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 study the pathology and possible mechanism of experimental hydrochloric acid（HCl） inhalation-indued pulmonary fibrosis in rats.Methods 120 male SD rats were randomly divided into a nomal control group，a bleomycin group，a high dose HCl group，a middle dose HCl group and a low dose HCl group.The bleomycin group was intratracheally injected with bleomycin once to induce pulmonary fibrosis.The three HCl groups were intratracheally injected with HCl once per week.The control group was given saline by the same way.Six rats of each group were randomly sacrificed on day 7，14，28 and 42 respectively.The histological changes of lung tissue were studied by HE and Masson’s trichrome staining.Hydroxyproline level in lung tissue was measured by digestion method.Protein and mRNA expression of transforming growth factor-β1（TGF-β1） were assayed by immunohistochemistry and RT-PCR respectively.Results Alveolitis in three HCl groups was significantl compared to control group，most severe at the second week，then remained at a high level which was equivalent to or exceeded the level of the bleomysin group after 28 days.Pulmonary fibrosis in three HCl groups was also significantly more severe than that in the control group，but milder than that in the bleomysin group.The high-dose and middle-dose HCl groups were not significantly different from the bleomysin group on day 42.There was no difference between three HCl groups in the earlier period，but the high-dose HCl group has a significantly difference from low-dose group on day 42.The content of hydroxyproline in high-dose and middle-dose HCl groups was also significantly higher than that in the control group.On day 42 hydroxyproline content in high-dose HCl dose rather middle –or low dose group was similiar with the level of bleomysin group.Content of TGF-β1 mRNA in three HCl groups was comparable to the level of bleomysin group on day 28 and exceeded on day 42.The expression of TGF-β1 in three HCl groups was not significantly different from the bleomysin group on day 42.Conclusion Experimental acid aspiration might contribute to pulmonary fibrosis in rats.Acid induced alveolar epithelial cell damage，abnormal proliferation and repair and fibrosis could be involved..
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 investigate the expression of vascular endothelial growth factor-A （VEGF-A） and the phenotypic transition after the activation of fibroblasts by the supernatant of cultured tumor cells．Methods The growth tendency of fibroblasts was tested by the MTT assay．The expressions of alpha-smooth muscle actin （α-SMA） and VEGF-A mRNA were tested by RT-PCR．The expressions of α-SMA and VEGF-A protein were tested by immunohistochemistry and Western blot．Results The MTT assay indicated that the conditional medium which contained tumor cells supernatant could obviously promote the growth of the fibroblasts． RT-PCR and Western blot manifested that α-SMA expressed by the fibroblasts which cultured by normal medium reached its peak on day 5，then decreased to a low level on day 7．When the medium contained 2 ng/ml transforming growth factor-β1 （TGF-β1），the fibroblasts could steadily express more α-SMA．But the above two mediums could not make the fibroblasts express the VEGF-A. When using the conditional medium，the α-SMA peak advanced on the third day and maintained at a high level，so as the expression of the VEGF-A．Conclusions The results suggested that fibroblasts can be activated to be myofibroblasts when using the conditional medium．The best activation time of the fibroblasts is consistent with the time of the VEGF-A expression at the highest level by the activated fibroblasts．The fibroblasts which activated at the best time are expected to become a kind of cells which can be used for promoting revascularization．
Objective To study the influence of transforming growth factor-β1(TGF-β1), dentin non-collagen proteins(dNCPs) and their complexon tissue engineering pulp system. Methods Collagen I and dentin powder were used to construct the system of pulp cells in 3dimensional culture, dentin powder was added in the gel. The tissue engineering pulp were divided TGF-β1 group, dNCPs group, TGF-β1/dNCPsgroup and control group.After3, 6 and 14 days, the appearance and the differentiation of pulp cells were observed by HE staining and immunohistochemical staining -respectively. Results Collagen I could form netted collagen gel construction. Growing condition of pulp cells in gel was similar to that of pulp cells in vivo. After the TGF-β1 and dNCPswere added, the pulp cells had some characteristics of odontoblasts and had unilateral cell process after culture 6 days. Pulp cells arranged with parallel columnar and form dentin-pulp-like complex after 14 days. Immunohistochemical staining showed dentin salivary protein(DSP) began to express in some cells.The number of positive cell was most in the TGF-β1 group. No positive cells were detected in the control group. Conclusion The transforming growth factor-β1 and noncollagen proteins can stimulate the pulp cells to transform into odontoblasts to some extent, which promote the formation of tissue engineering pulp.
