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AIM: To investigate the mechanism of pyrrolidine dithiocarbamate(PDTC)on transforming growth factor-beta 2(TGF-β2)-induced epithelial-mesenchymal transition(EMT)in human lens epithelial cells(LECs).METHODS: LECs were treated with various doses of PDTC chemicals following TGF-β2 caused EMT on these cells. Cell proliferation and lateral migration were discovered using the CCK-8 and cell scratch test. The markers of EMT, including E-cadherin, α-SMA and nuclear factor-κB(NF-κB)signaling pathway-related expression, were tested by Western Blot as well as the changes in the expression of the apoptosis-related proteins BAX, BCL-2, Caspase-3, and Cyclin D1.RESULTS: The proliferation and migration viability of cells in the TGF-β2 treated group was increased compared to the group without TGF-β2, and the expression of α-SMA increased whereas the E-cadherin expression decreased. With the effect of TGF-β2, NF-κB p65 and phosphorylated NF-κB p65 expression increased, the concentration of TGF-β2 that had the greatest capacity for proliferation and migration was 10 ng/mL(P<0.05). Mechanism study of PDTC-induced EMT reversal and apoptosis showed that cell viability and migratory capability were both significantly reduced after PDTC intervention; PDTC prevents IκB phosphorylation, thus inhibiting NF-κB nuclear translocation. Protein associated to the NF-κB signaling pathway, and protein expression of NF-κB/IκBα/p-IκBα/Iκκ-α/p-Iκκ-α was decreased(P<0.05), PDTC increased the expression of the pro-apoptotic protein BAX/Caspase-3, expression of the inhibitor of apoptosis protein BCL-2 and the cell cycle protein Cyclin D1 was reduced. The expression of NF-κB/IκB mRNA was reduced, expression of the apoptosis-related mRNA BAX increased, while BCL-2 reduced.CONCLUSION: The EMT in LECs cells induced by TGF-β2 can be significantly reversed by PDTC, which may be related to the decreased expression of NF-κB p65/IκB/Iκκ-α and activation of apoptosis-related protein. PDTC can reverse EMT by inhibiting NF-κB signaling pathway and induce apoptosis of abnormally proliferated cells, which will provide new potential therapeutic agents for posterior capsular opacification(PCO)treatment.
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Objective: To investigate the targeting relationship between microRNA-153-3p and transforming growth factor-beta 2 (TGFβ2) and its effects on invasion, migration and epithelial-mesenchymal transformation of cultured glioma cells. Methods: Real-time fluorescence quantitative PCR and Western blotting were used to detect the effects of miR-153-3p on the expressions of TGFβ2 mRNA and protein. Bioinformatics software was used to predict the binding sites of miR-153-3p to TGFβ2. Luciferase reporter assay was used to analyze the targeting relationship between miR-153-3p and TGFβ2. Transwell and wound healing assays were used to detect the effects of miR-153-3p on SHG-44 cells invasion and migration. Western blotting was used to detect the expressions of proteins related to invasion, migration and epithelial-mesenchymal transition. Results: miR-153-3p mimic inhibited the expressions of TGFβ2 mRNA and protein in SHG-44 cells. Software predicted results showed a continuous binding region between miR-153-3p and TGFβ2. miR-153-3p mimic co-transfected with TGFβ2 wild-type reporter vector significantly decreased the activity of luciferase in the cells (P<0.01). miR-153-3p mimic co-transfected with TGFβ2 mutant reporter showed no significant change in luciferase activity. The number of invasive cells and scratch closure rate of SHG-44 cells transfected with miR-153-3p mimic were significantly decreased. The expression levels of MMP-2, MMP-9 and VEGF were significantly decreased. The expression levels of N-cadherin, Snail-2, Vimentin and Twist were significantly decreased, and the expression level of E-cadherin was significantly increased (P<0.01). The high expression of TGFβ2 alleviated the effect of miR-153-3p on the cell invasion and epithelial mesenchymal transition related proteins in SHG-44. Conclusion: miR-153-3p inhibits the invasion and migration of cultured glioma cells and epithelial mesenchymal transition by targeting TGFβ2.
