ObjectiveTo investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. MethodsTSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). ResultsThe final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). ConclusionInhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.
OBJECTIVE To investigate the different expression of actin, myosin II in hypertrophic scars, keloids and normal skins, and to understand the relationship of actin, myosin II and the scar contracture. METHODS Fifteen cases with hypertrophic scars, 10 cases with keloids and 15 cases with normal skins were chosen randomly. The expression of actin and myosin II were detected by immunohistochemical method in the hypertrophic scars, keloids and normal skins. The fibroblasts isolated from three types of tissue were cultured in vitro, then actin and myosin II in three different fibroblasts were measured using flow cytometry. RESULTS The immunohistochemical staining of myosin II in hypertrophic scars was positive, while the staining in keloids and normal skins were negative. The positive rate of myosin II expression in hypertrophic scars, keloids and normal skins were (95.11 +/- 2.78)%, (16.86 +/- 7.11)%, and (5.31 +/- 1.79)% respectively. There were significant difference between keloids and the two others(P lt; 0.01). The actin expression in three difference tissues were positive, there were no significant difference in hypertrophic scars, keloids and normal skins(P gt; 0.05). The positive rate of actin expression in hypertrophic scars, keoids and normal skins were(77.77 +/- 15.43)%, (88.89 +/- 10.29)%, and (82.92 +/- 13.48)% respectively, and there were no significant difference(P gt; 0.05). CONCLUSION Myosin II may play an important role in the scar contracture. Actin is the contractile protein of cell, it plays
OBJECTIVE: To study the relationship between intracellular actin and scar contracture. METHODS: Fibroblasts from 10 cases of hypertrophic scar and 5 cases of keloid were cultured in vitro. Total actin, filamentous actin(F actin), globular actin (G actin) and the ratio of F to G actin(F/G) were measured by densitometry after differential extraction and separation by polyacrylamide gel electrophoresis in the presence of sodium sulfate. RESULTS: Total actin, F actin, G actin and F/G in hypertrophic scar fibroblasts were 2.38 ng/10(4) cells, 0.98 ng/10(4) cells, 1.42 ng/10(4) cells and 0.68 respectively, while in keloid fibroblasts were 1.68 ng/10(4) cells. 0.46 ng/10(4) cells, 1.26 ng/10(4) cells, and 0.36 respectively. There was significant differences between two tissues fibroblasts in the items of total actin, F actin, G actin, and F/G (P lt; 0.01), while no significant difference in G actin (P gt; 0.05). CONCLUSION: Total intracellular actin, F actin, and F/G may play an important role in the scar contracture. The hypertrophic scar and keloid can be distinguished by the contents of total intracellular actin, F actin and F/G.
Objective To investigate the effects of platelet-derived growth factor(PDGF) on the expression of α-smooth muscle actin(α-SMA) of cultured human retinal pigment epithelium cells(RPE). Methods Cultured human RPE cells of the 4-6 th passages were divided into two groups: Delbecco′s modified Eagle′s medium (DMEM) and 2%DMEM (20 g/L foeta calf serum+DMEM). PDGF (0,1,50 ng/ml) was added to medium.The expression of α-SMA was detected and quantitatively analyzed by image process of immunofluorescence.Results PDGF stimulated the expression of α-SMA of human RPE cells.In group of DMEM, The rate of RPE of α-SMA expression was 40%-50% and the intension of fluorescence was 8.08 without PDGF. After stimulated by PDGF(1 ng/ml,50 ng/ml), the rates were 80% and 90% respectively, and the intension of fluorescence were 12.35 and 17.23. In 2%DMEM group, The rates of RPE of α-SMA expression were 85% without PDGF, and 95% ,100% respectively treated with PDGF (1 ng/ml,50 ng/ml). The intension of fluorescence was 14.79 without PDGF, and after stimulated by PDGF, they were 16.28 at 1 ng/ml and 21.36 at 50 ng/ml，which was 2 .7 times ber than that in DMEM group without PDGF. Conclusion PDGF could stimulate RPE cells to express α-SMA. (Chin J Ocul Fundus Dis，2003，19：201-268)
【Abstract】ObjectiveTo compare the effects of newcastle disease virus (NDV) and adriamycin (ADM) on surface structure and actin of hepatocellular carcinoma cell lines SMMC-7721. Methods SMMC-7721 carcinoma cell lines were divided into 2 groups. NDV was added into one group, while ADM was added into the other group. The cells were then cultured at 5 time phases (8, 16, 24, 36 and 48 h). Intracellular actin and Ca2+ were examined by using immunofluorescence method. CD44 and intercellular adhesion molecule-1 (ICAM-1) were detected by using immunochemical method and flow cytometry, respectively. The change of cellular surface structure was observed by scan electron microscope. Results Cells gradually contracted and turned round over time. It was observed that actin was segmented and cells alignment became disordered. The mean fluorescence intensity of actin decreased in both groups, but it was obvious in NDV group. There were significant differences of fluorescence intensity between 2 groups at the phases of 16 h (P＜0.05), 24 h (P＜0.05), 36 h (P＜0.01) and 48 h (P＜0.05), except the one after 8 h. Intracellular Ca2+ concentration increased gradually in both groups, and the amplifications in NDV group were significantly higher at the phases of 24 h, 36 h and 48 h than those in ADM group (P＜0.01， P＜0.05 and P＜0.01, respectively). There were also differences at 8 and 16 h, but there were no statistical significance. The expression of CD44 in cells decreased. The mean fluorescence intensity of ICAM-1 raised gradually, and then came to peaking at 36 h, but there was no significant difference between two groups. All the above indices between different phases in the same group showed significant differences (P＜0.05). Conclusion Both NDV and ADM could make tumor cells degenerate and rupture, but the effect of NDV is more intensive. It could increase the fragility of cells and hasten the process of cell rupture. Disintegrated cancer cell and changes of adhesion molecule could lead cancer cells be identified, encapsulated, and killed by immune cells under static condition.
