Objective To investigate the mechanismof lung injury caused by paraquat poisoning by observing the changes of fibrogenic cytokines in acute paraquat poisoned rats and the effects of pyrrolidine dithiocarbamate ( PDTC) . Methods Sprague-Dawley rats were randomly divided into three groups, ie. acontrol group ( n =6) , a PDTC group ( n =36) , a paraquat group ( n = 36) , and a paraquat + PDTC group( n =36) . The rats in the PDTC group, the paraquat group, and the paraquat + PDTC group were subdivided into 6 subgroups sacrificed respectively on 1st, 3rd,7th,14th, 28th and 56th day after the treatment. The levels of transforming growth factor-β1( TGF-β1 ) , platelet-derived growth factor ( PDGF) , insulin-like growthfactor-1 ( IGF-1) in serum were measured. Meanwhile the expression of connective tissue growth factor ( CTGF) and hydroxyproline in lung tissues were detected. The relationship of above cytokines with hydroxyproline was analyzed. Results The destructive phase in early ( 1 ～7 d) was characterized by hemorrhage, alveolar edema, and inflammatory cell infiltration. The proliferous phase in later stage ( 14 ～56 d) was characterized by diffused alveolar collapse with fibroblast proliferation and patchy distribution of collagen fibers. Compared with the control group, the level of TGF-β1 on all time points, the level of PDGF from7th to 56th day, the level of IGF-1 from3rd to 56th day in the paraquat group all significantly increased ( P lt;0. 01) . Immunohistochemistry results showed CTGF positive cells mainly located in aleolar epithelialcells, endothelial cells,macrophages in early stage, and fibroblasts were main positive cells on the 28th and the 56th day. The expression of CTGF in the paraquat group increased gradually compared with the control group on different time points ( P lt; 0. 05 or P lt; 0. 01) . Meanwhile, the levels of above cytokines were positively correlated with the level of hydroxyproline. Noteworthy, PDTC treatment led to significant decreases of above cytokines compared with the paraquat group in corresponding time points ( P lt;0. 05 or P lt;0. 01) .Conclusions Over expressions of IGF-1, TGF-β1 , PDGF, IGF-1 and CTGF may play important roles in lung fibrosis of paraquat poisoned rats. PDTC, as a b NF-κB inhibitor, may inhibits NF-κB activity and further significantly decreases expressions of cytokines, leading to significantly attenuated pulmonary inflammation and fibrosis. However, the mechanisms of PDTC intervention still remain to be explored.
Objective To study the function of platelet-derived growth factor (PDGF) in inducing phosphorylation extracellular signalregulated kinase 1/2 (pERK1/2) localization in osteoblasts. Methods Primary osteoblasts were isolated and cultured from cranial bone of 10 mice atthe age of 3 days, weighting 6-9 g without limitation in male and female.The sixth passage osteoblasts were incubated in 1% serum for 12 hours and divided into 2 groups: treated with DMSO(control group) or with PP2(experimentalgroup) for 30 minutes. Each group was further divided into 2 subgroups according to with or without PDGF (20ng/ml) stimulation for 10 minutes. pERK1/2 localization was analysized by immunofluorescence staining in osteoblasts pretreated with or without Src inhibitor PP2. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or with PP2 (experimental group) for 30 minutes. Each group was further divided into two subgroupsaccording to with or without PDGF (20 ng/ml) stimulation for 10 mintues. The ability of osteoblast migration was determined by wound healing assay. The sixth passage osteoblasts were divided into 2 groups treated with DMSO (control group) or 10 μmol/L PP2 (experimental group) for 30 mintues. Each group was further divided into 2 subgroups according to with or without PDGF (20 ng/ml) stimulation.The pERK1/2 was determined by Western blot in osteoblastic cytoskeleton inducedby PDGF. Results Immunofluorescence staining showed pERK1/2 localization in osteoblastic nuclears and focal adhesions after PDGF stimulation. PP2 significantly inhibited ERK1/2 localization in focal adhesions, but not in nuclears. The wound healing assay results showed that PP2 significantly inhibited osteoblast migration induced by PDGF. The result of Western blot demonstrated that pERK1/2 in osteoblastic cytoskeleton was significantly inhibitedSrc activation is required for pERK1/2 translocalization to focal adhesions and osteoblasts migration.
Objective To investigate the possible signaling mechanisms by which recombinant human plateletderived growth factor (rhPDGF) accelerated healingof cutaneous wound in diabetic rats. Methods Four full-thickness skin woundswere incised in the back of 26 male Wistar diabetic rats. The wounded rats were divided into 3 groups (7 or 8 rats each group). One group without treatmentwas used as a control, and the other 2 groups were treated with rhPDGF at a dose of 7.0 μg/cm2 wound or vehicle (DMSO/09% NaCl, vol/vol 1∶1) from 1 to14 days. The wound healing was evaluated by the measurements of the wound volume and area. Immunofluorescent and immunohistochemical staining were used to examine the phosphorylation of extracellular signalregulated kinase 1/2 (ERK1/2) andthe expression of proliferative cell nuclear antigen (PCNA), respectively. Results Granulation tissue appeared in the bed of wound after injury. The number of blood capillary buds and fibroblasts was greater in the rhPDGF-treated group than that in the other 2 groups. A lot of inflammatory cells infiltration and collagen deposition were observed in the wound. The wound-volume in the rhPDGF-treated group was smaller than that in control group (Plt;0.05). The reepithelialization rate in rhPDGF-treated group was higher than that inthe other 2 groups at 7 days after injury (Plt;0.05). The expression of PCNA in reparative cells was higher in rhPDGF-treated group than in control group or vehicle-treated group at 3,7 days after injury(Plt;0.05). The phosphorylation of ERK1/2 was ber in rhPDGF-treated group than that in control group or vehicle group at 7 and 14 days after injury(Plt;0.05). Conclusion These results suggest that rhPDGF accelerates wound healing and improves healing quality by increasingthe phosphorylation of ERK1/2.
