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 investigate the effect of bone marrow mesenchymal stem cell (MSCs) transp1antation combined with transmyocardial drilling revascularization (TMDR) and degradable stent on myocardium revascu1arization after acute myocardial infarction(AMI), and to provide the experimental evidence for surgical treatment of myocardial infarction. Methods After established models of AMI, the 24 pigs were divided into four groups with random number table, 6 pigs each group. Control group: only established models of AMI; MSCs group: AMI immediately followed by MSCs implantation; TMDR combined with stent group: AMI followed by TMDR and absorbable basic fibroblast growth factor (bFGF) stent implantation; MSCs combined with TMDR and stent group: AMI followed by TMDR and absorbable bFGF stent implantation, and then MSCs implantation. Three months after operation, the infarcted areas and vessel density in infarcted zone were detected by histopathology method. Results Three months after operation, the histopathological examination showed that infarcted areas in MSCs group, TMDR combined with stent group, and MSCs combined with TMDR and stent group were decreased as compared with control group （27.9%±3.1% vs. 48.9%±2.7%，P=0.000；20.3%±1.7% vs. 48.9%±2.7%，P=0.000；12.5%±1.9% vs. 48.9%±2.7%，P=0.000）; and vessel density was further increased （8.4±1.2/HP vs.4.5±14/HP，P=CM(1583mm] 0.001；11.5±2.6/HP vs.4.5±1.4/HP，P=0.001;15.6±1.4/HP vs.4.5±1.4/HP，P=0.000）. Conclusion [CM)]MSCs transplantation combined with TMDR and absorbable bFGF stents implantation could significantly reduce the infarction areas, increase the vessel density. This method may enhance the efficacy of MSCs transplantation in acute cardiac infarction model, which provide a new ideas for the surgical treatment of myocardial infarction.
Objective To explore the impact of basic fibroblast growth factor (bFGF) and parathyroid hormone-related protein (PTHrP) on early and late chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) induced by transforming growth factor β1 (TGF-β1). Methods BMSCs were isolated from 3 healthy Japanese rabbits (2-month-old, weighing 1.6-2.1 kg, male or female), and were clutured to passage 3. The cells were put into pellet culture system and were divided into 5 groups according to different induce conditions: TGF-β1 group (group A), TGF-β1/bFGF group (group B), TGF-β1/21 days bFGF group (group C), TGF-β1/PTHrP group (group D), and TGF-β1/21 days PTHrP group (group E). At the beginning, TGF-β1 (10 ng/mL) was added to all groups, then bFGF and PTHrP (10 ng/mL) were added to groups B and D respectively; bFGF and PTHrP (10 ng/mL) were added to groups C and E at 21 days respectively. The gene expressions of collagen type I (Col I), Col II, Col X, matrix metalloproteinases (MMP)-13, and alkaline phosphatase (ALP) activity were detected once every week for 6 weeks. The 1, 9-dimethylmethylene blue (DMMB) staining was used to observe the extracellular matrix secretion at 6 weeks. Results The expression of Col I in groups C and E showed a significant downward trend after 3 weeks; the expression in group A was significantly higher than that in groups C and E at 4 and 5 weeks (P lt; 0.05), and than that in groups B and D at 3-6 weeks (P lt; 0.05); and significant differences were found between groups B and C at 3 and 4 weeks, and between groups D and E at 3 weeks (P lt; 0.05). After 3 weeks, the expressions of Col II and Col X in groups C and E gradually decreased, and were significantly lower than those in group A at 4-6 weeks (P lt; 0.05). Groups B and D showed no significant difference in the expressions of Col II and Col X at all time points, but there was significant difference when compared with group A (P lt; 0.05). MMP-13 had no obvious expression at all time points in group A; significant differences were found between group B and groups A, C at 3 weeks (P lt; 0.05); and the expression was significantly higher in group D than in groups A and E (P lt; 0.05). ALP activity gradually increased with time in group A; after 4 weeks, ALP activity in groups C and E obviously decreased, and was significantly lower than that in group A (P lt; 0.05); there were significant differences between groups B and C, and between groups D and E at 2 and 3 weeks (P lt; 0.05). DMMB staining showed more cartilage lacuna in group A than in the other groups at 6 weeks. Conclusion bFGF and PTHrP can inhibit early and late chondrogenic differentiation of BMSCs by changing synthesis and decomposition of the cartilage extracellular matrix. The inhibition is not only by suppressing Col X expression, but also possibly by suppressing other chondrogenic protein.