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Objective To prepare a new(alcoxyle cyanoacrylate)-based nanosphere for brain targeting gene delivery and evaluate its physicochemical properties,capability of delivery of transforming growth factor beta 2(TGF-β2)antisense oligonucle?otides(ASON),and its potential use on tumor cell suppression in vitro.Methods The cationic nanospheres(NS)were prepared by emulsion polymerization method with DEAE-dextran as cationic stabilizer.The ASON were adsorbed by charge interaction,and poly?sorbate-80 was used as brain-targeting modification.The morphology was observed by transmission electron microscopy(TEM).The average particle size and Zeta potential were determined by dynamic light scattering(DLS). The ultraviolet spectrophotometry was used to determine the entrapment efficiency and drug loading.Agarose gel electrophoresis was used to analyze the optimal loading ratio of ASON-NS,and also the protection of ASON in DNaseⅠand serum containing environment.The release rate of ASON was deter?mined by dialysis.The cytotoxicity on L929 cells and the anti-tumor activity on A172 cells were evaluated by MTS.Results The TEM showed a typical round nanospheres morphology,and no adhesion was detected.The particle size was(79.04±4.33)nm,the disper?sion coefficient was 0.04 ± 0.03,the Zeta potential was(33.60 ± 0.60)mV. The encapsulation efficiency of ASON-NS was(83.14 ± 1.90)%,and the drug loading of ASON-NS was(11.59±0.56)%.The NS provided ASON protection against the Dnase I and serum containing environment. The NS-ASON could effectively deliver ASON into A172 cells and show anti-tumor activity. Besides,little L929 cytotoxicity was detected.Conclusion A new cyanoacrylate nanosphere with alcoxyle side group for brain targeting gene deliv?ery was prepared successfully. It had good ASON loading and delivery capability,providing new carrier materials for nucleic acid drugs.
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@#AIM:To investigate the relationship of matrix metalloproteinase-2(MMP-2)and transforming growth factor-beta 2(TGF-β2)levels in human vitreous with axial length(AL)of patients with high myopia.<p>METHODS: The concentrations of MMP-2 and TGF-β2 levels were tested by Enzyme-Linked Immunosorbnent Assay(ELISA). Fifty-five human vitreous samples of 55 patients were collected during vitrectomy surgery, and were divided into two groups according to their spherical equivalent(SE)and axial length(AL). High myopia group(25 cases): SE>-6.00D, AL≥26.00mm, and control group or non-high myopia group(30 cases): SE≤-6.00D, AL<26.00mm.<p>RESULTS: The MMP-2 levels in vitreous of high myopia group(96.87±55.95ng/mL)was significantly higher than that of control group(77.24±41.81ng/mL, <i>P</i><0.05), but not correlated with AL(<i>r</i>=0.088, <i>P</i>=0.544). While the TGF-β2 vitreous concentration was negatively correlated with AL(<i>r</i>=-0.344, <i>P</i>=0.014), and there was significant difference of TGF-β2 vitreous levels between high myopia group(3729.08±1890.88pg/mL)and control group(3926.00±1333.88pg/mL, <i>P</i><0.05).<p>CONCLUSION: MMP-2 and TGF-β2 in human vitreous may play a critical role in human high myopia development, and the TGF-β2 appears to be associated with axial length.
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Objective To investigate the expressions and clinical significances of breast cancer stem cell markers such as aldehyde dehydrogenase 1 (ALDH1) and transforming growth factor-β2 (TGF-β2) in patients with triple negative breast cancer.Methods Samples of 60 triple negative breast cancer tissues were investigated for the expressions of ALDH1 and TGF-β2 proteins by immunohistochemical staining.The correlation analysis,disease-free survival analysis and overall survival analysis were performed.Results The positive expressions of ALDH1 protein and TGF-β2 protein in the 60 breast cancer primary lesions were 23 cases (38.33％) and 38 cases (63.33％) respectively.The expression of ALDH1 protein was not correlated with tumor size (x2 =0.307,P =0.580),histological grade (x2 =4.244,P =0.120),clinical stage (x2 =0.982,P =0.612) or lymph node metastasis (x2 =1.111,P =0.292).The expression of TGF-β2 protein was not correlated with histological grade (x2 =4.651,P =0.098),lymph node metastasis (x2 =3.513,P =0.061),clinical stage (x2 =1.310,P =0.519) or tumor size (x2 =0.629,P =0.428).The disease-free survival time [(38.43±3.86) months vs.(53.38 ±2.58) months] and the overall survival time [(42.00±3.11) months vs.(53.84 ± 2.19) months] of ALDH1-positive patients were significantly shorter than those of ALDH1-negative patients,and the differences were statistically significant (x2 =8.490,P =0.004;x2 =11.270,P =0.001).The disease-free survival time [(42.81 ±3.32) months vs.(54.72 ±2.50) months] and the overall survival time [(44.74 ± 2.68) months vs.(57.18 ± 1.55) months] of TGF-β2 positive patients were significantly shorter than those of TGF-β2-negative patients,and the differences were statistically significant (x2 =4.300,P =0.038;x2 =8.900,P =0.003).The expression of ALDH1 protein was positively correlated with the expression of TGF-32 protein (r =0.360,P =0.005).Conclusion The ALDH1 phenotype is an independent predictor of poor prognosis.The activation of TGF-32 signaling pathway may be involved in the regulation of triple-negative breast cancer stem cells.
