OBJECTIVETo prepare semiconductor quantum dots (QDs)-Smad2 monoclonal antibody fluorescent probes, to detect the optical qualities and the ability to specific recognition of Smad2 in rat dental papillae cells (RDPC) of quantum dots-Smad2 monoclonal antibody fluorescent probes.

METHODS(1) QDs were chemically modified with Smad2 proteins to prepare water soluble QDs-Smad2 monoclonal antibody fluorescent probes which were purified after preparation. (2) The absorption band and emission band of these probes were obtained through ultraviolet spectrophotometer and fluorospectrophotometer, the shape, fluorescence intensity and photochemistry stability of these probes were studied through confocal laser scanning fluorescence microscopy. (3) Before the location of Smad2 proteins in RDPC was studied with anti-Smad2 immunocytochemical method and direct immunofluorescence imaging, RDPC were incubated with transforming growth factor-beta1 (TGF-beta1), and the related optical qualities of quantum dots-Smad2 monoclonal antibody fluorescent probes in RDPC were detected.

RESULTSQDs and monoclonal antibody linked together through covalent bond to form the fluorescent probes which could specifically and effectively recognize Smad2 proteins in RDPC. These fluorescent probes still had good properties, including broad excite spectra, narrow emission spectra, high fluorescence intensity and photostability.

CONCLUSIONQDs and monoclonal antibody could link together through covalent bond to form the nanometer molecular probes with distinct optics character and photostability, which provides the scientific evidence that QDs can visualize the molecular movement in living cells in long-term, in situ and in real time.

Animals ; Antibodies, Monoclonal ; Fluorescent Dyes ; Quantum Dots ; Rats ; Semiconductors ; Smad2 Protein

OBJECTIVETo determine the TGF-beta/Smads signal pathway in different human prostate cancer cell lines, and to explore the role of the TGF-beta/Smads pathway in the progression and metastasis of prostate cancer and its possible mechanisms.

METHODSWe detected the expressions of the key proteins involved in the TGF-beta/Smads pathway, TbetaR II, Smad2/3, p-Smad2 and Smad4, in prostate cancer cell lines LNCaP, PC-3, DU145, IF11 and IA8 with different metastatic potentials by Western blotting.

RESULTSTbetaR II was expressed highly in PC-3, DU145, IF11 and IA8, but extremely lowly in LNCaP. Smad2/3 was highly expressed in all the cell lines with different intensity, while p-Smad2 only in PC-3 and DU145. The expression of Smad4 was detected in LNCaP, PC-3 and DU145, but not in IF11 and IA8.

CONCLUSIONThe status of the TGF-beta/Smads pathway differs in the cell lines with different metastatic potentials, only active in PC-3 and DU145. The TGF-beta/Smads pathway may be involved in the invasion and metastasis of prostate cancer through altering the expressions of the key proteins in it.

Cell Line, Tumor ; Humans ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Signal Transduction ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo observe the effect of single herb pilose antler (PA) on the expression of Smad2 and Smad3 in the cartilage of osteoarthritis (OA) rats.

METHODSOne hundred 3-month old female healthy SD rats, (200 +/- 20) g, were recruited and routinely fed for 1 week. They were randomly divided into 5 groups, i.e., the low dose PA group, the high dose PA group, the normal saline control group, the model group, and the normal control group, 20 in each group. The model was prepared using classic Hulth method except the normal control group. After 6-week modeling, the model was confirmed successful by pathologic observation. PA at 0.021 g/100 g and 0.084 g/1 00 g was given by gastrogavage to rats in the low dose PA group and the high dose PA group respectively. Normal saline was administered to those in the normal saline control group. No treatment was given to rats in the normal control group and the model group. Bilateral knee cartilages were harvested at week 2,4, and 6. mRNA and protein expressions of Smad2 and Smad3 were detected by immunohistochemical assay, fluorescent quantitative PCR, and Western blot.