Objective To study the effect of serum rich in growth factors (SRGF) derived from plateletrich plasma (PRP) on the biological function of human and rat osteoblast.Methods PRP and platelet-poor plasma (PPP) obtained from healthy human and SD rat were activated by thrombin toget SRGF and serum poor in growth factors (SPGF). The level of TGFβ1 and PDGF-AB in human-SRGF and SPGF were assayed by enzyme-linked immunoassay(ELISA). Rat and human osteoblast were cultured and identified. Rat osteoblasts were treated with 5% rat-SRGF, 5% rat-SPGF and serumfree F12 medium, respectively. And human osteoblast were treated with 5% human-SRGF, 5% human-SPGF and serumfree DMEM. Cellular mitogenic activity was evaluated by thiazoly blue (MTT) colorimetric assay at 24, 48, 72 and 96hours.Results The level of TGF-β1 in human-SRGF was 307.67±35.57 ng/ml, and that of PDGF-AB was 52.76±7.89 ng/ml. The proliferation of rat and human osteoblast were promoted after treated with rat-SRGFand human-SRGF, respectively. In rat osteoblast groups, there were significant differences in absorbency between ratSPGF group and rat-SRGF group at 48 and 96 hours(Plt;0.05). In human osteoblast groups, the differences between human-SPGF group and human-SRGF group were significant at 48, 72 and 96 hours(Plt;0.05). The proliferation of these two kinds of osteoblasts almost stopped in serum-free medium, and the differences in absorbency , compared with othergroups，were significant (Plt;0.05). Conclusion High quality of PRP can be achieved by the improved method and SRGF is capable of up-regulating the proliferation of rat osteoblast and human osteoblast.
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)
ObjectiveTo summarize the pathogenesis and epidemiology features of gastrointestinal stromal tumor(GIST), explore its diagnosis and therapy, and analyze its prognosis. MethodThe pertinent literatures about the pathogenesis, epidemiology features, diagnosis, therapy, and prognosis of GIST in recent years were reviewed. ResultsGIST was non-epithelial tumor which derived from interstitial cells of Cajal, was the most common mesenchymal tumor about accounting for 1%-3% in the digestive tract tumor. The median onset age of patients with GIST was 40-60 years. The gastric stromal tumor was about 60% in all the digestive tract tumor. The current consensus statement was that there was a relation between the pathogenesis of the GIST and proto-oncogene c-kit or platelet-derived growth factor receptor alpha(PDGFRα)gene mutation. But the mutations of PDGFRαand c-kit gene did not emerge at the same time in the same patient. The clinical manifestations of GIST were not specific, and the diagnosis mainly depended on endoscope and image technology, the correct diagnosis depended on pathological examination. The treatment of GIST was given priority of surgery and molecular targeted drug therapy, and the prognosis was closely related to risk assessment stratify of GIST. ConclusionsGISTs are mesenchymal tumors that has a potential of malignant transformation, the risk classification criteria for aggressive clinical course of primary GIST is an important indication for guiding the clinical therapy and prognostic evaluation. Further research would be needed in prevention, diagnosis, treatment, and relapse prevention of GIST.
Objective To study the effect of mechanical stretch on the microenvironment of BEAS-2B on macrophage polarization and the role of polarized macrophages in the epithelial-mesenchymal transition (EMT) of BEAS-2B. Methods Using enzyme linked immunosorbent assay to detect the changes in the levels of cytokines such as interferon-γ, granulocyte-macrophage colony stimulating factor, tumor necrosis factor-α, interleukin (IL)-4, IL-6, IL-10 in the supernatant of lung epithelial cells cultured statically and mechanically stretched. The M0 macrophages (derived from THP-1) were stimulated by stretch/static conditioned medium of BEAS-2B. The surface markers of M1 (CD197) /M2 (CD206) macrophages were detected by flow cytometer. Stretch/static conditioned medium were used to stimulate the co-culture system of macrophages and BEAS-2B in the presence or absence of platelet-derived growth factor receptor inhibitor (PDGFRi), then the protein expression level of EMT makers was examined by Western blot. Results Exposure of BEAS-2B to mechanical stretch resulted in significantly higher production of the pro-M1/M2 polarized factor. The EMT of the co-culture system of M0 and BEAS-2B could be induced by stretch conditioned medium, epithelial marker cytokeratin (CK)-8 and E-cadherin were decreased, while mesenchymal marker α-smooth muscle actin, N-cadherin and vimentin were increased in stretch conditioned medium group. The expression of platelet-derived growth factor (PDGF) was significantly higher in stretch conditioned medium group. The PDGFRi can block the EMT in stretch conditioned medium group. Conclusions The lung epithelial cell supernatant induced by mechanical stretch can promote the polarization of macrophages to M1 and M2. Polarized macrophages promote EMT in human lung epithelial cells via PDGF, and blocking PDGF might attenuate the VILI-associated lung fibrosis.