OBJECTIVE: To explore the expression of alpha-smooth muscle actin (alpha-SMA) induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: Five samples of hypertrophic scars and three samples of normal mature scars were collected as the experimental and control groups respectively. The fibroblasts were isolated from scars, and cultured in 2-dimension or 3-dimension culture system. The immunohistochemical staining method of LSAB were used to investigate the expression of alpha-SMA in fibroblasts in the different concentration of TGF-beta 1. RESULTS: The expression of alpha-SMA in 3-dimension culture system were markedly lower than those in 2-dimension culture system with respect to the fibroblasts in the experimental group. The expression of alpha-SMA in fibroblasts were different in response to various TGF-beta 1 concentration, it was more effective at the concentration of 5 ng/ml. The expression of alpha-SMA in the fibroblasts from hypertrophic scars seemed to be more sensitive to TGF-beta 1 compared to that of the normal mature scars. CONCLUSION: There are concentration-dependent in the expression of alpha-SMA induced by TGF-beta 1 in scar fibroblasts in vitro. The biological characteristics of the fibroblasts from hypertrophic scars and normal mature scars and their sensitivity to the inducement of TGF-beta 1 were different. The inducement of TGF-beta 1 may be depressed by extracellular matrix components and that may decrease the expression of alpha-SMA.
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.
OBJECTIVE: To study the effect of overexpression of truncated type II TGF-beta receptor on transforming growth factor-beta 1(TGF-beta 1) autoproduction in normal dermal fibroblasts. METHODS: In vitro cultured dermal fibroblasts were treated with recombinant human TGF-beta 1(rhTGF-beta 1) (5 ng/ml) or recombinant adenovirus containing truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-beta 1 were observed with Northern blotting. RESULTS: rhTGF-beta 1 up-regulated the gene expression of TGF-beta 1 and type I procollagen. Overexpression of truncated receptor II down-regulated the gene expression of TGF-beta 1. CONCLUSION: Overexpression of the truncated TGF-beta receptor II decreases TGF-beta 1 autoproduction via blocking TGF-beta receptor signal. The results may provided a new strategy for scar gene therapy.
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.
OBJECTIVE To study the relationship between the changes of mRNA expression in wound tissues of diabetic ulcers and tissue repair. METHODS The mRNA expression of TGF-beta 1 and IL-6 in eight bioptic samples of diabetic ulcers were detected by RT-PCR and pathologic methods, and the surrounding normal skins from the same patients were measured as control group. RESULTS The mRNA expression levels of TGF-beta 1 were markedly decreased in the diabetic ulcers compared with control group, while the mRNA expression levels of IL-6 were increased at the same reaction conditions. CONCLUSION The different changes of mRNA expression level of TGF-beta 1 and IL-6 in wound tissue result in low production and decreased activity of TGF-beta 1 and IL-6, which lower the reparative ability of wound tissue.
Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) gene transfer on the biological characteristics of osteoblasts. Methods The expression of TGF-β1 in the transfected osteoblasts was detected by in situ hybridization and assay of TGF-β1 activity in the supernatant (minklung epithelium cell growth -inhibition test). The effects of gene transfer andsupernatant of the transfected osteoblasts on the proliferation and alkaline phosphatase(ALP) activity of osteoblasts were detected by 3 H-TdR and MTT. Results The results of in situ hybridization analysis suggested that the osteoblasts transfected by TGF-β1 gene could express TGF-β1 obviously. The complex medium, which was the mixture of serum-free DMEM and the activated supernatant according to 1∶1, 1∶2, 1∶4, could inhibit growth of Mv-1-Lu evidently and the ratios ofinhibition were 16.3%, 22.7%, 28.2% respectively. TGF-β1 gene transfer hadno effect on the biological characteristics of osteoblasts, but the activated supernatant of transfected osteoblasts stimulated proliferation and inhibited ALPactivity of osteoblasts. Conclusion TGF-β1 gene transfer promotes the expression of TGF-β1 and the biological characteristics of trasfected osteoblasts are stable, which is helpful for gene therapy of bone defects in vivo.
Objective To study the effect of serum rich in growth factors (SRGF) derived from plateletrich plasma (PRP) on the biological function of human and rat osteoblast.Methods PRP and platelet-poor plasma (PPP) obtained from healthy human and SD rat were activated by thrombin toget SRGF and serum poor in growth factors (SPGF). The level of TGFβ1 and PDGF-AB in human-SRGF and SPGF were assayed by enzyme-linked immunoassay(ELISA). Rat and human osteoblast were cultured and identified. Rat osteoblasts were treated with 5% rat-SRGF, 5% rat-SPGF and serumfree F12 medium, respectively. And human osteoblast were treated with 5% human-SRGF, 5% human-SPGF and serumfree DMEM. Cellular mitogenic activity was evaluated by thiazoly blue (MTT) colorimetric assay at 24, 48, 72 and 96hours.Results The level of TGF-β1 in human-SRGF was 307.67±35.57 ng/ml, and that of PDGF-AB was 52.76±7.89 ng/ml. The proliferation of rat and human osteoblast were promoted after treated with rat-SRGFand human-SRGF, respectively. In rat osteoblast groups, there were significant differences in absorbency between ratSPGF group and rat-SRGF group at 48 and 96 hours(Plt;0.05). In human osteoblast groups, the differences between human-SPGF group and human-SRGF group were significant at 48, 72 and 96 hours(Plt;0.05). The proliferation of these two kinds of osteoblasts almost stopped in serum-free medium, and the differences in absorbency , compared with othergroups，were significant (Plt;0.05). Conclusion High quality of PRP can be achieved by the improved method and SRGF is capable of up-regulating the proliferation of rat osteoblast and human osteoblast.
