ObjectiveTo investigate the effects of transforming growth factor-β (TGF-β) in choroidal neovascularization (CNV) induced by laser in mice. Methods Eighty male C57BL/6J mice at the age of 6-8 weeks old were randomly divided into the normal control, photocoagulation model, photocoagulation with phosphate buffered saline (PBS control group) and photocoagulation with TGF-β receptor inhibitor groups (TGF-β receptor inhibitor group), twenty mice of each group. Fundus argon laser photocoagulation was performed in the photocoagulation model group, PBS control group and TGF-β receptor inhibitor group to induce CNV. One week, two, three and four weeks after the laser procedure, fundus fluorescein angiography (FFA) was carried out in the normal control or photocoagulation model groups to observe CNV formation dynamically. Western blot was used to analyze the expressions of TGF-β in the retina from the mice of normal control or photocoagulation model groups, and VEGF or TNF-α in the retina of normal control, PBS control or TGF-β receptor inhibitor groups. The CNV areas of each group were evaluated by using fluorescein stain on retinal pigment epithelium (RPE)/choroid flat mounts after two weeks of photocoagulation. ResultsThe FFA results showed the retinal vessels centered on the optic disc and arranged radially, while the choroidal vascular present network distribution in the normal control mice. Significant leakage of fluorescein showed discoid strong fluorophore in photocoagulation sites of retina at one week after photocoagulation. The quantitative analysis results of Western blot demonstrated that the TGF-β protein expression levels in retina of photocoagulation model mice gradually increased with time passing. The protein expression levels of TGF-β were significant differences in the photocoagulation model group comparing with the normal control group (F=13.042, P < 0.05). The protein expression levels of TNF-α (F=14.721, 17.509) and VEGF (F=18.890, 11.251) increased significantly in retina of PBS control or TGF-β receptor inhibitor groups when compared with that of normal control group at one week, two, three and four weeks after photocoagulation, and the differences were both statistically significant (P < 0.05). Compared with PBS control group, the protein levels of TNF-α and VEGF in retina from TGF-β receptor inhibitor group were significantly reduced, the differences was statistically significant (F=21.321, 16.160, P < 0.05). Two weeks after laser photocoagulation, a distinct reduction in CNV lesion size in the TGF-β receptor inhibitor group mice when compared to PBS or normal control groups, the differences was statically significant (F=4.482, P < 0.05). ConclusionTGF-β may promote CNV formation by up-regulating both TNF-α and VEGF protein expressions, the application of its specific inhibitor is able to reduce CNV progression.
Objective To determine whether the transforminggrowth factor β1 (TGF-β1) is a key regulatory molecule required for an increase or a balance of extracellular matrix (ECM) and DNA synthesis in the goat passaged nucleus pulposus (NP) cells. Methods The NP cells isolated from the goat intervertebral discs were cultured in vitro for a serial of passages and transfected with the replicationincompetent adenoviral vectors carrying the human TGF-β1 (hTGF-β1) or lacZ genes. Then, they were cultured in monolayer or alginate bead 3dimensional (3-D) systems for 10 days.The changes in the production and the molecular components of ECM that occurredin the NP cells transfected with Ad/hTGF-β1 or the controls were evaluated by Westernblot and absorbance of glycosaminoglycan (GAG)-Alcian Blue complexes. Differences of DNA synthesis in the variant cells and culture systems were assessed by fluorometric analysis of the DNA content. ResultsA quantitation in the variant culture systems indicated that in monolayers the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher cell viability and more DNA synthesis(P＜0.05); however, in the alginate 3-D culture system, the NP cells transfected with Ad/hTGF-β1 did not have any significant difference from the controls(P＞0.05). The Western blotting analysis ofthe protein sample isolated from the variant cells for TGF-β1, type Ⅱ collagen, and Aggrecan expression indicated that in the monolayers and alginate 3-D culture systems the NP cells at Passage 3 transfected with Ad/hTGF-β1 revealed much higher protein levels than the controls(P＜0.05)； whereas the type Ⅰcollagen content was much lower than the controls (P＜0.05), but a significatly increased ratio of type Ⅱ/type Ⅰ collagen was found in both of the cell culture systems(P＜0.05). The GAG quantification also showed a positive result in both the cell culture systems and the NP cells at Passage 3 transfected with Ad/hTGF-β1 had a much higher GAG content than the controls(P＜0.05). Conclusion To a greaterextent, hTGF-β1 can play a key role in maintaining the phenotype of the NP cells and can still have an effect of the phenotypic modulation after a serial of the cell passages. The NP cells that are genetically manipulated to express hTGF-β1 have a promising effect on the restoration of the intervertebral disc defects. The NP cells transfected with Ad/hTGF-β1 cultured in the 3-D alginate bead systems can show a nearly native phenotype.
Objective To study the influence of transforming growth factor-β1(TGF-β1), dentin non-collagen proteins(dNCPs) and their complexon tissue engineering pulp system. Methods Collagen I and dentin powder were used to construct the system of pulp cells in 3dimensional culture, dentin powder was added in the gel. The tissue engineering pulp were divided TGF-β1 group, dNCPs group, TGF-β1/dNCPsgroup and control group.After3, 6 and 14 days, the appearance and the differentiation of pulp cells were observed by HE staining and immunohistochemical staining -respectively. Results Collagen I could form netted collagen gel construction. Growing condition of pulp cells in gel was similar to that of pulp cells in vivo. After the TGF-β1 and dNCPswere added, the pulp cells had some characteristics of odontoblasts and had unilateral cell process after culture 6 days. Pulp cells arranged with parallel columnar and form dentin-pulp-like complex after 14 days. Immunohistochemical staining showed dentin salivary protein(DSP) began to express in some cells.The number of positive cell was most in the TGF-β1 group. No positive cells were detected in the control group. Conclusion The transforming growth factor-β1 and noncollagen proteins can stimulate the pulp cells to transform into odontoblasts to some extent, which promote the formation of tissue engineering pulp.
