【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
ObjectiveTo investigate the effect of transforming growth factorβ1 (TGF-β1) and basic fibroblast growth factor 1 (bFGF-1) on the cellular activities, prol iferation, and expressions of ligament-specific mRNA and proteins in bone marrow mesenchymal stem cells (BMSCs) and ligament fibroblasts (LFs) after directly co-cultured.
MethodsBMSCs from 3-month-old Sprague Dawley rats were isolated and cultured using intensity gradient centrifugation. LFs were isolated using collagenase. The cells at passage 3 were divided into 6 groups: non-induced BMSCs group (group A), non-induced LFs group (group B), non-induced co-cultured BMSCs and LFs group (group C), induced BMSCs group (group D), induced LFs group (group E), and induced co-cultured BMSCs and LFs group (group F). The cellular activities and prol iferation were examined by inverted contrast microscope and MTT; the concentrations of collagen type Ⅰ and type Ⅲ were determined by ELISA; and mRNA expressions of collagen types I andⅢ, fibronectin, tenascin C, and matrix metalloproteinase 2 (MMP-2) were measured by real-time fluorescent quantitative PCR.
ResultsA single cell layer formed in the co-cultured cells under inverted contrast microscope. Group F had fastest cell fusion ( > 90%). The MTT result indicated that group F showed the highest absorbance (A) value, followed by group D, and group B showed the lowest A value at 9 days after culture, showing significant difference (P < 0.05). Moreover, the result of ELISA showed that group F had the highest concentration of collagen type Ⅰ and type Ⅲ (P < 0.05); the concentration of collagen type Ⅲ in group E was significantly higher than that in group D (P < 0.05), but no significant difference was found in the concentration of collagen type Ⅰ between 2 groups (P > 0.05). The ratios of collagen type Ⅰ to type Ⅲ were 1.17, 1.19, 1.10, 1.25, 1.17, and 1.18 in groups A-F; group D was higher than the other groups. The real-time fluorescent quantitative PCR results revealed that the mRNA expressions of collagen type Ⅰ and type Ⅲ and fibronectin were highest in group F; the expression of tenascin C was highest in group D; the expression of MMP-2 was highest in group E; and all differencs were significant (P < 0.05).
ConclusionDirectly co-cultured BMSCs and LFs induced by TGF-β1 and bFGF-1 have higher cellular activities, proliferation, and expressions of ligament-specific mRNA and protein, which can be used as a potential source for ligament tissue engineering.
Inflammatory myofibroblastic tumor (IMT) occurring in the pancreas was rare and few clinical cases had been reported. The specificity of clinical presentation of IMT was lacked and its CT imaging manifestations were diverse. The enhanced CT images of a rare patient with IMT of the pancreas with liver metastases were presented to the readers, and the pathophysiological mechanisms associated with the CT imaging manifestations were briefly described, so as to enhance the readers’ understanding and knowledge of the CT imaging manifestations and raise the diagnostic awareness of the disease.
ObjectiveTo investigate the effect of Wnt5a derived from tumor-associated fibroblasts （CAFs） on the migration and invasion of gastric cancer cells. MethodsThe differentially expressed genes Wnt5a between CAFs and normal gastric fibroblasts (NGFs) in gastric cancer tissues and their corresponding normal gastric tissues using the GEO database GSE194261 dataset were screened. Immunohistochemical method was used to detect the expression of Wnt5a protein in tissue samples of clinical gastric cancer patients, and the relationship between Wnt5a protein expression and clinicopathological features of gastric cancer was analyzed. CAFs and NGFs were extracted from fresh surgical specimens of gastric cancer patients, and the expression of Wnt5a in CAFs was detected by real-time fluorescence quantitative-polymerase chain reaction and Western blot experiment. Transwell invasion and migration experiment was used to observe the effects of CAFs, inhibition of Wnt5a expression in CAFs and different concentrations of recombinant Wnt5a protein on the migration and invasion ability of gastric cancer MGC-803 and MKN-28 cell lines in vitro. ResultsThrough the screening of GEO database GSE194261 data set, it was found that Wnt5a was more expressed in CAFs than NGFs (P<0.05). Immunohistochemical results showed that the expression of Wnt5a protein in gastric cancer tissues was significantly stronger than that in normal gastric tissues (P<0.05), and the expression of Wnt5a protein was related to T stage of tumor (χ2=5.035, P<0.05), but not related to gender, age, degree of tumor differentiation, lymph node metastasis, vascular invasion and nerve invasion (P>0.05). Inhibiting Wnt5a derived from CAFs could inhibit the invasion and migration of gastric cancer cells. By stimulating gastric cancer cells with different concentrations of human recombinant Wnt5a protein, it was found that when the concentration of human recombinant Wnt5a protein was greater than 100 ng/mL, the invasion and migration abilities of MGC-803 and MKN-28 gastric cancer cells were significantly increased (P<0.05). ConclusionWnt5a is highly expressed in CAFs derived from the interstitial tissue of gastric cancer, which is related to the invasion depth of gastric cancer and can promote the invasion and migration of gastric cancer cells.
