【Abstract】Objective To investigate the apoptosis induced by TGF-β1 in human hepatic carcinoma cell lines and its relationship with p53 gene and Smad. Methods Three human hepatic carcinoma cell lines which involving in various status of the p53 gene were used in this study. TGF-β1-induced apoptosis in hepatic carcinoma cell lines was measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. To study the mechanism of TGF-β1-induced apoptosis, these cell lines were transfected with a TGF-β1-inducible luciferase reporter plasmid containing Smad 4 binding elements (SBE) and luciferase gene using Lipofectamine 2000, then treated with TGF-β1, relative luciferase activity was assayed. Results Of three cell lines studied with TUNEL assay, TGF-β1 induced apoptosis was observed in HepG2 cells (wild type p53). Huh-7 (mutant p53) and Hep3B (deleted p53) cell lines showed less apoptosis. Luciferase activity assay indicated that the response to TGF-β1 induction in HepG2 cells was increased dramatically but was not significant in Huh-7 and Hep3B cell lines. Conclusion HepG2 cells seem to be highly susceptible to TGF-β1-induced apoptosis compared with Hep3B and Huh7 cell lines. Smad 4 is a central mediator of the TGF-β1 signal transduction pathway.
Objective To observe the expression and distribution of transforming growth factor-β1 (TGF-β1) in the healing process of bile duct and discuss its function and significance in the process of benign biliary stricture formation. Methods An injury to bile duct of dog was made and then repaired. The expression and distribution of TGF-β1 in the tissue at different time of the healing process were studied after operation with immunohistochemical SP staining. Results TGF-β1 staining was observed in the granulation tissue, fibroblasts and endothelial cells of blood vessels. High expression of TGF-β1 was observed in the healing process lasting for a long time. Conclusion The high expression of TGF-β1 is related closely with the fibroblast proliferating activity, extracellular matrix overdeposition and scar proliferation in the healing process of bile duct.
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 observe the influence of the transforming growth factor β1(TGF-β1) on the denervated mouse musclederived stem cells（MDSCs） producing the connective tissue growth factor（CTGF）at different time points in vitro. Methods MDSCs from the primarycultureof the denervated mouse skeletal muscle were isolated and purified by the preplate technique, and they were identified before the culture and after the culturein vitro with TGF-β1 (10 ng/ml) for 24 hours. Then, MDSCs were randomlydivided into 6 groups (Groups A, B, C, D, E and F) according to the different time points, and were cultured in vitro with TGF-β1 (10 ng/ml) for 0, 3, 6, 12, 24 and 48 hours, respectively. The levels of CTGF mRNA in MDSCs were measured by the real time RT-PCR and the expression of CTGF protein was detected by the CTGF Western blot. Results The immunohistochemistry revealed that before the adding of TGF-β1, MDSCs highly expressed Sca-1, with a positivityrate of 96%; however, after the adding of TGF-β1, the positive expression of Sca-1 decreased greatly, with a negativity rate gt;99%. The Western blot test showed that the ratios of CTGF to the average absorbance of βactin in Groups A-F were 0.788±0.123, 1.063±0.143, 2.154±0.153, 2.997±0.136, 3.796±0.153 and 3.802±0.175, respectively. In Groups AD，the absorbance increased gradually, with a significant difference between the abovementioned groups (Plt;0.05). However, in Groups D-F, there was no significant difference between the groups as the promotive tendency became less significant (P＞0.05). The RT-PCR test showed that the △Ct values in GroupsA-F were 1.659±0.215, 1.897±0.134, 2.188±0.259, 2.814±0.263,2.903±0.125 and 3.101±0.186, respectively. In Groups A-D, the increase in the △Ct value was gradual, but the differences were significant between the groups (Plt;0.05). But in Groups E and F, the promotive tendency became less significant（Pgt;0.05）. Conclusion TGF-β1 can promote the production of CTGF inthe mouse MDSCs cultured in vitro and the time-dependent relation exists for 3-12 hours.