RESULTSOA model was successfully prepared by pathological observation. Results of immunohistochemical assay showed that Smad2 and Smad3 expressed extensively in the cartilage, and located inside the chondrocyte membrane. Compared with the model group, mRNA expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 2, 4, and 6, showing statistical difference (P < 0.05). Compared with the same group at week 4 after gastrogavage, mRNA expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.05). Compared with the model group, protein expression of Smad2 and Smad3 obviously increased in the chondrocytes of the low dose PA group and the high dose PA group at week 2 and 4, showing statistical difference (P < 0.01). Compared with the same group at week 2 after gastrogavage, protein expression of Smad2 and Smad3 obviously increased in the low dose PA group and the high dose PA group at week 4, showing statistical difference (P < 0.01). Compared with the same group at week 4 after gastrogavage, protein expression of Smad2 and Smad3 obviously decreased in the low dose PA group and the high dose PA group at week 6, showing statistical difference (P < 0.01).

CONCLUSIONS(1) The pilose antler could repair cartilages by regulating mRNA and protein expressions of Smad2 and Smad3. (2) Up-regulating mRNA and protein expressions of Smad2 and Smad3 might be one of important mechanisms for the pathogenesis of OA.

Animals ; Antlers ; chemistry ; Cartilage ; cytology ; metabolism ; Chondrocytes ; drug effects ; metabolism ; Female ; Medicine, Chinese Traditional ; Osteoarthritis ; drug therapy ; metabolism ; Rats ; Rats, Sprague-Dawley ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism

OBJECTIVETo detect the expression of important proteins associated with transforming growth factor-beta (TGF-beta)/Smad signaling pathway in Peutz-Jeghers syndrome (PJS) and investigate the correlation of these proteins to LKB1 gene expression.

METHODSThe expression and localization of LKB1, TGFbeta1 and pSmad2 proteins in 20 PJS polyp samples and normal intestinal mucosal tissues were detected with immunohistochemical staining.

RESULTSThe expressions of LKB1, TGFbeta1 and pSmad2 were lower in PJS polyps than in normal mucosa, and the differences in LKB1 and TGFbeta1 proteins were significantly different between them (P<0.05). In PJS polyps, positive correlations were found between LKB1 and TGFbeta1 and between TGFbeta1 and pSmad2 expressions.

CONCLUSIONTGFbeta/Smad pathway is probably subjected to the regulation by LKB1 and may play a role in the occurrence of PJS.

Humans ; Immunohistochemistry ; Peutz-Jeghers Syndrome ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Signal Transduction ; Smad2 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism

OBJECTIVETo explore the effects of Sini Decoction (SD) on the expressions of Samd2 and Smad7 isoproterenol (Iso) induced myocardial fibrosis rats.

METHODSTotally 19 Wistar rats were randomly divided into 3 groups, i.e., the control group, the model group, and the SD group. Iso was injected to rats in the model group and the SD group, while normal saline was injected to rats in the control group. SD was given to rats in the SD group by gastrogavage, while normal saline was administered to rats in the control group and the model group by gastrogavage. Four weeks later Masson staining and electron microscopic analysis were performed in each group. The protein and mRNA expressions of Smad2 and Smad7 were detected using immunohistochemical assay and RT-PCR.

RESULTSMasson staining showed the IOD value of the myocardial collagen fiber was 9 303 in the model group, 2 459 in the SD group, and 4 224 in the control group, indicating the myocardial fibrosis was more obvious in the model group than in the SD group and the control group. The IOD value of Smad2 protein was 20 275 and the mRNA IOD of Smad2 protein was 0. 919 in the model group, while they were respectively 9 949 and 0. 561 in the SD group, indicating the protein and mRNA expressions of Smad2 were obviously higher in the model group than in the SD group (P < 0.05). The IOD value of Smad7 protein was 25 667 and the mRNA IOD of Smad7 protein was 0.222 in the model group, while they were respectively 93 147 and 0. 412 in the SD group, indicating the protein and mRNA expressions of Smad7 was obviously lower in the model group than in the SD group (P < 0.05).

CONCLUSIONSD could effectively inhibit Iso induced myocardial fibrosis, and its mechanism may be associated with down-regulating the expression of Smad2 and up-regulating the expression of Smad7.

