OBJECTIVETo investigate the effect of HMME-PDT on the proliferation and cell distribution of hypertrophic scar fibroblast (HSF).

METHODSHSF were cultured and only 4-6th passages were used in this study. Argyrophilic protein in nucleolar organizer regions(AgNORs) were calculated by I. S% after argyrophilic staining. Flow cytometry was applied to analyze the cell cycle and proliferation index (PI).

RESULTS1) I. S% of HSF after HMME-PDT was reduced markedly. 2) HMME-PDT inhibited HSF entering S stage from G, stage, cell percentage in S stage was decreased to (11.2 +/- 2.3)%. 3) PI in HMME-PDT group was less than that in control group [(35.0 +/- 3.4)% vs (27.2 +/- 3.1)%, P < 0.05].

CONCLUSIONSHMME-PDT can inhibit proliferation of HSF, and chang cell distribution.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; drug therapy ; pathology ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans ; Photochemotherapy

OBJECTIVEThe aim of this study was to investigate the effects of SiO2 on fibrocytes and whether fibrocytes participate in silicosis in vivo.

METHODSA macrophagocyte (AM)/fibrocyte coculture system was established, and AMs were treated with 100 μg/mL SiO2. Flow cytometry was used to detect the number of fibrocytes. Real-time PCR was performed to measure the expression of collagen I, collagen III, and α-SMA mRNA. The levels of collagen I, collagen III, and TGF-β1 protein were determined by ELISA. Immunohistochemical staining was performed to measure α-SMA protein expression. A rat silicosis model was induced by intratracheal instillation of SiO2. Lung histopathological evaluation was conducted using HE and Masson's trichrome staining after 1 and 9 weeks. The number of fibrocytes in peripheral blood or lung tissue of rat was detected by flow cytometry. Double-color immunofluorescence was applied to identify fibrocytes in the lung tissue.

RESULTSPeripheral blood monocytes were found to differentiate into fibrocytes in vitro in a time-dependent manner, and exposure to crystalline silica might potentiate fibrocyte differentiation. In addition, fibrocytes were able to migrate from peripheral blood to the lung tissue, and the number of fibrocytes was increased after SiO2 exposure.

CONCLUSIONSilica exposure potentiates fibrocyte differentiation, and fibrocytes may participate in silicosis in vivo.

Animals ; Cell Differentiation ; drug effects ; Collagen ; metabolism ; Fibroblasts ; drug effects ; Lung ; metabolism ; pathology ; Male ; Rats ; Silicon Dioxide ; toxicity ; Silicosis ; metabolism ; pathology

OBJECTIVETo explore the expression of integrin-linked kinase (ILK) in fibroblasts (Fbs) of scar induced by cobalt chloride (CoCl2) and its effect on cell proliferation.

METHODSThe human hypertrophic scar Fbs of seven patients were isolated and cultured in vitro. Cells from the 5th to the 6th passages were used in the experiment. Six bottles of Fbs were obtained from each of the seven patients, and they were respectively cultured with DMEM nutrient solution containing CoCl2 in the concentration of 0, 50, 100, 150, 200, and 250 µmol/L for 24 h. The expression of ILK mRNA was determined with real-time fluorescence quantitative PCR. Fbs were stimulated by CoCl2 in the most suitable concentration (100 µmol/L) and the protein expression of ILK was determined 0, 1, 2, 4, 12, and 24 h after the stimulation. Then the Fbs were divided into control group (cultured with nutrient solution), negative control group (transfected with con-siRNA), and ILK siRNA group (transfected with ILK siRNA). They were cultured with nutrient solution containing CoCl2 in different concentrations 24 h after transfection, with 4 wells for each concentration in each group. The cell proliferation was detected by XTT assay. Data were processed with one-way analysis of variance (ANOVA) and ANOVA for repeated measurement, and LSD method was used in multiple comparisons.