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 localize the distribution of basic fibroblast growth factor (bFGF) and transforming growth factor-beta(TGF-beta) in tissues from dermal chronic ulcer and hypertrophic scar and to explore their effects on tissue repair. METHODS: Twenty-one cases were detected to localize the distribution of bFGF and TGF-beta, among them, there were 8 cases with dermal chronic ulcers, 8 cases with hypertrophic scars, and 5 cases of normal skin. RESULTS: Positive signal of bFGF and TGF-beta could be found in normal skin, mainly in the keratinocytes. In dermal chronic ulcers, positive signal of bFGF and TGF-beta could be found in granulation tissues. bFGF was localized mainly in fibroblasts cells and endothelial cells and TGF-beta mainly in inflammatory cells. In hypertrophic scar, the localization and signal density of bFGF was similar with those in granulation tissues, but the staining of TGF-beta was negative. CONCLUSION: The different distribution of bFGF and TGF-beta in dermal chronic ulcer and hypertrophic scar may be the reason of different results of tissue repair. The pathogenesis of wound healing delay in a condition of high concentration of growth factors may come from the binding disorder of growth factors and their receptors. bFGF may be involved in all process of formation of hypertrophic scar, but TGF-beta may only play roles in the early stage.
The expressions and significance of c-met oncoprotein and transforming growth factor-α (TGF-α) were studied by immunohistochemical method in 50 cases of breast cancer (BC) and 12 cases of benign lesions of breast (BL). The positive rate of c-met, TGF-α in BC was 26.0% and 25.0% respectively, in BL was 8.3% and 25.0% respectively. The positive rate of c-met oncoprotein was lower in the cases of histologic Grade Ⅰ, positive of ER and PR or CEA than that of histologic Grade Ⅲ, negative of ER and PR or CEA. The positive rate of TGF-α was lower in the cases of histologic Grade Ⅰ, negative of ER and PR or CEA than that of histologic Grade Ⅲ, positive of ER and PR or CEA. These results suggest the expression of c-met and TGF-α might be related to the carcinogenesis and development or endocrine state of BC.
Objective To observe the effects of cigarette smoke extract ( CSE) on the proliferation and secretion of hydrogen peroxide ( H2O2 ) in human lung fibroblasts ( HLFs) induced by transforming growth factor-β1 ( TGF-β1 ) . Methods Cultured HLFs were divided into a normal group and a model group induced by TGF-β1 ( 5 ng/mL) , then intervened with CSE at different concentrations ( 0% , 2. 5% , 5% ,10% , respectively) . Brdu ELISA assay was used to detect cell proliferation. H2O2 release from cultured cells was assayed using a fluorimetric method. Cellular localization of H2O2 and expression of α-SMA were performed using a fluorescent-labeling strategy. Results TGF-β1 stimulated group showed positive expression of α-SMA, implying TGF-β1 had induced fibroblasts to differentiate into myofibroblasts. In TGF-β1 stimulated group, 2. 5% and 5% CSE promoted cell proliferation ( P lt; 0. 01 or 0. 05) , while 10% CSE inhibited cell proliferation ( P lt; 0. 01) . In the normal group, both low and high concentration of CSE inhibited cell proliferation ( P lt; 0. 01 or P lt; 0. 05) , and the inhibition effect was dose-dependent. HLF induced by TGF-β1 generated low constitutive levels of extracellular H2O2 that was markedly enhanced by CSE treatment ( P lt; 0. 01) . Pretreatment with DPI, an inhibitor of NADPH oxidase, abolished secretion of H2O2 . Cellular localization of H2O2 by a fluorescent-labeling strategy demonstrated that extracellular secretion of H2O2 is specific to the myofibroblast. Conclusions Low concentration of CSE can promote myofibroblast proliferation, and markedly increase extracellular secretion of H2O2 . CSE possibly take part in the development and progress of idiopathic pulmonary fibrosis by increasing oxidative stress.