Animals ; Cardiomyopathies ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Isoproterenol ; adverse effects ; Male ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Wistar ; Smad2 Protein ; metabolism ; Smad7 Protein ; metabolism

OBJECTIVETo study the effect of Tangshenkang Granule (TG) containing serum on renal mesangial cells' (RMCs) proliferation and TGF-β1/Smad2/3 pathway in the high glucose condition.

METHODSTwelve SD rats were randomly divided into four groups, i.e., the low dose TG group, the middle dose TG group, the high dose TG group, and the blank control group, 3 in each group. After 7-day gastrogavage via portal vein blood, rats were sacrificed and their serum samples were collected. RMCs were cultured in common rat serum and TG containing serum respectively. The proliferation of mesangial cells was determined by methly thiazolyl tetrazolium (MTT) assay to determine the optimal TG containing serum concentration. Expression levels of TGF-β1 mRNA and protein were determined by real time quantitative PCR and ELISA. Smad2/3 protein expression and phosphorylation were determined by Western blot and immunofluorescence.

RESULTSTG containing serum at different doses could inhibit high glucose induced RMC cells' proliferation, TGF-β1 over-expression and Smad2/3 phosphorylation.

CONCLUSIONTG containing serum could inhibit high glucose induced RMC cells' proliferation, and its mechanism might be possibly associated with inhibiting TGF-β1/Smad2/3 signaling pathway.

Animals ; Cell Proliferation ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; Mesangial Cells ; Phosphorylation ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Serum ; Signal Transduction ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo elucidate the role of transforming growth factor-beta1(TGF-β1) in epithelial-mesenchymal transition of mesothelial cells and peritoneal metastasis of gastric cancer.

METHODSHMrSV5 cells, a human peritoneal mesothelial cell line, were incubated with TGF-β1, and their morphological changes were observed by phase contrast microscopy. Expressions of α-smooth muscle actin (α-SMA), vimentin, cytokeratin, E-cadherin, phosphorylated-Smad2 and Smad2 were examined by Western blotting. After fibroblastic-like mesothelial cells were co-incubate with HSC-39 cells(gastric cancer cell line), the adhesion and invasion potential of HSC-39 were evaluated by adhesion and invasion assay in vitro.

RESULTSFew mesothelial cells converted to spindle fibroblast-like morphology for 24 h, and remarkable phenotypic changes were observed at 72 h of TGF-β1 activation. TGF-β1 could induce α-SMA and vimentin expression, and down-regulate cytokeratin and E-cadherin expression in mesothelial cells (P<0.05). TGF-β1 induced phosphorylation of Smad2 within 15 min of stimulation, reached a maximum at 30 min after treatment and remained high level during the experiment without affecting total Smad2 expression(P>0.05). The percentage of HSC-39 gastric cancer cells adhered were significantly increased as compared to the control. When the mesothelial cells were treated by TGF-β1 for 72 h, the increased adhesion percentage was(146±17)%(P<0.05). After fibroblastic-like mesothelial cells co-incubated with HSC-39 cells for 48 h, more cancer cells [(61.1±11.4) cells/view field] invaded the coated membrane as compared to the control group [(31.9±8.1) cells/view field] (P<0.05).

CONCLUSIONTGF-β1 can induce the transition of mesothelial cells into myofibroblasts and Smad2 signal pathway may play a role in this transition, which is associated with increased adhesion and invasiveness of gastric cancer cells, and provides favorable environment for the dissemination of gastric cancer.

Cadherins ; Cell Line, Tumor ; Epithelial Cells ; Epithelial-Mesenchymal Transition ; Epithelium ; Fibroblasts ; Humans ; Peritoneal Neoplasms ; Signal Transduction ; Smad2 Protein ; Stomach Neoplasms ; Transforming Growth Factor beta1 ; Vimentin

OBJECTIVETo investigate the expressions of phosphorylated Smad2 (P-Smad2) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in hepatocellular carcinoma (HCC) tissues.

