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 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.
Objective To investigate the effects of adenovirus-mediated melanoma differentiation-associated gene-7 (mda-7)/IL-24 and/or adriamycin (ADM) on transplanted human hepatoma in nude mice and to explore a new way for hepatoma gene therapy combined with chemotherapy. Methods The recombinant adenovirus vector carrying Ad.mda-7 was constructed; Ad.mda-7 and/or ADM were injected into the tumor-bearing mice. Their effects on the growth of the tumor and the survival time of the mice were observed. The expressions of VEGF and TGF-β1 were detected by an immunohistochemistry method. Results Ad.mda-7 was constructed and expressed in vivo successfully. Compared with other three groups 〔control group (43.4±1.67) d, ADM group (64.2±4.14) d, Ad.mda-7 group (61.4±1.67) d〕, the mice treated with Ad.mda-7 combined with ADM had longer average survival time 〔(83.8±4.82) d, P＜0.01〕; the average size of tumor treated with Ad.mda-7 combined with ADM diminished significantly compared with that treated with ADM or Ad.mda-7 separately (P＜0.01). VEGF and TGF-β1 expressions of Ad.mda-7 group were (56.2±7.7)%, (35.2±4.5)%, respectively, and were lower than those in ADM group (VEGF: P＜0.05; TGF-β1: P＜0.01). VEGF expression of Ad.mda-7+ADM group was (37.3±5.0)%, and was significantly lower than that in other three groups (P＜0.01). TGF-β1 expression of Ad.mda-7+ADM group was (31.2±3.1)% and significantly lower than that in control group and ADM group (P＜0.01), however, there was no significant difference compared with Ad.mda-7 group (Pgt;0.05). Conclusion Ad.mda-7 combined with ADM has b antitumor potency and synergistic effects and suppresses the growth of human HCC xenograft in nude mice, possibly by inducing the apoptosis of hepatoma cell lines and suppressing tumor angiogenesis.
Objective To validate the mechanism of effect of hepatic artery ischemia on biliary fibrosis after liver transplantation and the prevention method. Methods Eighteen male dogs were established into the concise auto orthotopic liver transplantation models and assigned into three groups randomly: hepatic artery ischemia (HAI) group, TBB group (transferred the blood by a bridge duct ) and control group, each group contained 6 dogs. After opening portal vein, the samples were cut from liver in each group at the time of 6 h, 3 d and 14 d. The pathological modifications of intrahepatic bile ducts were observed and expression of transforming growth factor-β1 (TGF-β1) were detected in the three times. Expressions of Smad3 and phosphate-Smad3 as well as mRNA of α-smooth muscle actin (α-SMA) in intrahepatic bile ducts were detected 14 d after opening portal vein.Results Compared with control group, the collagen deposition and lumens stenosis in biliary vessel wall were more obviously in HAI group. In TBB group, the pathological modifications were slighter compared with HAI group. The positive cell index of TGF-β1 reached peak on day 3 after opening portal vein, then decreased in TBB group, and which in HAI group kept increase and was significantly higher than that in TBB group （Plt;0.05）. The expression level of phosphate-Smad3 and transcriptional level of α-SMA mRNA were 1.04±0.13 and 1.12±0.55 in TBB group on day 14 after opening portal vein, which were significantly higher than those in control group (0.59±0.09 and 0.46±0.18) and lower than those in HAI group (1.82±0.18 and 1.86±0.73), the diversities among three groups were significant (Plt;0.05). There was not significant difference of expression of Smads among three groups (Pgt;0.05). Conclusions Hepatic artery ischemia could increase the deposition of collagen fibers and the transdifferentiation of myofibroblast in bile duct and result in the biliary fibrosis by activating the TGF-β1/Smads signaling pathway. The bridging bypass device could lessen the biliary fibrosis caused by hepatic artery ischemia by inhibiting the activation of TGF-β1/Smads signal transduction passageway.