RESULTSThe expression level of ILK mRNA was highest in Fbs cultured with 100 µmol/L CoCl2 for 24 h, with significant difference compared with those of Fbs cultured with other concentrations of CoCl2 (F = 50.958, P < 0.001). The expression of ILK protein in Fbs cultured with 100 µmol/L CoCl2 for 1 h (0.243 ± 0.009) was lower than that cultured for 0 h (0.387 ± 0.017), and it started to increase from 2 h (0.361 ± 0.010), and exaggerated at 4 h (0.584 ± 0.028), 12 h (0.730 ± 0.029), and 24 h (0.785 ± 0.031). The expression levels of ILK protein at 1, 4, 12, 24 h were statistically different from that at 0 h (P values all below 0.05). XTT showed that cell proliferation level was highest in control group when cultured with 100 µmol/L CoCl2 (F = 488.026, P < 0.001), which decreased from 150 µmol/L. The cell proliferation level in control group cultured with 250 µmol/L CoCl2 was significantly lower than that with 0 µmol/L (P values all below 0.05). There was no significant change in cell proliferation in ILK siRNA group among different concentrations of CoCl2 (F = 2.542, P = 0.056). The cell proliferation level in ILK siRNA group was significantly lower than that in control group and negative control group (F = 2519.542, P < 0.001).

CONCLUSIONSILK may be a key protein in response of hypoxia in Fbs. The mild hypoxia can stimulate the expression of ILK and promote the proliferation of Fbs, while severe hypoxia can reduce the expression of ILK and inhibit cell proliferation.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix ; metabolism ; pathology ; Cobalt ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans ; Protein-Serine-Threonine Kinases ; metabolism

OBJECTIVETo seek an effective drug for treatment of human hyperplastic scar through studying the effects of curcumin on fibroblast growth and collagen synthesis.

METHODSFibroblasts derived from scar tissue and from normal epidermal tissue were isolated and cultured separately with tissue-block method, their morphology were observed under invert phase contrast microscope, their growth curve was drawn respectively to determine the speed of growth. Then, fibroblasts from scar were stimulated with curcumin in different concentrations (0, 12.5, 25, 50 and 100 micromol/L) for detecting the inhibitory effect of curcumin on growth of fibroblasts using MTT methods and that on activity of procollagen alpha-1 gene transcription in fibroblast was detected by RT-PCR.

RESULTSThe cell growth curve showed that double-multiplying time was 5 days in fibroblasts from scar and 4 days in those from normal dermis, showing significant difference between them (P < 0.05). MTT showed that curcumin in 12.5 micromol/L showed a cell proliferation enhancing trend, and its absorbance value was significantly higher than that in the normal group, but the effect turned to inhibition when concentration increased to over 25-100 micromol/L, and became significant inhibition at concentration of 50 and 100 micromol/L. Besides, curcumin also showed markedly inhibition on collagen type I synthesis in fibroblasts (P < 0.01).

CONCLUSIONHigh concentration curcumin can inhibit effectively the fibroblast proliferation and collagen I synthesis in hyperplastic scar, therefore, may has therapeutic effect on the disease in human being.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagen Type I ; biosynthesis ; Curcumin ; pharmacology ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans

OBJECTIVETo study the effect of melatonin on proliferation and apoptosis of fibroblasts in human hypertrophic scar and its mechanism.

METHODSFibroblasts from human hypertrophic scar were isolated and cultured with DMEM medium containing 10% FBS, and then they were divided into control (C, added with ethanol), low concentration (LC, added with 1 × 10(-5) mmol/L melatonin), middle concentration (MC, added with 1 × 10(-3) mmol/L melatonin), and high concentration (HC, added with 1 mmol/L melatonin) groups according to the random number table. After being cultured for 24 hours, cell morphologic change was observed under microscope; XTT-PMS assay was used to examine cell proliferative activity; cell cycle and apoptosis were assessed with flow cytometry after double staining of FITC and PI, and the levels of cyclin E, p53, and Fas mRNA were determined with fluorescence quantitative RT-PCR. Data were processed with analysis of variance and LSD test.