METHODSThe expressions of P-Smad2 and PTEN were detected using Envision immunohistochemical technique in 31 cases of HCC tissues, 25 cases of HCC adjacent liver tissues and 13 cases of non-hepatocellular carcinoma tissues.

RESULTSThe positive expression and staining intensity of PTEN in the cytoplasm of HCC cells (64.5%, 4.19+/-3.31) was significantly lower than those of the cells of the cancer adjacent tissues and non-cancerous tissues (96.0%, 7.88+/-0.93; 100%, 7.77+/-0.93). The staining intensity of PTEN in the cytoplasm of Edmondson pathologic grade III HCC cells was lower than those of the Edmondson grade I. The expression of PTEN was negatively correlated with intrahepatic vascular cancer thrombi (r=-0.43) and the expression of PTEN in the nuclei or cytoplasm of liver cells was negatively correlated with the liver disease progressions (r=-0.34). The positive rate and expression intensity of phosphorylated Smad2 in nuclei of HCC cells were the same as those in cancer adjacent and non-tumor liver tissues. The expression was mostly in the nucleus and cytoplasm of Edmondson grade I HCC cells, cancer adjacent liver tissue cells and non-tumor liver tissues, but its expression was only in the nuclei of Edmondson grade II and III HCC cells. The phosphorylated Smad2 expression appeared in the nuclei and in the cytoplasm of liver cells and it was positively correlated with the severity of the tumor pathology (r=0.22). Spearman correlation analysis revealed a significant inverse correlation between PTEN and phosphorylated Smad2 in HCC tissues (r=-0.73).

CONCLUSIONSThe aberrant expressions of PTEN and phosphorylated Smad2 and their interaction may play an important role in the pathogenesis of hepatocellular carcinoma.

Adult ; Aged ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Female ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Staging ; Oxidative Phosphorylation ; PTEN Phosphohydrolase ; metabolism ; Smad2 Protein ; metabolism

Animals ; Epithelial-Mesenchymal Transition ; Lung/metabolism* ; Male ; Pulmonary Disease, Chronic Obstructive ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad2 Protein/metabolism* ; Syndecan-1 ; Transforming Growth Factor beta1/metabolism*

OBJECTIVETo observe Smads protein expression in lung tissue of quartz exposed mice and to explore its association with pulmonary fibrosis in silicosis.

METHODSThe experimental mice were divided into control and quartz groups. 0.2 g/kg weight of quartz was injected intratracheally in quartz group. Samples were collected at the 1st, 3rd, 5th, 7th, 14th and 28th day after injection. Immunohistochemical methods with quantitative image analysis were used to assay the protein expression of transforming growth factor beta(1) (TGF-beta(1)), Smad 2/3, Smad 4, and Smad 7 protein levels. Protein expression level is presented by positive unit (PU).

RESULTSSmad 2/3 protein expression increased from day 3, reaching its peak level in day 14 [(42.2 +/- 2.4) PU], and decreased gradually. The elevation of Smad 4 protein level began from day 5, and the highest degree came into day 14 [(40.0 +/- 1.8) PU], decreased thereafter. The expression of Smad 7 presented a decreasing tendency at the beginning and reaching the lowest level in day 14 [(33.5 +/- 3.3) PU]. It seemed to elevate in day 28, but was still lower than the controls. There were positive correlation between Smad 2/3, Smad 4 and TGF-beta(1) (r = 0.91, r = 0.71, respectively, P < 0.05) and also between Smad 2/3 and hydroxyproline contents of lung tissue (r = 0.85, P < 0.05) except Smad 7.

CONCLUSIONSmad protein may have certain association with pulmonary fibrosis in silicosis.

Animals ; DNA-Binding Proteins ; immunology ; metabolism ; Lung ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Pulmonary Fibrosis ; chemically induced ; metabolism ; Quartz ; toxicity ; Smad2 Protein ; Smad3 Protein ; Smad4 Protein ; Smad7 Protein ; Trans-Activators ; immunology ; metabolism ; Transforming Growth Factor beta ; metabolism