ObjectiveTo investigate the effect of peroxisome proliferator-activated receptorγ(PPARγ) agonist on bile duct fibrosis induced by transforming growth factor-β1 (TGF-β1). MethodsPrimary cultures of bile duct fibroblast were treated with different concentrations of pioglitazone (PGZ) or 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) and then treated with TGF-β1. The mRNA levels of collagenⅠ(COLⅠ)、fibronectin (FN)、α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) were determined by RT-PCR. ResultsPGZ and 15-d-PGJ2 could down regulated the mRNA levels of COLⅠ, FN, α-SMA and CTGF induced by TGF-β1 (3 ng/mL). The same concentration of 15d-PGJ2 was a more potent inhibitor ofα-SMA and less potent inhibitor of COLⅠthan PGZ. But PGZ had the same effect as 15d-PGJ2 on CTGF. ConclusionPPARγagonist can prevent fibrosis induced by TGF-β1.
ObjectiveTo investigate the effect of dust fine particles on tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP), transforming growth factor-β1 (TGF-β1), and collagens in the lung tissue of rats.MethodsAccording to random number table method, 96 male Wistar rats were divided into an untreated control group, a treated control group and an experimental group, with 32 rats in each group. The experimental group was exposed to the wind tunnel simulation of sandstorm (5 days per week, 5 hours per day); the untreated control group was put in the standard living environment next to the wind tunnel; the treated control group was exposed to the same wind tunnel simulation of sandstorm for 5 hours every day, the speed of wind was the same as the experimental group, but without dust; On the 30th, 60th, 90th, and 120th day, the levels of TNF-α, MMP-2, MMP-9, TGF-β1, lung collagen type Ⅰ and Ⅲ in the lung tissue of rats were determined by enzyme linked immunosorbent assay.ResultsCompared with the untreated control group and the treated control group, the content of TNF-α was higher in the experimental group on 30th, 60th, 90th and 120th day (all P<0.05). The contents of MMP-9 and MMP-2 in the experimental group on 60th and 90th day were significantly higher than those in the untreated group and the treated control group, respectively (all P<0.05). On the 30th, 60th, 90th, and 120th day, the content of TGF-β1 in the experimental group was significantly higher compared with the two control groups (all P<0.05). The contents of lung collagen type Ⅰ and type Ⅲ were higher in the experimental group on 60th, 90th and 120th day, respectively, compared with the two control groups (all P<0.05).ConclusionsThe strong sandstorm environmental exposure to a certain period of time can promote lung interstitial collagen deposition in rat. With the prolonged exposure time, the deposition of collagen increases. TNF-α, MMP-2, MMP-9 and TGF-β1 may all participate and induce the process of pulmonary fibrosis.
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.
【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.
Abstract： Objective To explore the association between transforming growth factor-β receptor typeⅡ (TGFBR2) gene rs6785358 and rs764522 polymorphisms and rheumatic heart disease (RHD) in Chinese Han People. Methods The research design was a case-control study. A total of 207 patients who were hospitalized in Nanjing First Hospital Affiliated to Nanjing Medical University between October 2008 and January 2011 with RHD served as RHD group while 225 age and gender matched healthy adults as control group. Polymerase chain reaction-restriction length polymorphism (PCR-RFLP) technique was used to determine TGFBR2 gene rs6785358 and rs764522 polymorphisms. Results The frequencies of genotype AA, AG and GG of rs6785358 in RHD group and control group were 72.0%, 25.1%, 2.9% and 68.9％, 28.0％, 3.1％,respectively. There was no significant difference in the distribution of genotype frequencies for rs6785358 between RHD group and control group（χ2=0.50，P=0.78）. The frequencies of allele A and G of rs6785358 in RHD group and control group were 84.5％, 15.5％ and 82.9％, 17.1％,respectively. There was no significant difference in the distribution of allele frequencies for rs6785358 between RHD group and control group（χ2=0.43，P=0.51）. The frequencies of genotype CC, CG and GG of rs764522 in RHD group and control group were 77.3%, 21.3%, 1.4% and 75.6％, 21.3％, 3.1％, respectively. There was no significant difference in the distribution of genotype frequencies for rs764522 between RHD group and control group（χ2=1.33，P=0.51）. The frequencies of allele C and G of rs764522 in RHD group and control group were 87.9％, 12.1％ and 86.2％, 13.8％,respectively. There was no significant difference in the distribution of allele frequencies for rs764522 between RHD group and control group（χ2=0.55，P=0.46）. Further analysis by sex stratification showed that no statistical significance was detected in the distribution of genotype and allele frequencies for rs6785358 or rs764522 between RHD patients and controls. Conclusion TGFBR2 gene rs6785358 and rs764522 polymorphisms are not associated with RHD in Chinese Han people.