To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.
Cells, Cultured ; DNA, Complementary/genetics ; Escherichia coli/genetics ; Eukaryotic Cells/metabolism ; Fibroblast Growth Factor 2/biosynthesis ; Fibroblast Growth Factor 2/*genetics ; Gene Transfer Techniques ; Osteoblasts/cytology ; Osteoblasts/*metabolism ; Plasmids ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; *Transfection ; Transformation, Genetic

OBJECTIVETo preliminarily investigate the mechanism of small interfering RNA (siRNA) induced apoptosis in glioma U251 cells by silencing basic fibroblast growth factor (bFGF).

METHODSU251 cells were divided into the normal control group, the mock group and experiment group, the mock and experiment group were transfected with mock vector (Ad-null) and the recombinant adenovirus carrying bFGF-siRNA (Ad-bFGF-siRNA) respectively at a multiplicity of infection (MOI) of 100. After 72 hours, the expression of related proteins was revealed by the method of Western blot. Mitochondrial transmembrane potential (ΔΨm) was measured with flow cytometry and confocal microscopy, Groups were compared using single factor analysis of variance (One-way ANOVA).

RESULTSAfter U251 cells were transfected with bFGF-siRNA, the results of Western blot showed that after 72 hours of transfection the bFGF protein in the experiment group decreased obviously, meanwhile Cytochrome C, Caspase-3 and Bax showed increased expression while in the Bcl-xl and Bcl-2 proteins decreased expression. The proportion of high mitochondrial membrane potential of cells by flow cytometry, the experimental group was 74.4% ± 4.7% decreased significantly compared with the control group 92.1% ± 2.5%, the mock group 90.9% ± 1.8% (F = 28.805, P < 0.05); laser scanning confocal microscopy results showed that the red fluorescence and green fluorescence ratio of the experimental group was 0.83 ± 0.12 decreased significantly compared with 1.36 ± 0.40 of the control group and 1.32 ± 0.35 of the mock group(F = 7.920, P < 0.05).

CONCLUSIONsiRNA targeting bFGF induced U251 cell apoptosis may be achieved through the mitochondrial pathway.

Adenoviridae ; genetics ; Apoptosis ; Cell Line, Tumor ; Fibroblast Growth Factor 2 ; genetics ; Glioma ; pathology ; Humans ; RNA Interference ; RNA, Small Interfering ; Transfection

Adult ; Aged ; Female ; Fibroblast Growth Factor 1 ; genetics ; Fibroblast Growth Factor 2 ; genetics ; Humans ; Middle Aged ; Neoplasm Staging ; Neoplasms, Glandular and Epithelial ; metabolism ; pathology ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; analysis ; Receptor Protein-Tyrosine Kinases ; genetics ; Receptor, Fibroblast Growth Factor, Type 1 ; Receptors, Fibroblast Growth Factor ; genetics

OBJECTIVETo investigate the relationship between expression of angiogenic factors and invasion and proliferation of pancreatic cancer cell.

METHODSThe three pancreatic cancer cell lines of SW1990, Panc-1 and PCT-3 were divided into four groups respectively: control group, VEGF siRNA group, bFGF siRNA group and VEGF siRNA + bFGF siRNA group. The expression and the secretion of VEGF and bFGF in the three cell lines were inhabited by VEGF siRNA and bFGF siRNA. The proliferation and the invasion of the three cell lines were determined by CCK-8 and Boyden Chamber invasion tests.

RESULTSExpressions of VEGF and bFGF in three cell lines were significantly inhibited by VEGF siRNA and bFGF siRNA. The proliferation was inhabited by VEGF siRNA and bFGF siRNA in SW1990 and Panc-1 (P < 0.05), while was not in PCT-3 (P > 0.05). The invasion was inhabited significantly by VEGF siRNA and bFGF siRNA in the three cell lines (P < 0.05). Combination of VEGF siRNA and bFGF siRNA resulted in more efficient influence in inhibition of invasion in Panc-1 and proliferation in PCT-3 and SW1990 than VEGF siRNA or bFGF siRNA individually.

CONCLUSIONThe decreased expression of VEGF and bFGF can inhabited the ability of invasion and proliferation of pancreatic cancer cell.