RESULTS(1) Fibroblasts in C group were spindle-shaped with growth in colonies. Along with the increase in melatonin concentration, fibroblasts in LC, MC, and HC groups gradually dispersed, deformed and atrophied, with shrunk cellular membrane, and decrease in ratio of nucleus and cytoplasm. (2) Proliferative activity of fibroblasts in LC, MC, and HC groups decreased along with an increase in melatonin concentration (1.49 ± 0.15, 1.24 ± 0.20, and 0.92 ± 0.09), which were lower that in C group (1.79 ± 0.10, F = 67.61, P < 0.05). Cell ratios of S and G2/M phases in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were all lower than those in C group [(10.6 ± 1.1)%, (6.1 ± 1.2)%, (3.2 ± 0.8)% vs.(16.9 ± 1.3)%, F = 286.10, P < 0.05; (13.5 ± 1.1)%, (9.8 ± 1.0)%, (6.0 ± 0.7)% vs. (16.7 ± 1.6)%, F = 162.69, P < 0.05]. Apoptotic rates in early and late stages of LC, MC, and HC groups increased along with an increase in melatonin concentration, all higher than those in C group (with F value respectively 424.05, 236.44, P values all below 0.05). The expressions of cyclin E mRNA in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were lower than that in C group (1.58 ± 0.21, 0.90 ± 0.20, and 0.24 ± 0.12 vs. 2.90 ± 0.30, F = 266.79, P < 0.05), while the expressions of p53 mRNA and Fas mRNA showed opposite tendency (with F value respectively 10.11, 12.03, P values all below 0.05).

CONCLUSIONSMelatonin can inhibit proliferation and induce apoptosis of fibroblasts in hypertrophic scar through regulating the gene expressions of cyclin E, p53, and Fas.

Adult ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Cyclin E ; metabolism ; Female ; Fibroblasts ; drug effects ; metabolism ; pathology ; Humans ; Male ; Melatonin ; pharmacology ; Oncogene Proteins ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo observe the effect of tetrandrine on activity of collagenase derived from human hypertrophic scar for the sake of clarifying the mechanism as tetrandrine acting on scar.

METHODSThe experimental concentration was controlled below that of cell proliferation inhibited, SDS-PAGE electrophoresis was adopted to separate collagenase from extracellular matrix, and then activated by trypsin analyzed the activity of collagenase with density scanning apparatus. At the same time quantity of extracellular collagen was measured using improved chloraseptine T oxidizing assay, moreover analyzed correlation between activity of collagenase and quantity of extracellular collagen.

RESULTSIn the concentration below the lever of inhibiting fibroblast proliferation, the total activity of collagenase could be significantly increased by tetrandrine with dosage-dependence associated with quantity of extracellular collagen reduced, which was much greater than that of triamcinolone.

CONCLUSIONIncreasing activity of collagenase on degradation of collagen even in a lower concentration was one of the mechanisms of tetrandrine treating hypertrophic scar.

Benzylisoquinolines ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Collagenases ; metabolism ; Fibroblasts ; cytology ; Humans

OBJECTIVETo investigate the effects of ethanolic extract from Capparis spinosa (ECS) on the fibroblast proliferation and type I collagen production in normal and progressive systemic sclerosis (PSS).

METHODCellular activity was determined by the MTT method. Apoptosis was detected by flow cytometry analysis of Annexin V-stained cells. The expression levels of type I collagen messenger RNA and protein were analyzed by RT-PCR and western blot analysis.

RESULTECS could significantly inhibit the proliferation of fibroblast and reduced the expression of alpha2 (I) collagen mRNA and type I collagen protein in PSS in a dose-and time-dependent manner. ECS did not affect the proliferation of fibroblast and expression of type I collagen mRNA and protein in normal human. ECS could counteract the harmful effects on fibroblast by H2O2.

CONCLUSIONECS can effectively inhibit the fibroblast proliferation and type I collagen production in PSS.

