Objective To observe the expression of adenovirus vector coding for mouse endostatin gene（Ad-mES） in lung cancer cells and its antiangiogenic activity in human umbilical vein endothelial cells（ECV304） in vitro.Methods Lewis lung cancer（LLC） cells were transfected with Ad-mES at different multiplicity of infection（MOI）.The expression of mES in LLC cells and supernatant after 48 hours was detected by immunohistochemical staining and Western blot respectively.The inhibitory effect of supernatant at different MOI on ECV304 non-stamulated and stimulated by basic fibroblast growth factor（bFGF） was measured by methyl thiazolyl tetrazolium（MTT） assay.Results After transfected for 48 hours，endostatin was identified in the cell plasma of infected LLC and negative result was founded in non-infected LLC.Western blot revealed band of endostatin in 20 kDa in culture supernatant of infected LLC and negative results in non-infected LLC.The inhibitory effects on ECV304 cell proliferation were ber at higher MOI，and the difference was significant between stimulated and non-stamulated cells by bFGF（Plt;0.05）.Conclusion Ad-mES can transfect and express endostatin effectively in LLC with biological activity
Objective To observe the impact of collagen patches using 1-ethyl-3- （3-dimethylaminopropyl) carbod-iimide hydrochloride chemistry （EDC） to conjugate vascular endothelial growth factor （VEGF） ＋ basic fibroblast growth factor （bFGF） or VEGF alone on the survival rate of transplanted human bone morrow mesenchymal stem cells （hBM-MSCs）in vitro and in vivo. Methods Collagen patches which were activated by EDC were used as the control group，and EDC activated collagen patches that were conjugated with VEGF or VEGF ＋ bFGF were used as the experiment groups（VEGF group and VEGF ＋ bFGF group）. hBM-MSCs （0.5×106/patch） were used as seeding cells to construct engineered heart tissue （EHT）. MTT assay was performed to assess in vitro proliferation of hBM-MSCs on 3 different collagen patches. Ventricular aneurysm model after myocardial infarction was created by left anterior descending artery （LAD） ligation in male SD rats，and EHT which were constructed with 3 different patches were used for ventricular plasty. Four weeks later，immunofluorescence staining was used to examine arteriole density （anti-α-SMA staining） and transplanted cell survival （anti-h-mitochondria staining）. Results （1） hMSCs proliferation in VEGF group and VEGF ＋ bFGF group was significantly better than that in the control group on the 2nd and 4th day after cell transplantation （P＜0.05）； （2） Four weeks afterEHT implantation，immunofluorescence staining for α-SMA revealed that arteriole density of VEGF group and VEGF ＋ bFGF group was significantly higher than that of the control group （P＜0.05）； （3） Immunofluorescence staining forh-mitochondria showed that survival rates of transplanted hBM-MSCs of VEGF group and VEGF ＋ bFGF group were significantly higher than that of the control group （P＜0.05）； （4） There was a significantly positive correlation between survival rate of hBM-MSCs and arteriole density （r 2=0.99，P=0.02）. Conclusion VEGF or VEGF ＋ bFGF conjugated collagen patch can significantly improve hBM-MSCs proliferation in vitro and enhance survival rate of transplanted hBM-MSCs by accelerating revascularization of EHT in vivo.
