCOL 2.5, containing -2.5 kb of 5’f lank sequence of human alpha 1(I) procollagen gene and CAT reporter gene by FuGENE transfection reagent; and treated thereafter by 16 ng/ml bFGF, 2 ng/ml TGF-beta 1 and 16 ng/ml bFGF + 2 ng/ml TGF beta 1 for 24 hours. The relative CAT expression values were determined by CAT-ELISA. RESULTS: TGF-beta 1 bly induced the CAT expression level, however, bFGF not only inhibited the basal CAT expression but also reduced the CAT expression up-regulated by TGF-beta 1 in normal skin and hypertrophic scar fibroblasts (P lt; 0.05). CONCLUSION: bFGF can reduce the promoter activities of human alpha 1(I) procollagen gene and antagonize the role of TGF-beta 1 in up-regulating the promoter activities of human alpha 1(I) procollagen gene in normal skin and hyertrophic scar fibroblasts.
OBJECTIVE: To localize the distribution of basic fibroblast growth factor (bFGF) and transforming growth factor-beta(TGF-beta) in tissues from dermal chronic ulcer and hypertrophic scar and to explore their effects on tissue repair. METHODS: Twenty-one cases were detected to localize the distribution of bFGF and TGF-beta, among them, there were 8 cases with dermal chronic ulcers, 8 cases with hypertrophic scars, and 5 cases of normal skin. RESULTS: Positive signal of bFGF and TGF-beta could be found in normal skin, mainly in the keratinocytes. In dermal chronic ulcers, positive signal of bFGF and TGF-beta could be found in granulation tissues. bFGF was localized mainly in fibroblasts cells and endothelial cells and TGF-beta mainly in inflammatory cells. In hypertrophic scar, the localization and signal density of bFGF was similar with those in granulation tissues, but the staining of TGF-beta was negative. CONCLUSION: The different distribution of bFGF and TGF-beta in dermal chronic ulcer and hypertrophic scar may be the reason of different results of tissue repair. The pathogenesis of wound healing delay in a condition of high concentration of growth factors may come from the binding disorder of growth factors and their receptors. bFGF may be involved in all process of formation of hypertrophic scar, but TGF-beta may only play roles in the early stage.
OBJECTIVE To investigate the effects of basic fibroblast growth factor(bFGF) on repairing transected sciatic nerves in rats. METHODS The animal models of the transected sciatic nerve of 40 SD rats were established, which divided into 4 groups: normal saline (NS) group, nerve growth factor (NGF) group, bFGF group and normal control group. The epineurium of the transected sciatic nerve was sutured under microscope, then bFGF or NGF was dropped into local sites and injected intramuscularly once a day for 30 days after operation. Functional repair for the transected sciatic nerves was studied by nerve conductive velocity (NCV) and sciatic nerve function index (SFI). RESULTS As a criterion, the level of the normal control group was regarded as zero, SFI of NS group, NGF group and bFGF group were -114.30 +/- 10.34, -70.50 +/- 11.01, -50.45 +/- 7.82 respectively at 1 month after operation, and they were -54.96 +/- 16.46, -35.21 +/- 10.80, -27.53 +/- 11.23 respectively in 3 months after operation. NCV of bFGF group was significantly faster than NS group and NGF group. CONCLUSION bFGF can significantly promote the functional repair of injured peripheral nerve, and its effects are better than NGF.
OBJECTIVE: To explore the expression of basic fibroblast growth factor(bFGF) during the wound healing of human fetal and adult skin and its significance. METHODS: We established the animal model of fetal scarless healing by transplanting full-thickness skin grafts from human fetus to a subcutaneous location on the athymic mouse recipient, and then making the linear incisions. The expression of bFGF was observed in the normal adult skin, normal fetal skin and during wound healing by immunohistochemical method. The positive staining cells were counted under selected high-power focus randomly. RESULTS: bFGF staining was not observed in the normal fetal skin and the wounded one. However, bly positive staining was shown around the vessels in normal adult skin. Moreover, the positive straining became ber in the wounded skin, especially in dermal fibroblasts and endotheliocytes. The number of positive staining cell was 2.1 +/- 0.1 in normal fetal skin, and 2.2 +/- 0.1, 2.1 +/- 0.3, 2.1 +/- 0.3 and 2.0 +/- 0.1 in the fetal skins after 12 hours, 1 day, 3 days and 7 days of wound respectively. The number of positive staining cell were 23.2 +/- 4.2 in normal adult skin and 40.5 +/- 3.6 in the wound adult skin. There was significant difference between the fetal skin and adult skin (P lt; 0.01). CONCLUSION: The negative expression of bFGF in the fetal skin may be one of the important reasons for fetal scarless healing.
Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.