【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at 【Abstract】 Objective Sonic hedgehog (Shh) signaling pathway is involved in an important part of regulating angiogenesis. To investigate the effects of recombinant Shh N-terminant (rShh-N) on the expression and secretion of angiogenesis-related factor—vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Methods Bone marrow mesenchymal stem scells (BMSCs) were isolated from 3-day-old healthy Sprague Dawley rats and cultured to passage 3 in vitro. rShh-N at the concentrations of 0, 10, 100, and 200 ng/mL were applied to culture BMSCs in groups A, B, C, and D, respectively. At 12, 24, 48, and 72 hours of culture, the expressions of VEGF and bFGF mRNA and the levels of VEGF and bFGF in supernatant were measured with real-time quantitative PCR and ELISA, respectively. Results At the gene level, compared with group A, the expressions of VEGF and bFGF mRNA were enhanced in group D (P lt; 0.05) and the upregulation was more significant at 12 and 48 hours than 24 and 72 hours (P lt; 0.01). In group C, bFGF mRNA expression was substantially promoted at 12-72 hours (P lt; 0.05) and VEGF mRNA level was upregulated at 24-72 hours (P lt; 0.05), and both reached peak at 72 hours (P lt; 0.01). In group B, VEGF mRNA expression was inhibited at 12 hours (P lt; 0.05), but the level increased at 48 and 72 hours (P lt; 0.05); bFGF mRNA expression was obviously promoted at 12-48 hours (P lt; 0.05) and the maximum appeared at 48 hours (P lt; 0.01). At the protein level, the secretion of VEGF and bFGF in group D was significantly increased at 12-72 hours, as compared with group A (P lt; 0.05). In group C, VEGF and bFGF secretion was increased at 24-72 hours (P lt; 0.05). The secretion of VEGF in group B was inhibited at 12 and 48 hours (P lt; 0.05) and was promoted at 24 hours (P lt; 0.05); bFGF secretion was up-regulated at 24 and 48 hours (P lt; 0.05). The secretion of VEGF and bFGF in supernatant at
Objective To study the release properties of basic fibroblast growth factor (bFGF) chitosan microspheres prepared by cross-linking-emulsion method using chitosan as a carrier material so as to lay a foundation for further study. Methods Using 0.6% sodium tripolyphosphate solution as a crosslinking agent and 1.5% solution of chitosan as a carrier material, bFGF chitosan microspheres were prepared by cross-linking-emulsion method. Laser particle size analyzer and Zeta electric potential analyzer were used to measure the particle diameter distribution, scanning electronic microscope to observe the morphology, and ELISA to determine the drug loading, the encapsulation rate, and the drug release properties. Results The particle size of bFGF chitosan microspheres ranged 20.312-24.152 μm. The microspheres were round with a smooth surface and uniform distribution, and it had no apparent porosity. The drug loading and encapsulation rate of microspheres were (7.57 ± 0.34) mg/g and 95.14% ± 1.58%, respectively. The bFGF chitosan microspheres could continuously release bFGF for 24 days; the bFGF level increased gradually with time and reached (820.45 ± 21.34) ng/mL at 24 days; and the microspheres had a burst effect, and the burst rate was 18.08%, and the accumulative release rate of the microspheres reached 82.05% during 24 days. Conclusion It is easy-to-operate to prepare the bFGF chitosan microspheres with the cross-linking-emulsion method. The bFGF chitosan microspheres have smooth surface, uniform distribution, and no apparent porosity.
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.