【Abstract】Objective To investigate the changes of myoepithelial cells in mammary atypical hyperplasia and breast cancer. MethodsSP immunohistochemistry was used to detect actin expression in normal breast tissue, grade Ⅰ, Ⅱ and Ⅲ atypical hyperplasia and breast cancer. Electromicroscopy was used to observe the changes of ultrastructure of myoepithelial cells. Results Actin was only detected in myoepithelial cells of normal breast tissue and grade Ⅰand Ⅱ atypical hyperplasia. The positive expression rates of actin in grade Ⅲ atypical hyperplasia（70%） and breast cancer（90%） were significantly higher than that in grade Ⅱ atypical hyperplasia（10%），P＜0.01. In mammary atypical hyperplasia, the number of myoepithelial cells increased with disturbed alignment and abnormal ultrastructure. The changes included that the protrusions on the cell surface diminished, myofilaments and pinosomes in the myoepithelial cells of grade Ⅱ, Ⅲ atypical hyperplasia decreased, and the irregularity of the nuclear morphosis and the increase of nuclear heterochromosome were found. ConclusionThe changes of actin expression in atypical hyperplasia are possibly correlated with carcinogenesis of breast cancer, and myoepithelial cells may play a role in carcinogenesis of breast cancer.
ObjectiveTo improve the knowledge of pulmonary actinomycosis. MethodsOne case of pulmonary actinomycosis with positive blood culture diagnosed on December 2013 in North Huashan Hospital was analyzed,and related literature from CNKI and Medline after 1990 were reviewed. ResultsA 57-year-old male had recurrent fever for 24 days.Chest CT showed pneumonia in the right middle lobe.Actinomyce culture was twice positive by anaerobic blood culture.The patient's temperature was normal after large doses of penicillin and doxycycline therapy.Literature review revealed that the incidence of pulmonary actinomycesis is common in males.Poor oral hygiene is the main predisposing factors.The common clinical presentations include cough,sputum production,chest pain and hemoptysis.The peripherally progressive enhancement on CT has a certain diagnostic value for pulmonary actinomycosis.Pulmonary actinomycosis can increase FDG uptake on 18F-FDG PET scan and can mimic lung cancer.Accurate diagnosis is generally made by histopathological examination of lung biopsy or surgical samples.High-dose intravenous penicillin therapy is preferred for pulmonary actinomycosis followed by prolonged oral antibiotics for 6 to 12 months.Surgical intervention may be necessary for refractory hemoptysis or patients who do not respond to antibiotic therapy. ConclusionPulmonary actinomycosis is a rare disease.Symptoms of pulmonary actinomycosis are atypical.This patient is the first reported case of actinomyces with positive blood cultures.
ObjectiveTo explore the effects of cytoskeleton depolymerizing agent and stabilizer on the clathrin/caveolae-mediated endocytosis, the expression of membrane vascular endothelial cadherin (VE-cad), and the vascular permeability by the transformation of cytoskeleton structure after lipopolysaccharide (LPS) treatment. MethodsCRL-2922 cells were used in the experiments. Indexes were tested at corresponding time point according to name of group, but in blank control group indexes could be tested at any time point. CRL-2922 cells were divided into blank control group, LPS-1 h group, and LPS-4 h group to observe cytoskeleton structure; CRL-2922 cells were divided into LPS-1 h group, Cyt D+LPS-1 h group, LPS-4 h group, and Jasp+LPS-4 h group to determine the expression of membrane VE-cad, and to determine the expression of its co-immunoprecipitation with clathrin and caveolin-1 (Cav1); besides, CRL-2922 cells were divided into blank control group, LPS-1 h group, Cyt D+LPS-1 h group, LPS-4 h group, and Jasp+LPS-4 h group to detect the cumulative infiltration rate. Results①The cytoskeleton showed a dynamic change after LPS treat-ment, the F-actin polymerized and stress fibers formed at 1 hour after LPS treatment, but depolymerized at 4 hours after LPS treatment. ②Compared with LPS-1 h group, the level of co-immunoprecipitation of VE-cad with clathrin in Cyt D+ LPS-1 h group decreased (P<0.05), the level of co-immunoprecipitation of VE-cad with Cav1 increased (P<0.05), and expression level of VE-cad in plasma membrane decreased (P<0.05); compared with LPS-4 h group, there was no significant difference in the level of co-immunoprecipitation of VE-cad with clathrin of Jasp+LPS-4 h group (P>0.05), but the level of co-immunoprecipitation of VE-cad with Cav1 decreased in Jasp+LPS-4 h group (P<0.05), and expression level of VE-cad in plasma membrane increased (P<0.05). ③Compared with blank control group, the cumulative infiltration rates of LPS-1 h group and LPS-4 h group were both higher (P<0.05); compared with LPS-1 h group, the cumulative infiltration rate of Cyt D+LPS-1 h group was higher (P<0.05); compared with LPS-4 h group, the cumulative infiltration rate of Jasp+LPS-4 h group was lower (P<0.05). ConclusionActin cytoskeleton shifts from polymerization to depoly-merization after LPS treatment, the structural change of actin cytoskeleton is an important reason for the transformation of VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated after LPS treatment.