Objective To observe the influences of estradiol (E2), basic fibroblast growth factor (bFGF), and tamoxifen (TAM) on the proliferation of hemangioma vascular endothelial cell (HVEC). Methods Two strawberry hemangioma from 2 infants (case 1 and case 2) were prepared for HVEC culture. The HVEC on passage 3 were cultured in estrogenfree improved minimum essential medium (IMEM) and subjected to various treatments with 100 pg/ml 17-β-E2, 10 ng/ml bFGF, and 1×10-6 mol/L 4-OH-tamoxifen(4-OH-TAM). The experiment was divided into 5 groups: group 1(IMEM, control group), group 2(17-β-E2), group 3(bFGF), group 4(17-β-E2／bGFG) and group 5(17-β-E2／bGFG／4-OH-TAM). The cell count(CC) and DNA proliferation index (PI) were determined. Results Two cases of HVEC were successfully cultured in vitro. The HVEC showed cobblestoneslike under microscopy and factor Ⅷrelated antigen(also named as von Willebrand factor，vWF) was positive by immunochemical staining. At 9 days in case 1: CC and PI remained unchanged in the control group; CC and PI were slightly increased in group 2, being 1.4 and 1.6 times as much as those in the control group respectively (P＜0.05); CC and PI significantly increased in group 3, being2.6 and 2.3 times as much as those in the control group respectively (P＜0.01); CC and PI increased remarkably in group 4, being 3.7 and 2.9 times as much as those in thecontrol group respectively (P＜0.01); CC and PI were down to the levels of controls in group 5(P＞0.05). The results in case 2 were similar to those in case 1. Conclusion In vitro, the promoting effect of bFGF on HVEC proliferation is much ber than that of estrogen. Estrogen and bFGF enhance this proliferation in a synergistic manner, which can be inhibited by tamoxifen.
Objective To investigate the effects of the insulin-like growth factor 1 (IGF-1), the transforming growth factor β1(TGFβ1), and the basic fibroblast growth factor (bFGF) on proliferation and cell phenotype of the human fetal meniscal cells, and to find out the best combination and concentration of the growth factors for the meniscus tissue engineering. Methods The fetus came from the healthy woman accidental abortion and the procedure had got her approval.The human fetal meniscal fibrochondrocytes were cultured in vitro. The cell phenotype was identifiedby the collagen type Ⅱ immunohistochemistry and Aggrecan immunofluorescence. Inthe growth factor groups, the 3rd passage meniscal cells synchronized by the serum starvation method and were mixed with IGF-1 (1, 10, 50, 100 μg/L), TGF-β1 (0.1, 1.0, 5.0, 10.0, 50.0 μg/L), and bFGF (5, 10, 50, 100, 200 μg/L), respectively, and in the combination groups, the combinations of bFGF and TGF-β1, bFGF and IGF-1, TGF-β1 and IGF-1 were established at their optimal effect concentrations. The control group was also established for comparison. The dose-response relationship was studied at 48 h and 72 h bythe MTT colorimetric method. Results The 3rd passage meniscalcells could express collagen type Ⅱ and Aggrecan before and after the addition of the three growth factors. The proliferating effects of the growth factors (IGF-1 50 μg/L，TGF-β1 5 μg/L，bFGF 50 μg/L) on the 3rd passage cells at 48 h and 72 h were significantly better in the growth factor groups than in the control group (Plt;0.05)，and the combination groups of bFGF 50 μg/L and IGF-1 50 μg/L, IGF-1 50 μg/L and TGF-β1 5 μg/L showed a significantly higher proliferatingeffect than that in the single growth factor group (Plt;0.05). bFGF 50 μg/L and TGF-β1 5 μg/L had no synergetic effect (Pgt;0.05). Conclusion IGF-1, TGF-β1 and bFGF can promote the proliferation of the human fetal meniscal cells, respectively, and the combinations of bFGF and IGF-1, IGF-1 and TGF-β1 at their optimal concentrations can have better proliferating effects than the single growth factor. They can be used for the in vitro amplification of the meniscal seed cells.
Objective To investigate effects of the basic fibroblast growth factor (bFGF) and fibronection (FN) on the osteoblast adhesion on the bio-derived bone. Methods The third generation of the osteoblast was treated with bFGF 0.1, 1, 10, and 100 ng/ml, respectively, and then was seeded in the bioderived bone, which had been modified with FN 0.1, 1, 10, and 100 μg/ml, or Polylysine, respectively. The cell adhesion was measured by the MTT assay. The cell density and the cell appearance were observed by the scanning electron microscope. The abovementioned procedures were repeated by an application of the GRGDS peptide. Results Both FN and bFGF could enhance the osteoblast adhesion efficiency on the bioderived bone (Plt;0.05). However, the osteoblast adhesion efficiency could be significantly strengthened bya combined use of FN and bFGF. FN and bFGF had a significant synergistic effectin statistics (Plt;0.01), but Polylysine and bFGF had no such synergistic effect (P＞0.05). The combined use of FN and bFGF had a better effect on the cell density and the cell appearance than either of them when observed with the scanning electron microscope. Adhesion efficiency generated by the combined use of FN and bFGF was significantly blocked by the application of the GRGDS peptide. Conclusion The combined use of FN and bFGF has a significant synergistic effect on the osteoblast adhesion efficiency on the bioderived bone. This effect is probably mediated by the RGD-integrin α5β1 pathway.