Objective To assess the effect of basic fibroblast growth factor (bFGF) and 5-fluorouracil (5-FU) appl ied topically on the tendon adhesion and the heal ing process after the flexor tendon repair in Leghorn chickens. Methods Ninety male Leghorn chickens (weighing 3.0-3.5 kg) were randomly divided into 3 groups, with 30 chickens in each group. The flexordigitorum profundus tendons of the third right toes were transected and sutured directly. The repair site in group A was given 0.6 μL fibrin sealant (FS). In group B, the repair site was given 0.6 μL FS containing 500 ng bFGF. In group C, before the tendons were transected, they had been soaked in 5-FU solution, and then the same treatment as group B was given. Six specimens of the third toe were harvested to perform the macroscopical and histological examinations at 1, 2, 4, and 8 weeks, respectively, and to perform the biomechanical test at 8 weeks. Results All animals survived until the experiment was completed. All incisions healed smoothly. No rupture occurred in the reparied tendon. At 8 weeks, the adhesion degree was l ighter in group C than in group B (P lt; 0.05), but there was no significant difference in the adhesion degree between group A and groups B, C (P gt; 0.05). At 1, 2, and 4 weeks after operation, the number of fibroblast cells of group A was significantly less than that of group B (P lt; 0.05), and the number of fibroblast cells of group C was significantly less than that of group A and group B in the tendon sheath and epitenon (P lt; 0.05); however, it was significantly more than that of group A in the tendon parenchyma (P lt; 0.05), and no significant difference was observed when compared with that of group B (P gt; 0.05). At 8 weeks, no difference was found among 3 groups (P gt; 0.05). The collagen fiber content of group A was significantly less than that of group B at 4 and 8 weeks (P lt; 0.05). In the sheath and epitenon, the collagen fiber content of group A was significantly more than that of group C at 4 weeks (P lt; 0.05); however, no significant difference was found between 2 groups at 8 weeks (P gt; 0.05). The collagen fiber content of group A wassignificantly less than that of group C in the parenchyma at 4 and 8 weeks (P lt; 0.05). At all time points, the collagen fiber content of group B was significantly more than that of group C in the sheath and epitenon (P lt; 0.05), but no significant difference in the parenchyma was observed between 2 groups (P gt; 0.05). The biomechanical tests showed that the gl iding excursion of the tendon in groups A, B, and C was (3.51 ± 0.56), (2.84 ± 0.42), and (4.56 ± 0.59) mm, respectively; the work of flexion was (14.08 ± 1.85), (20.62 ± 3.52), and (10.91 ± 1.53) N.mm, respectively; and the ultimate tensile strength of the tendon was (11.26 ± 1.83), (15.02 ± 2.20), and (14.40 ± 1.57) N, respectively. There were significant differences in the gl iding excursion of the tendon and the work of flexion among 3 groups (P lt; 0.05) and in the ultimate tensile strength of the tendon between group A and groups B, C (P lt; 0.05), but there was no significant difference in the ultimate tensile strength of the tendon between group B and group C (P gt; 0.05). Conclusion Local single-use bFGF and 5-FU can not only effectively promote the heal ing of flexor tendon, but also significantly reduce tendon adhesion.