Cell Line, Tumor ; Cell Proliferation ; Fibroblast Growth Factor 2 ; genetics ; Humans ; Neoplasm Invasiveness ; Pancreatic Neoplasms ; pathology ; RNA, Small Interfering ; genetics ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVE: To investigate the expression of fibroblast growth factor receptor 2 (FGFR2) in clear cell renal cell carcinoma (ccRCC; or kidney renal clear cell carcinoma, KIRC), to analyze the relationship between the expression of FGFR2 and the clinical pathological features and prognosis of ccRCC, to study the relationship between the expression of FGFR2 and other molecules, and to explore its role in the development of ccRCC. METHODS: Gene expressional and clinical information of ccRCC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus(GEO) database. Next, the data were transformed and collated. In the study, 104 clinical ccRCC samples and corresponding paracancerous normal tissue samples were collected from Department of Urology, Peking University First Hospital. Immunohistochemistry (IHC) was performed and the staining results were scored, so as to compare the expression of FGFR2 in ccRCC and paracancerous normal tissues. Besides, quantify real-time polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of FGFR2 in normal renal epithelial cell lines (293) and ccRCC cell lines (786-O, 769-P, OSRC-2, Caki-1, ACHN, and A498). In addition, the relationship between FGFR2 expression and clinical pathological characteristics (including TNM staging and pathological grading) and survival prognosis in ccRCC patients was further analyzed. Furthermore, the relationship between FGFR2 expression and B cells, T cells, natural killer (NK) cells and neutrophil infiltration in the ccRCC patients was analyzed, and the Biological General Repository for Interactionh Datasets (BioGRID) was used to builds protein-protein interaction (PPI) networks to study molecules that interacted with the FGFR2 protein. RESULTS: In the TCGA database, the expression of FGFR2 was down-regulated in ccRCC tissue samples compared with normal tissue samples, and the expression in the GEO database also showed this differences. Furthermore, FGFR2 expression was downregulated in ccRCC clinical samples and ccRCC cell lines, compared with corresponding paracancerous normal tissue or normal renal epithelial cell lines. In addition, FGFR2 high expression was associated with earlier, lower-level ccRCC and was associated with a better prognosis in the patients with ccRCC. Moreover, FGFR2 expression was not significantly related to B cells, T cells, NK cells and neutrophil infiltration, and the PPI network showed that FGFR2 protein interacted with certain molecules. CONCLUSION Our work sheds light on the potential role of FGFR2 in the development of ccRCC, suggesting that FGFR2 may serve as a prognostic marker and potential therapeutic target for patients with ccRCC.
Biomarkers, Tumor/analysis* ; Carcinoma, Renal Cell/pathology* ; Humans ; Kidney Neoplasms/pathology* ; Prognosis ; Receptor, Fibroblast Growth Factor, Type 2/genetics*

OBJECTIVETo investigate the expression of bFGF, ET-1 and SCF in different passages of cultured dermal papilla cells (DPC), and their possible effect on biological behaviour of DPC.

METHODSThe expression of bFGF, ET-1 and SCF in different passages of cultured DPC was detected by immunocytochemistry and in situ hybridization.

RESULTThe expression of ET-1 and SCF in early passages of cultured DPC was stronger, but became negative in late passages (>6 passages). The stronger the expression of ET-1 and SCF in DPC, the higher ability of DPC to induce hair follicle regeneration.

CONCLUSIONThe expression strength of ET-1 and SCF is related to the ability of DPC inducing hair follicle regeneration.

Endothelin-1 ; analysis ; genetics ; Fibroblast Growth Factor 2 ; analysis ; genetics ; Hair Follicle ; chemistry ; cytology ; physiology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Stem Cell Factor ; analysis ; genetics

OBJECTIVETo investigate the mechanism and the accelerating effect of rhEGF and rhbFGF on wound healing.

METHODSTwelve New Zealand rabbits with 72 incised wounds on ventral side of 24 ears were randomly divided into two therapeutic groups (rhEGF of 10 ug/cm(2) and rhbFGF of 100 AU/cm(2)) and a control group (1% silver sulfadiazine cream, SD-Ag). The general conditions of the wound healing was observed grossly. Biopsies were harvested at different time points for the pathomorphological examination, the electron microscopic examination, and for assessment of integrin beta1 mRNA expression by in situ hybridization.

RESULTSThe expressions of integrin beta 1 mRNA in two therapeutic groups were significantly higher than that of control group. The quality of the wound healing was improved in therapeutic group with its healing time shortened when compared with that in control group (P < 0.05). There was an obvious difference in the number of fibroblasts and capillary gemmules between the therapeutic and control groups (P < 0.05).