ObjectiveTo discuss whether human amniotic mesenchymal stem cells (hAMSCs) possesses the characteristic of mesenchymal stem cells, and could differentiate into ligament cells in vitro after induction.
MethodsThe hAMSCs were separated through enzyme digestion, and the phenotypic characteristics of hAMSCs were tested through flow cytometry. The cells at passage 3 were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1)+basic fibroblast growth factor (bFGF) (group A), containing hyaluronic acid (HA) (group B), containing TGF-β1+bFGF+HA (group C), and simple L-DMEM/F12 medium (group D) as control group. The morphology changes of cells in each group were observed by inverted phase contrast microscope at 21 days after induction; the cellular activities and proliferation were examined by sulforhodamine (SRB) colorimetric method; and specific mRNA and protein expressions of ligament including collagen type I, collagen type III, and tenascin C (TNC) were measured by real-time fluorescence quantitative PCR and immunohistochemical staining.
ResultsThe flow cytometry result indicated that hAMSCs expressed mesenchymal stem cell phenotype. After 21 days of induction, the cells in groups A, B, and C grew like spindle-shaped fibroblasts under inverted phase contrast microscope, and cells showed single shape, obvious directivity, and compact arrangement in group C. The SRB result indicated that the cells in each group reached the peak of growth curve at 6 days; the cellular activities of groups A, B, and C were significantly higher than that of group D at 6 days after induction. Also, the immunohistochemical staining results showed that no expressions of TNC were detected in 4 groups at 7 days; expressions of collagen type I in groups A, B, and C were significantly higher than that in group D at 7, 14, and 21 days (P<0.001); the expressions of collagen type III in groups A, B, and C were significantly higher than that in group D at 14 and 21 days (P<0.001). There was an increasing tendency with time in collagen type I of group B, in collagen type III and TNC of groups A and C, showing significant difference among different time points (P<0.001). The real-time fluorescence quantitative PCR results revealed that the mRNA expressions of collagen type I and TNC in group C were significantly higher than those in groups A and B (P<0.05), and the mRNA expression of collagen type III in group B were significantly higher than that in groups A and C at 21 days (P<0.05). The mRNA expressions of collagen type I and TNC in groups A and C and mRNA expression of collagen type III in group C had an increasing tendency with time, showing significant difference among different time points (P<0.001).
ConclusionThe hAMSCs possesses the characteristics of mesenchymal stem cells and excellent proliferation capacity. After in vitro induction, the expressions of ligament specific genes can be up-regulated and the synthesis of ligament specific proteins can be also strengthened. As a result, it can be used as one of ligament tissue engineering seed cell sources.
Human fibroblasts and human epidermal keratinocytes were used for culture. Chitosan solution were added in the culture solution(DMEM). After 72 hours, the fibroblasts showed rapid growth in the control culture without Chitosan, But the numbers of human fibroblasts from growth was decreased as the concentration of Chitosan was increasing. On the contrary the human epidermal keratinocytes growed more rapidly in the culture with Chitosan than in the culture without Chitosan. The results showed that Chitosan inhibited the growwth of human fibroblast and stimulated the growth of human epidermal keratinocyte .
ObjectiveTo study the effects of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (hBMSCs).