Objective To study the expression changes of vascular endothel ial growth factor (VEGF), basic fibroblast growth factor (bFGF), and bone morphogenetic protein 2 (BMP-2) in femoral neck fracture, traumatic, and non-traumatic avascular necrosis of femoral head (ANFH), and to study the relationshi p between the expressions of VEGF, bFGF, BMP-2mRNA and bone mass so as to explore the pathogenesis of ANFH and provide the exprimental basis for individual treatment of ANFH. Methods Femoral head specimens were obtained from 59 donors undergoing total hip replacement, including 22 cases of traumatic ANFH (group A, 13 cases of Ficat stage III and 9 cases of Ficat stage IV), 19 cases of non-traumatic ANFH (group B, 11 cases of Ficat stage III and 8 cases of Ficat stage IV; 10 cases of steroid-induced ANFH, 7 cases of alcohol ic ANFH, and 2 cases of unexplained ANFH), and 18 cases of fresh femoral neck fracture (group C). There was no significant difference in the general data among 3 groups (P gt; 0.05). The bone mineral density (BMD) at weight-bearing area of the femoral head was measured with dual energy X-ray absorptiometry. The pathological changes were observed by using optical microscope and scanning electron microscope. The percentage of empty bone lacuna and the percentage of trabecular bone area were calculated. The expressions of VEGF, bFGF, and BMP-2 mRNA in femoral head were detected by use of in-situ hybridization technique. Results The BMD in groups A and B were significantly lower than that in group C (P lt; 0.05), and there was significant difference between group A and group B (P lt; 0.05). In the necrosis area of groups A and B, the bone trabecula was rarefactive and not of integrity, with a great number of empty bone lacuna. In healthy area, more fiber hyperplasia was observed in group A, the prol iferated and hypertrophic fat cells in the medullary cavity in group B. Scanning electron microscope showed that many osteocytes underwent fatty degeneration and necrosis, and that the prol iferation of fat cells in bone matrix was observed in groups A and B. While in group C, the femoral head had intact articular cartilage and intact bone trabeculae, and osteocytes were clearly seen. The percentage of empty bone lacuna was significantly higher (P lt; 0.05) and the percentage of trabecular bone area was significantly lower (P lt; 0.05) in groups A and B than group C; and there was significant difference in the percentage of empty bone lacuna between groups A and B (P lt; 0.05). The expressions of VEGF, bFGF, and BMP-2 mRNAwere significantly lower in groups A and B than group C (P lt; 0.05), and the expressions of BMP-2 and bFGF mRNA in group A were significantly higher than those in group B (P lt; 0.05). There were positive l inear correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and the BMD and percentage of trabecular bone area, respectively. While there were significantly negative correlation between the expressions of VEGF mRNA, bFGF mRNA, BMP-2 mRNA and percentage of empty bone lacuna. Conclusion The repair capacity of local femoral head in traumatic ANFH is ber than that in non-traumatic ANFH. The expressions of VEGF mRNA, bFGF mRNA, and BMP-2 mRNA decl ine in traumatic and nontraumatic ANFH.
Objective To investigate the efficacy of basic fibroblast growth factor (bFGF) combined with topical oxygen therapy for deep II degree burn wounds, by comparing the effects of bFGF combined with topical oxygen therapy and bFGF with routine therapy. Methods From February 2004 to July 2009, 85 patients with deep II degree burn wounds (117 wounds) were enrolled and divided into 4 groups randomly according to different treatments. There was no significant difference in sex, age, disease course, wound size, and wound treatment size among 4 groups (P gt; 0.05). In group A, 18 patients (28 wounds) were treated routinely; in group B, 23 patients (30 wounds) were treated with routine methods and topical oxygen therapy; in group C, 19 patients (25 wounds) were treated with routine methods and bFGF therapy; and in group D, 25 patients (34 wounds) were treated with routine methods and bFGF/topical oxygen therapy. Topical oxygen therapy was administered to the wound for 90 minutes per day for 3 weeks. The bFGF therapy was appl ied everyday (150 U/ cm2) for 3 weeks. Results All cases were followed up 6-12 months (9 months on average). The wound heal ing times in groups A, B, C, and D were (27.3 ± 6.6), (24.2 ± 5.8), (22.2 ± 6.8), and (18.2 ± 4.8) days, respectively; showing significant difference between group A and group D (P lt; 0.05). The wound heal ing rates in groups A, B, C, and Dwere 67.8% ± 12.1%, 85.1% ± 7.5%, 89.2% ± 8.3%, and 96.1% ± 5.6%, respectively; showing significant differences between group A and groups B, C, D (P lt; 0.05). The therapic effective rates in groups A, B, C, and D were 75%, 90%, 92%, and 100%, respectively; showing significant difference between group A and group D (P lt; 0.05). The Vancouver scar scale scoring of group D 6 months after treatment was better than that of group A (P lt; 0.05). Conclusion The bFGF combined with topical oxygen therapy can enhance deep II degree burn wound heal ing. Furthermore, the therapy method is simple and convenient.