Objective To explore the effectiveness of the transforming growth factor-β1(TGF-β1) and tumor necrosis factor-α(TNF-α) inducing human bronchial epithelial(HBE) cells to optimize epithelia-mesenchymal transformation(EMT) model. Methods Blank control, TGF-β1 10 ng/ml, TNF-α 10 ng/ml, TGF-β1 10 ng/ml+TNF-α 10 ng/ml induced human epithelial cells for 24 hours. Then the change of morphological alteration were observed by applying CCK8, cells migration assay and Western blot technique. Results When TGF-β1 plus TNF-α induced human epithelial cells for 24 hours, most of HBE cells traits changed including morphological alteration from cobblestone to fusiform, connection between cells vanishing, intercellular space broadening. In the experiments of checking cell migration capacity by the vitro scratch test, the group spacing was 420.06±10.38 μm in the blank control group, 499.86±34.00 μm in the TGF-β1 10 ng/ml group, 514.93±10.56 μm in the TNF-α 10 ng/ml group, 569.68±33.58 μm in the TGF-β1 10 ng/ml+TNF-α10 ng/ml group. TGF-β1 cooperated with TNF-α led to scratch spacing narrowing significantly. Western blot analysis showed that expression of E-cadherin and Vimentin varied significantly in the TGF-β1+TNF-α group. Conclusion Inducing human bronchial epithelial cell by TGF-β1 cooperated with TNF-α optimizes EMT model.
ObjectiveTo observe the expression of heat shock protein 47 (HSP47) and transforming growth factor-β2(TGF-β2) in vitreous specimens and epiretinal membranes of patients with proliferative vitreoretinopathy diseases. MethodsVitreous specimens and epiretinal membranes were obtained from 48 patients (48 eyes) with proliferative vitreoretinopathy (PVR) and 50 patients (50 eyes) with proliferative diabetic retinopathy (PDR). Vitreous specimens and internal limiting membranes were collected from 20 patients (20 eyes) with idiopathic macular hole (IMH) as control group. The expression of HSP47 and TGF-β2 in the vitreous specimens was evaluated using enzyme linked immunosorbent assay. The expression of HSP47, TGF-β2, typesⅠandⅢcollagen in epiretinal membrane and internal limiting membrane specimens were observed for immunohistochemical staining method. The correlation between the positive expression of HSP47 and TGF-β2, typesⅠandⅢcollagen in epiretinal membrane specimens of patients with PVR and PDR were analyzed. ResultsThe expression of HSP47 in vitreous specimens of patients with PVR, PDR and IMH were (212.35±23.32), (231.30±26.79), (171.06±28.91) pg/ml, respectively. The expression of TGF-β2 in vitreous specimens of patients with PVR, PDR and IMH were (1919.96±318.55), (1939.39±177.57), (1194.61±234.20) pg/ml, respectively. The expression of HSP47, TGF-β2 in the vitreous specimens of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=12.952, 34.532;P < 0.01). The epiretinal membrane of patients with PVR and PDR showed markedly increased expression of HSP47, TGF-β2, typesⅠandⅢcollagen in the cytoplasm and extracellular matrix. The expression of HSP47 and typeⅢcollagen was negative and the expression of TGF-β2 was weakly positive and the expression of typesⅠcollagen was positive in internal limiting membrane of patients with IMH. The expression of HSP47, TGF-β2, typesⅠandⅢcollagen in the epiretinal membrane of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=13.469, 18.752, 12.875, 20.358; P < 0.01). The expression of HSP47 was positively correlated with the positive expression of TGF-β2, typesⅠandⅢcollagen in epiretinal membrane specimens of patients with PVR (r=0.475, 0.556, 0.468; P < 0.05) and PDR (r=0.484, 0.589, 0.512; P < 0.05). ConclusionsThis study showed increased consistent expression of HSP47 and TGF-β2 in vitreous and epiretinal membrane specimens of patients with PVR and PDR. Both HSP47 and TGF-β2 were expressed in the cytoplasm and extracellular matrix. HSP47 and TGF-β2 may be involved in the pathological process of PDR and PVR by promoting collagen synthesis.