MethodshBMSCs at passage 4 were divided into 4 groups according to different culture conditions:cells were treated with complete medium (α-MEM containing 10%FBS, group A), with complete medium containing 10 ng/mL LIF (group B), with complete medium containing 10 ng/mL bFGF (group C), and with complete medium containing 10 ng/mL LIF and 10 ng/mL bFGF (group D). The growth curves of hBMSCs at passage 4 in different groups were assayed by cell counting kit 8; cellular morphologic changes were observed under inverted phase contrast microscope; the surface markers of hBMSCs at passage 8 including CD44, CD90, CD19, and CD34 were detected by flow cytometry.
ResultsThe cell growth curves of each group were similar to the S-shape; the cell proliferation rates in 4 groups were in sequence of group D > group C > group B > group A. Obvious senescence and differentiation were observed very early in group A, cells in group B maintained good cellular morphology at the early stage, with slow proliferation and late senescence; a few cells in group C differentiated into nerve-like cells, with quick proliferation; and the cells in group D grew quickly and maintained cellular morphology of hBMSCs. The expressions of CD44 and CD90 in groups A and C at passage 8 cells were lower than those of groups B and D; the expressions of CD19 and CD34 were negative in 4 groups, exhibiting no obvious difference between groups.
ConclusionLIF combined with bFGF can not only maintain multiple differentiation potential of hBMSCs, but also promote proliferation of hBMSCs.
Objective
To investigate the role and mechanism of S100 calcium binding protein B (S100B) in osteoarthritis (OA) cartilage damage repair.
Methods
Twenty New Zealand rabbits were randomly divided into control group and model group, with 10 rabbits in each group. Rabbits in the model group were injured by the right knee joint immobilization method to make the artilage injury model, while the control group did not deal with any injury. After 4 weeks, the levels of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) in synovial fluid were detected by ELISA method; the mRNA and protein expressions of S100B, fibroblast growth factor 2 (FGF-2), and FGF receptor 1 (FGFR1) in cartilage tissue were examined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot assay. Human synovial fibroblasts (SF) were isolated and cultured in vitro. The effects of S100B overexpression and knockdown on the levels of IL-1β and TNF-α (ELISA method) and the expressions of FGF-2 and FGFR1 gene (qRT-PCR) and protein (Western blot) were observed. Moreover, the effects of FGFR1 knockdown in above S100 overexpression system on the levels of IL-1β and TNF-α (ELISA method) and the expressions of FGF-2 and FGFR1 gene (qRT-PCR) and protein (Western blot) were observed.
Results
ELISA detection showed that the expressions of IL-1β and TNF-α in the synovial fluid of the model group were significantly higher than those of the control group (P<0.05); qRT-PCR and Western blot detection showed that the mRNA and protein expressions of S100B, FGF-2, and FGFR1 in cartilage tissue were significantly higher than those of the control group (P<0.05). Overexpression and knockdown S100 could respectively significantly increase and decrease lipopolysaccharides (LPS) induced IL-1β and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1 (P<0.05); whereas FGFR1 knockdown could significantly decrease LPS induced IL-1β and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1 (P<0.05).
Conclusion
S100B protein can regulate the inflammatory response of SF and may affect the repair of cartilage damage in OA, and the mechanism may be related to the activation of FGF-2/FGFR1 signaling pathway.
Objective To review the research progress on acid fibroblast growth factor (aFGF) in promoting tissue injury repair and its mechanism of action. Methods By searching and reviewing the basic and clinical studies on aFGF published in recent years, the roles of aFGF in tissue injury including full-thickness skin, mucous and skin barrier, bone and nerve fiber were summarized. Results As a key member of the FGF family, aFGF exhibits potent mitogenic activity, it can regulate various cells proliferation and migration, accelerate extracellular matrix synthesis, promote angiogenesis and nerve fiber repair, upregulate tight junction protein expression, and therefore exert dual regulatory effects on dermal and epidermal regeneration and repair. It demonstrates promising clinical application for full-thickness healing and skin and mucosal barriers repair. Additionally, it mediates the regeneration and differentiation of osteoblasts, cardiomyocytes, and follicle cells, exhibiting potential for repairing multiple tissues and organs. Furthermore, the aFGF's functions in regulating energy metabolism, immune-inflammatory responses, and alleviating aging have revealed in recent years, indicating a broad clinical application. Conclusion aFGF is a valuable member of the FGF family. It is widely used in various kinds of wound healing, besides, it also holds promising application in multiple tissue and organ regeneration and repair.
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.