CONCLUSIONThe wound healing and quality could be improved by both rhEGF and rhbFGF, but rhbFGF seemed better to be employed during the early and middle stages of the wound repair for the growth of granulation tissue, while rhEGF should be applied at the late stage of wound repair to accelerate the re-epithelialization of the wound. Combined application of rhEGF with rhbFGF according to time effect could be more beneficial to the wound repair.

Animals ; Epidermal Growth Factor ; pharmacology ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; Integrin beta1 ; genetics ; Male ; Microscopy, Electron ; RNA, Messenger ; analysis ; Rabbits ; Recombinant Proteins ; pharmacology ; Wound Healing ; drug effects

OBJECTIVETo study the expressions of basic fibroblast growth factor (bFGF) and its receptor (bFGFR) in bone marrow of mice with acute radiation injury, and to evaluate the effect of Ligustrazine (Lt) on them.

METHODSFifty-six Kunming mice of clean grade were randomly divided into 3 groups, the normal group, the control group and the Lt group. Mice in the latter two groups were once homogeneously systemic irradiated with 6.0 Gy of 60Co, with the absorption dose rate of 0.56 Gy/min, then treated with saline (0.2 ml/mice) or Lt (2 mg/mice) respectively, twice a day through gastrogavage for successive 13 days. Mice were sacrificed in batch on the 3rd, 7th and 14th day by cervical dislocation to collect the bilateral femoral bone marrow for preparing bone marrow mono-nuclear cell (BMMNC) suspension. The bFGFR expression on surface of BMMNC was determined by flow cytometry; and the bFGF expression level in one side of femoral bone marrow tissue was detected by immunohistochemistry with SABC-AP assay.

RESULTSThe bFGF expression in bone marrow of mice on the 3rd, 7th and 14th day after acute radiation injury all were significantly lower than that of the normal mice (P < 0.05 or P < 0.01). The expressions of bFGF and bFGFR in the Lt group detected were significantly higher than that in the control group detected at the corresponding time points (P < 0.05 or P < 0.01).

CONCLUSIONBy way of enhancing bFGF expression in bone marrow and bFGFR expression on surface of BMMNC to accelerate the repairing of homopoietic micro-environment in bone marrow might be one of the mechanisms of Lt in promoting hemopoietic function reconstitution after acute radiation injury.

Animals ; Bone Marrow Cells ; metabolism ; Female ; Fibroblast Growth Factor 2 ; biosynthesis ; genetics ; Hematopoiesis ; drug effects ; Male ; Mice ; Pyrazines ; pharmacology ; Radiation Injuries, Experimental ; metabolism ; Random Allocation ; Receptors, Fibroblast Growth Factor ; biosynthesis ; genetics

OBJECTIVETo investigate the association of fibroblast growth factor receptor 2 gene (FGFR2) rs2981582 polymorphism with breast cancer in Chinese women.

METHODSA case-control study was performed in 936 breast cancer patients and 471 patients with benign breast diseases by using a novel fluorescent quantitative PCR method.

RESULTSThe numbers and frequencies of genotypes CC, CT, and TT in the control group were 234(49.68%), 181(38.43%) and 56(11.89%) respectively. The numbers and frequencies of genotypes CC, CT, and TT in the breast cancer group were 426(44.56%), 400(41.84%) and 130(13.60%) respectively. And no significant difference was found between the two groups (P=0.183). However, stratified analysis found that the numbers and frequencies of genotypes CC, CT, TT in the estrogen receptor(ER) positive subgroup of breast cancer patients were 189(41.27%), 202(46.12%) and 67(14.63%) respectively, and significant difference was observed compared with control group (P=0.035).

CONCLUSIONAssociation was found in the single nucleotide polymorphism(SNP) of the rs2981582 locus of intron 2 in FGFR2 gene between the ER positive breast cancer patients and control patients with benign breast diseases. The fluorescent quantitative PCR is a specific, easy-to-operate, low-expense method and is suitable for SNP detection in large scale samples.

Breast Neoplasms ; genetics ; Female ; Genetic Predisposition to Disease ; Humans ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; genetics ; Receptor, Fibroblast Growth Factor, Type 2 ; genetics ; Receptors, Estrogen ; genetics

Cell Line ; Epidermal Growth Factor ; biosynthesis ; genetics ; Fibroblast Growth Factor 2 ; biosynthesis ; genetics ; Hepatocytes ; metabolism ; Humans ; Interleukin-10 ; pharmacology ; Platelet-Derived Growth Factor ; pharmacology ; Proto-Oncogene Proteins c-sis ; RNA, Messenger ; biosynthesis ; genetics