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.
ObjectiveTo investigate the early diagnostic value of transforming growth factor-β1(TGF-β1) on acute rejection after liver transplantation in rhesus by detecting the expression of TGF-β1 in the liver tissue. MethodsLiver transplantation models in rhesus were constructed by the improved vascular dual cuff, supporting tube of biliary tract, and artery anastomosis method.The successful models were randomly divided into experimental group (no immunosuppressant treatment in perioperative period) and control group (treated by immunosuppressant in perioperative period).Then the blood samples and liver tissues were collected at 6, 12, 24, and 72 hours after surgery.Allograft rejections of liver tissue after liver transplantation were monitored by liver function test, hematoxylin-eosin staining and Banff score.Finally, the expression level of TGF-β1 was detected by Western blot analysis or immunohistochemistry technique. Results①The acute rejection happened in all the rhesus at 12 h, 24 h and 72 h after liver transplantation, especially at 72 h after liver transplantation in the experimental group, the Banff grade levels of acute rejection in the liver tissue was more severe than that in the control group (P < 0.05).②The levels of ALT, AST, and TBIL after liver transplantation was gradually increased, which were similar at 6 h and 12 h after transplantation between the two groups, but which at 24 h and 72 h after transplantation in the experimental group were significantly higher than those in the control group (P < 0.05).③The results of TGF-β1 protein expression using immunohistochemical detection:The percentage of positive area of TGF-β1 of liver tissue at 12 h in the experimental group was significantly higher than that in the control group (P < 0.05).With the extension of time, it was gradually increased and significantly higher than that in the control group at 24 h or 72 h (P < 0.05).④The semi-quantitative results of TGF-β1 protein expression using Western blot detection:The TGF-β1 protein expressions began to increase at 6 h after liver transplantation in the experimental group and the control group, and the magnitude of increase was more obvious in the experimental group.The TGF-β1 protein expressions at different time (6 h, 12 h, 24 h, and 72 h) in the experimental group were significantly higher than those in the control group (P value was 0.003, 0.001, 0.001, and 0.001, respectively). ConclusionsThe elevated level of TGF-β1 of liver tissue after liver transplantation might suggest the enhanced cellular immune function, it might have certain significance for early diagnosis of acute rejection after liver transplantation.
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.
【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 examine the effects of TGF-β1 on epithelial-myofibroblast transition ( EMT) of A549 cells and its relationship with extracellular regulating kinase1/2 ( ERK1/2) signaling system. Methods Cultured A549 cells were divided into one negative control group and four groups incubated with TGF-β1 for 48 hours at different concentration ( 0.05, 0. 5, 5, 10 μg/L, respectively) . The protein expressions of E-cadherin, α-smooth muscle actin ( α-SMA) , vimentin and fibronectin were assessed by indirect immunofluorescence and Western blot. In the other experiment, cultured A549 cells were incubated with TGF-β1 for different time. The protein and mRNA expressions of E-cadherin and α-SMA were assessed by Western blot and RT-PCR. The protein expressions of vimentin, fibronectin, ERK1 /2, and p-ERK1 /2 were detected by Western blot. Results By indirect immunofluorescence, Western blot, and RT-PCR analysis, E-cadherin expression significantly decreased and α-SMA expression significantly increased in A549 cells treated with TGF-β1 compared with negative controls in a time- and concentrationdependent manner ( Plt;0.05 ) . Vimentin and fibronectin protein expressions significantly increased simultaneously ( Plt;0.05) . The concentration of 5 ng/mL of TGF-β1 was most effective. The ratio of p-ERK1 /2 and ERK1/2 was significantly increased in the TGF-β1 treated cells in a time-dependent manner ( P lt;0. 05) . Conclusions TGF-β1 can induced EMT in A549 cells in vitro in a time- and concentrationdependant manner. This effect may involve in upregulation of ERK1/2 signaling system.