Objective Platelet-rich plasma (PRP) secretes many growth factors, including transforming growth factor β1 (TGF-β1), platelet derived growth factor, vascular endothl ial growth factor, insul in-l ike growth factor 1, and so on, which can promote cell prol iferation, chemotaxis, and collagen synthesis in wound heal ing. To investigate the effects of PRPon the tendon heal ing, and to explore the mechanism of action so as to provide the experimental basis for the tissue engineered tendons. Methods Forty healthy New Zealand white rabbits, weighing 2.5-3.0 kg and male or female, were randomly divided into the experimental group (n=20) and the control group (n=20). PRP was prepared from arterial blood of rabbit’s ears through twice centrifugation method of Landesberg. The platelet concentrations of whole blood and PRP were determined. The right achilles tendons of the rabbits were transected to make rupture models. In experimental group, the tendon was sutured after PRP (0.5 mL) was immediately appl ied at repair site. In control group, the tendon was sutured directly after transection. At 1, 2, 4, and 6 weeks after operation, the tendons of 5 rabbits in each group were harvested for morphological, histological, and immunohistochemical observations; the fibroblast counting, the content of collagen fibers, and the expression of TGF-β1 were detected. Results The concentration of platelet of PRP was 4.03 times of whole blood. All the animals survived till the end of the experiment, and the incision healed well. No death, infection, and other compl ications occurred. With time, the tendons almost healed in 2 groups, and the fibrous tissue at anastomosis site was more remarkable in control group than in experimental group. The histological observation showed significant differences in fibroblast counting at 1, 2, and 4 weeks after operation between 2 groups (P lt; 0.05), while no significant difference at 6 weeks (P gt; 0.05). The contents of collagen fibers in the parenchyma at repair site in experimental group were significantly higher than those in control group at each time point (P lt; 0.05). Immunohistochemistry staining showed the expression of TGF-β1 in experimental group was upregulated at 1 week and 2 weeks and reached the peak at the 2nd week, and subsequently downregulated at 4 and 6 weeks in comparison with the control group, showing signficant differences between 2 groups at each time point (P lt; 0.05). Conclusion PRP can facil itate rabbit’ s tendons heal ing and significantly improve the heal ing qual ity, which may be associated with its advancing the peak time of the TGF-β1 expression in tendon.
ObjectiveTo investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), a hypoxia-inducible factor-1α (HIF-1α) inhibitor, on hypoxia induced rat pulmonary arterial adventitial fibroblasts (AFs) proliferation and collagen synthesis, and explore the molecular mechanism.MethodsUnder hypoxic condition, rat AFs were cultured in DMEM medium supplemented with 10% fetal bovine serum in vitro. The cells were divided into five groups, ie. a normoxia group, a hypoxia group and three hypoxia+YC-1 groups (treated with YC-1 at concentration of 0.01, 0.05 and 0.1 mmol/L, respectively). The cells proliferation was determined by MTT method. Collagen synthesis of AFs was measured by 3H-proline incorporation assay. The expression of HIF-1α in AFs in different conditions was measured by Western blot, and the mRNA expression of transforming growth factor-β1 (TGF-β1) was measured by reverse-transcription polymerase chain reaction.ResultsThe proliferation rate and the incorporation data of 3H-proline in the hypoxia group were significantly increased as compared with those in the control group (both P<0.01). YC-1 significantly reduced the proliferation rate and incorporation data of3H-proline induced by hypoxia in a dose-dependent manner. YC-1 could also down-regulate the expressions of HIF-1α and TGF-β1 mRNA significantly (both P<0.01). Compared with the hypoxia group, the expressions of HIF-1α and TGF-β1 mRNA decreased respectively by 65% and 61% in the hypoxia+YC-1 (0.1 mmol/L) group (bothP<0.01).ConclusionsYC-1 can inhibit hypoxia-induced AFs proliferation and collagen synthesis in a dose-dependent manner. The mechanism may relate to YC-1’s inhibitory effect on expressions of HIF-1α and TGF-β1 mRNA.
Objective
To investigate the effects of advanced glycation endproducts (AGEs) on proliferation of pericytes of bovine retinal capillary vessels and expression of transforming growth factor beta;(TGF-beta;).
Methods
The proliferation of pericytes detected by methyl thiazolyl tetrazolium (MTT) colorimetric assay, cellular cycle of pericytes was analyzed by flow cytometry was used to analyze cell, and TGF-beta; protein expression of pericytes was observed by immunofluorescent staining.
Results
AGEs inhibited the proliferation of pericytes of bovine retinal capillary vessels, stopped the cellular cycle of pericytes in synthesis phase (S phase), increased the number of apoptotic cells obviously (Plt;0.01), and promoted the expression of TGF-beta; proteinof perycytes.
Conclusions
AGEs may promote the apoptosis of pericytes by inhibiting the proliferation of pericytes to lead the decrease of pericytes number, and may accelerate diabetic retinopathy by promoting the expression of TGF-beta; protein of pericytes.
(Chin J Ocul Fundus Dis, 2006, 22: 20-23)