Objective To explore the effects of exogenous basic fibroblast growth factor (bFGF) on insheathed tendon healing and adhesion formation. Methods Ninety Leghorn chickens were randomly divided into 3 groups (groups A, B and C), 30 animals for each group, and the right third digitorum longus tendon of the chicken was transected to make defect models. In group A, the tendon was sutured in situ after transection. In group B, the tendon was sutured after 0.6 μl fibrin sealant (FS) was applied at repair site. In group C, the tendon was sutured after 0.6 μl FS mixed with 500 ng bFGF was appliedat repair site. At 1, 2, 4 and 8 weeks after operation, the tendons of 6 chickens in each group were harvested for morphological and histological evaluation. Six specimens of each group was obtained for biomechanical test at 8 weeks. Results The gross observation showed that the differences of grading of tendon adhesion were not significant between groups A, B, and C 8 weeks after operation(Pgt;0.05). Histological evaluation showedthat there were no significant differences in fibroblast counting and the content of collagen fibers between groups A and B(P＞0.05). The angiogenesis, fibroblast proliferation and collagen production in the sheath, epitendon and parenchyma at repair site in group C occurred earlier and were more than those in groups A and B, showing significant differences (Plt;0.05). The biomechanical tests showed that the gliding excursionof the tendon in group A, B and C were 3.44±0.43、3.51±0.56 and 2.84±0.42 mm respectively; the work of flexion were 14.87±1.72、14.08±1.85 and 20.62±3.52 Nmm respectively; the ultimate tensile strength of the tendon was10.34±1.45，11.26±1.83 and 15.02±2.20 N respectively; showing no significant differences between groups A and B(Pgt;0.05), but showing significant differences between group C and groups A, B(Plt;0.05). Conclusion The exogenous bFGF at tendon repair site can facilitate insheathed tendon healing, but also increase the tendon adhesion formation.
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 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.
Objective To explore the effect of basic fibroblast growth factor（bFGF）and epidermal growth factor（EGF）on the growth of muscle derived stem cells(MDSCs). Methods MDSCs were isolated from hindlimb muscle of 15 new born Kunming mice through serial preplates. 2% fetal bovine serum-containing DMEM was used to induce MDSCs to differentiate into skeletal muscle lineage. The expressions of stem cell marker Sca-1 and skeletal musclecell marker αSarcomeric actin were examined by immunocytochemistry. The effect of bFGF and EGF on the proliferation of MDSCs was determined by MTT colorimetric microassay. The solo effect of bFGF or EGF at different concentrations (6.25,12.50, 25.00, 50.00, and 100.00 ng/ml) was examined at 96 h and the combined effect (100.00 ng/ml) was examined at 24,48,72 and 96 h.Results MDSCs were successfully isolated from the hindlimb of neonatal mice. Over 90% of MDSCs showed Sca-1 positive immunoreactivity. MDSCs could give rise to α Sarcomeric actin positive myotubes in differentiation cultures. The proliferative effect of bFGF and EGF on MDSCs increased with the elevated concentration.bFGF began to show significant proliferative effect at 12.50 ng/ml (P＜0.05). The effect increased significantly when the concentration reached 25.00 ng/ml from 12.50 ng/ml (P＜0.01) and reached a saturation point. The effect at 50.00 ng/ml or 100.00 ng/ml showed no significant increase when compared with thatat 25.00 ng/ml. EGF had a similar effect to bFGF except that the saturation concentration was 50.00 ng/ml. EGF showed significant effect at 72 h and bFGF at 96 h (Plt;0.01). When they were applied together, significant effect was shownat 24 h (Plt;0.01) and much higher effect was observed at 48, 72 and 96 h (Plt;0.05). Conclusion Both bFGF and EGF can promote the proliferation of MDSCs. The combined application reacts faster and ber.