Objective To review the recent advances in transforming growth factor-β(TGF-β) super family study and its role in new bone formation. Methods The latest original articles related to this subject were retrieved extensively,especially the effect of TGF-β, bone morphogenetic proteins(BMPs) and activin(ACT) on distractionosteogenesis. Results TGF-β, BMPs and ACT play important roles in prompting new bone formation and each of them has different effects. Among them, TGF-β can stimulate the proliferation of osteoblast and synthesis ofextra cellular medium; BMPs can initiate the differentiation of interstitial cell toosteocyte; then ACT displays the combine effect of above two factors. Conclusion TGF-β superfamily can regulate new bone formation and thus shorten the course of mandibular distraction osteogenesis.
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.
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 observe whether transforming growth factor-beta;2（TGF-beta;2）could promote the differentiation of retinal stem cells in rats cultured in vitro. Methods The retinal stem cells were separated from the embryonic ratsprime; eyes under the dissecting microscope, cultured, and subcultured. The cells were identified by nestin and Chx-10 immunofluorescence. The sixth generation of cells were induced and differentiated, immunofluorescent stained with anti-glial fibrillary acidic protein，anti-opsin, anti-b-tubulin, and anti-protein kinase C, and identified the final cells. Results The cultured cells after induced by TGF-beta;2 differentiated to the mature cells. The results of immunofluorescence showed that the differentiated cells induced by TGF-beta;2 were more than which induced by the embryonic bovine blood serum. Conclusion TGF-beta;2 may induce the retinal stem cell differentiating into retinal cells. The inductive and differentiating effect of TGF-beta;2 is ber than which of the blood serum. (Chin J Ocul Fundus Dis, 2007, 23: 104-107)
Objective To investigate the role of transforming growth factorβ3 (TGF-β3) on the amylase secretion of rat submandibular gland cells(RSGCs).Methods The RSGCs were cultured and identified. The expressions of CK 8.13, S100 and Vimentin in the RSGCs were examined by immunohistochemical staining. The experimental group was divided into 5 groups according to differentconcentrations of TGF-β3 (0.5, 1.0, 5.0, 10.0 and 25.0 ng/ml) and no TGF-β3 culture was used as control group. The effects ofTGF-β3 on the cell proliferation and amylase secretion were examined at the24th, the 48th, the 72nd and the 96th hour. MTT colorimetric method was used to estimate vital force of culture cells. Amylase protein was assayed by autobiochemistry equipment and Western blotting.Results The RSGCs were stained positively for CK 8.13 and S-100, but negatively for Vimentin. There were no significant differences in absorbency between the experimental groups and the control group(Pgt;0.05). Compared with the control group,TGF-β3 at concentrations of 0.5-10.0 ng/ml significantly stimulated the amylase secretion of RSGCs after 72 and 96 hours(Plt;0.01). But high concentration of TGF-β3 (25.0ng/ml) showed no stimulation. Western blotting demonstrated that the cultured RSGCs and submandibular gland had the same band of amylase electrophoresis.Conclusion TGF-β3 can stimulate RSGCs to differentiate and to secrete amylase, but TGF-β3 has no effect on proliferation ofRSGCs.
Objective To explore an experimental method of transfecting the marrow stromal stem cells (MSCs) with the reconstructed PGL3-t ransforming growth factor-β1 (TGF-β1) gene and to evaluate the feasibility of selfinduction of MSCs to the chondrocytes in vitro so as to provide a scientific and experimental basis for a further “gene enhanced tissue engineering” research. Methods The rabbit MSCs was transfected with the reconstructed PGL3-TGF-β1gene by the Liposo mesMethod, the growth of the cells were observed, and the growth curve was drawn. The living activity of the transfected cells in the experimental group was evalua ted by MTT, and the result was significantly different when compared with that in the control group. By the immunohistochemistry method (SABC), the antigens of TGF-β1 and collagen Ⅱ were examined at 2 and 7 days of the cell culture afte r transfe ction with PGL3-TGF-β1gene. The pictures of the immunohistochemistry slice were analyzed with the analysis instrument, and the statistical analysis was perfor med with the software of the SPSS 11.0, compared with the control group and the blank group. Results Transfection of the cultured rabbit MSCs in vitro with the reconstructed PGL3-TGF-β1gene by the Liposomes Method achie ved a success, with a detection of the Luceraferase activity. The result was significantly different from that in the control group (Plt;0.01). Tested by MTT, the living acti vity of the transfected cells was proved to be significantly decreased (Plt;0.01 vs. the control group). By the immunohistochemistry method (SABC) to study TGF-β1 positive particles were detected in the experimental group，but there were no positive particles in the control and the blank groups. There was a significant difference between the two groups of the experiment and the control group based on the analysis of the ttest (Plt;0.01). By the immunohistochemistry me thod (SABC) to study collagen Ⅱ, there were more positive particles in the transfected cells in t he experimental group than in the control and the blank groups, and there was a significant difference between the experimental group and the two other groups based on the t-test (Plt;0.01). Conclusion Transfection of the rabbit MSCs with the reconstructed PGL3-TGF-β1 gene by the Liposomes Method is successful. There may be some damage to the cells when transfection is performed. The transfecte d BMS cells with PGL3-TGF-β1 gene can express and excrete TGF-β1when cultured in vitro. The transfected MSCs that secret TGF-β1 can be self-induced into the chondrocytes after being infected for 7 days when cultured in vitro.