Capparis ; chemistry ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; cytology ; drug effects ; Humans ; Scleroderma, Diffuse ; drug therapy ; metabolism ; pathology

OBJECTIVETo investigate the effects of lanthanum chloride on the apoptosis of fibroblasts in trauma tissue.

METHODSFifty adult female SD rats were used and linear incisions were made on the back near the joints of extremities of the rats. One of the cuts receiving no treatment was designated as blank control (C). 0.25 ml of distilled water, lanthanum chloride (50 mmol/L) and the antibody of (TGFbeta(1)) transforming growth factor beta(1) (0.2 mg/ml) were respectively injected into the both sides of the other three wounds subcutaneously and the wounds were divided into simulating control (SC), lanthanum chloride (LC) and antibody (A) groups. The fibroblast apoptosis in the wound tissue samples and the change in intracellular calcium concentration (Ca(2+)) were determined by flow cytometry (FCM) and TUNEL methods on the 14th and 28th day after the injection.

RESULTSApoptosis of fibroblasts was enhanced significantly after 14 days of injection in LC and A groups compared with that in C and SC groups (P < 0.05 approximately 0.01). Furthermore, intracellular Ca(2+) was increased evidently in LC group (P < 0.01).

CONCLUSIONIt is indicated that lanthanum chloride might be effective in preventing scar development.

Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cicatrix ; prevention & control ; Female ; Fibroblasts ; drug effects ; pathology ; Flow Cytometry ; Lanthanum ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Wound Healing ; drug effects

OBJECTIVETo study the effect of silicosis alveolar macrophages (AM) restimulated by SiO(2) on expression of c-myc oncogene in human embryo lung fibroblasts.

METHODSThe bronchoalveolar lavage of silicosis patients was collected. AMs were divided into 2 groups: (1) SiO(2): AMs were stimulated with SiO(2) (30 microg/ml) for 1, 2, 6, 12, 24 and 36 h; (2) control: treated for the same time without SiO(2). Fibroblasts were cultured with different AMs supernatants for 2 h or 7 h respectively. The expression of c-myc mRNA was determined by RT-PCR and protein by Western Blot.

RESULTSThere was no c-myc expression when fibroblasts were static. The supernatants in the S6 group stimulated expression of c-myc mRNA and protein, with the peak expression at 2 h and 7 h respectively. In the control group, AMs supernatants cultured in different time stimulated expression of c-myc mRNA and protein with the most evident expression at 12 h. The ratios were 0.749 +/- 0.088 and 0.759 +/- 0.101 respectively. Compared with control in the same period, c-myc mRNA and protein expression were significantly stronger treated with the supernatants in which AMs were stimulated for 1 h, 2 h and 6 h by SiO(2) (P < 0.05 or P < 0.01).

CONCLUSIONAMs stimulated with SiO(2) has the ability to induce c-myc oncogene expression in human embryo lung fibroblasts.

Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; Lung ; drug effects ; metabolism ; pathology ; Macrophages, Alveolar ; drug effects ; metabolism ; Proto-Oncogene Proteins c-myc ; genetics ; metabolism ; RNA, Messenger ; genetics ; Silicon Dioxide ; toxicity ; Silicosis ; metabolism ; pathology

OBJECTIVETo study the interaction between transformation of human pulmonary epithelial cells and activation of fibroblasts induced by Yunnan tin mine dust.

METHODS(1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell line WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 degrees C and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 microg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA) agglutination and anchorage-independent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of alpha-SMA in fibroblasts were determined with immunocytochemistry.

RESULTS(1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce epithelial cells co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cultured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00 per thousand +/- 1.00 per thousand and 15.33 per thousand +/- 2.52 per thousand respectively, with the significant differences (P < 0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed alpha-SMA. Co-cultured with epithelial cell, the alpha-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding.

CONCLUSIONS(1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.

Cell Cycle ; drug effects ; Cell Line ; Cell Transformation, Neoplastic ; drug effects ; Coculture Techniques ; Dust ; Epithelial Cells ; pathology ; Fibroblasts ; metabolism ; pathology ; Humans ; Tin ; toxicity