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.
Objective To explore the effects of the basic fibroblast growth factor（bFGF） gene transfection on the meniscal fibrochondrocytes with the reconstructed lentivirus and to observe the response of the meniscal fibrochondrocytes to the bFGF gene transfection. Methods The cultured meniscal fibrochondrocytes were isolated from the same 3-monthold New Zealand rabbit. The cultured first-generation meniscal fibrochondrocytes were divided into 3 groups:Group A (experimental group), Group B (control group), and Group C (blank group). Each group comprised the cells in a 24hole flask in which each hole contained 2×104 cells. At the confluence of 60%, the fibrochondrocytes in Group A were cultured with the reconstructed lentivirus carrying the bFGF gene. The fibrochondrocytes in Group B were cultured with the lentivirus carrying no bFGF gene. The fibrochondrocytes in Group C were cultured without any intervention. After 48 h, the cell cycle, the collagen synthesis ability, the expression of bFGF, and the cell proliferation ability in each group were investigated. Results In Group A, the bFGF expression of 870±60 pg/ml was detected in the cells 48 h afterthe co-culture; however, in Group B and Group C, no expression of bFGF was found. After the co-culture for 6 days, the results of the MTT colorimetry revealed that the cells in Group A had an absorbtance of 0.427±0.037, which had a significant difference when compared with that in Group B and Group C (0.320±0.042,0.308±0.034,Plt;0.01). The cell cycle was significantly shorter in GroupA than in Group B and Group C (Plt;0.05); The durations of G1, S and G2M of the cells in Group A were 16.28, 12.60 and 11.04 h, but those in Group B and Group C were 23.61, 16.90, 21.33 h and 21.56, 19.80, 21.41 h, respectively. The disintegration per minute of the cells was significantly greater in Group A than in Group B and Group C (7281.69±805.50 vs 5916.40±698.11 and 5883.57±922.63，Plt;0.05). Conclusion The lentivirus vector can transfer the bFGF gene into the meniscal fibrochondrocytes, resulting in an increase of the cell proliferation and the collagen synthesis.
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 The biological effects of recombinant human epidermal growth factor (rhEGF) and recombinant human fibroblast growth factor (rhFGF) were evaluated on the model of incised wounds in mini pigs. METHODS Total of 160 incised wounds in 16 mini pigs were divided into two groups (rhEGF group and rhFGF group), each containing 80 wounds. In rhEGF group, 60 incised wounds were treated with different dosages of rhEGF (50, 10 and 0.5 micrograms/wound), and another 20 wounds were treated with solvent as control group. In rhFGF group, all wounds were treated in the same way as described in rhEGF group, the dosages of rhFGF were 150, 90 and 30 U/cm2 respectively. The measurements of cavity volume and area in wound, histological examination were used to evaluate the results of wound healing. RESULTS The results showed that wound healing was accelerated in all wounds treated with rhEGF and rhFGF. In rhEGF group, the velocity of re-epithelialization was faster than that of rhFGF group, however, new granulation tissue in rhFGF was more than that of rhEGF group. CONCLUSION The results indicate that rhEGF and rhFGF can stimulate wound healing, however, the mechanisms and the biological effects involved in these processes are quite different. It suggests that it is better to use rhFGF in those wounds which need more granulation tissue formation and use rhEGF in the wounds which mainly need re-epithelialization.
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 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 effects of liver X receptor agonist, T0901317, on the proliferation, migration and hydroxyproline production of human embryonic lung fibroblasts (HELF). MethodsHELF cells were devided into a control group, a growth factor group, a T0901317 group and three growth factor+T0901317 groups. The cells of the control group were treated with Dulbecco's modified Eagle medium. The cells of T0901317 group were treated with 1.00 μmol/L T0901317. The growth factor+T0901317 groups were incubated with different doses of T0901317 (0.25 μmol/L, 0.50 μmol/L and 1.00 μmol/L) for 2 h. Then the cells of the growth factor+T0901317 groups and the growth factor group were incubated with basic fibroblast growth factor and transforming growth factor-β1 for 24 h. The proliferation, migration and collagen production of HELF were determined by cell counting kit-8 method, transwell chamber, and hydroxyproline method. ResultsCompared with the control group, T0901317 had no effect on the proliferation, migration and hydroxyproline production of HELF. Growth factors could promote the proliferation, migration and hydroxyproline production of HELF significantly. T0901317 could inhibit those effects of growth factors with a dosage-dependent manner. ConclusionT0901317 may inhibit the proliferation, migration and hydroxyproline production of HELF induced by growth factors.
OBJECTIVE: To determine the influence of basic fibroblast growth factor (bFGF) on endothelial cell (EC) proliferation in vitro and its possible mechanisms, and to examine the effect of both TNP-470 and dexamethasone (Dex) on the EC proliferation induced by bFGF. METHODS: Human umbilical vein endothelial cells were cultured and the proliferation of EC was quantified by a colorimetric assay using MTT reagent. The expression of nuclear factor-kappa B (NF-kappa B) and ki-67 was detected with SABC immunohistochemical method. RESULTS: bFGF stimulated the EC proliferation and enhanced the expression of NF-kappa B and ki-67 in nucleus; TNP-470 and Dex suppressed EC proliferation induced by bFGF, and reduced the expression of NF-kappa B and ki-67 in nucleus. CONCLUSION: The above results indicate that the possible mechanisms of EC proliferation stimulated by bFGF come from that bFGF can activate NF-kappa B to promote the synthesis of DNA and EC mitosis. TNP-470 and Dex inhibited EC proliferation stimulated by bFGF by inhibiting NF-kappa B.