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Background Researches showed that transforming growth factor-β2 (TGF-β2) promotes the activity of human Tenon capsular fibroblasts (TFs),which plays a role in the scarring of filtering blebs after antiglaucoma surgery.However,its mechanism is not fully clear.Lysyl oxidases (LOXs) are important extracellular matrix proteases which can catalyze the cross-linking of collagen and elastin.Investigating the impact of TGF-β2 on the expression of LOXs has a great significance for the understanding of the pathogenesis of filtering bleb scarring and its prevention.Objective This study was to investigate the effect of TGF-β2 on the expression of LOXs in cultured human TFs.Methods The TFs at 4-8 generations were divided into normal control group and different concentrations of TGF-β2 treated-groups,and 100,200,400,800 μ1 of TGF-β2 with the final concentration of 2,4,8 and 16 ng/ml was added into the medium to treat human TFs respectively for 24 hours.The LOXs in the cells were detected by Western blot to determine the optimal dose of TGF-β2.The 4 ng/ml TGF-β2(200 μ1) was used to treat human TFs for 6,12,24 and 48 hours respectively,and the change of LOXs expression in the cells over time was assayed by Western blot.The expression and distribution of LOX protein in the normal cells and TGF-β2-treated cells was detected by using immunofluorescence technique.This study was approved by Daping Hospital of Third Military Medical University Ethic Commission.The guardians of the patients who offered the specimen knew the purpose of the study and signed informed consent.Results Western blot assay showed that the expressions of LOX,LOXL1,LOXL2,LOXL3 and LOXL4 in the cells were gradually elevated from the normal control group and 2,4,8,16 ng/ml TGF-β2-treated groups,showing significant differences among the groups (F =37.338,13.438,31.067,11.767,15.167,all at P＜0.01).The expression of LOXL2 protein in the cells was 0.68±0.07,1.09±0.10,1.32±0.07,1.50± 0.06 and 1.89±0.12 in the normal control group and 6-,12-,24-and 48-hour groups respectively after 4 ng/ml TGF-β2 treatment,with a significant increase over time (F =82.832,P=0.000).The expression of LOX was weak in the normal cultured TFs,while the fluorescence intensity of LOX expression was evidently enhanced in the cytoplasm of the cells in the TGF-β2-treated group.Conclusions TGF-β2 upregulates the expressions of LOXs in human TFs in a dose-and time-dependent manner,which probably offers a basis for the further study on the prevention of filtering bleb scarring after glaucoma surgery.
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Objective To investigate hydrogen peroxide (H2O2) and transforming growth factor-β2 (TGF-β2) induced fibronectin (FN), collagen 1 (COL1), nuclear factor (NF)-κB P65 proteins and interlukin (IL)-1βgene expression in human trabecular meshwork cells (HTMCs), and the interventional mechanism of resveratrol (RSV). Methods (1) HTMCs with 70 to 80%confluency were divided into 5 groups. The experimental groups were treated with serum-free medium and with H2O2 at concentrations of 150, 300, 450 and 800μmol/L. The control group was treated with 0μmol/L H2O2. The protein levels of FN, COL1, NF-κB P65 and NF-κB P65 phosphorylation (P-NF-κB P65) were measured by Western blot assay. The expression of IL-1βgene was measured by qPCR. (2) HTMCs were divided into 3 groups. The control group was treated withserum-free medium and without H2O2 and RSV. The H2O2 group was treated with 300μmol/L H2O2. The H2O2+RSV group was treated with 300μmol/L H2O2 and 25μmol/L resveratrol (RSV). The expressions of proteins and genes mentioned above were detected in three groups. NF-κB P65 nuclear translocation was assessed by immunofluorescence technique. (3) HTMCs were divided into 3 groups. The control group was treated with serum-free medium and without TGF-β2 and RSV. The TGF-β2 group was treated with 5μg/L TGF-β2. The TGF-β2+RSV group was treated with 5μg/L TGF-β2 and 25μmol/L RSV. The expressions of proteins and genes mentioned above were detected in three groups. Results (1) Compared with control group, the protein levels of FN and P-NF-κB P65 were significantly increased in 150, 300, 450 and 800μmol/L groups,the expression levels of COL1 protein and IL-1β gene were significantly increased in 300, 450 and 800 μmol/L groups (P <0.05). There were no statistical significances between other indicators. (2) The expression levels of FN, COL1, P-NF-κB P65 proteins and IL-1βgene were significantly higher in H2O2 group than those in control group, and which were significantly lower in H2O2+RSV group than those in H2O2 group. Compared with control group, only the expression of IL-1βgene was decreased in H2O2+RSV group (P < 0.05). NF-κB P65 was only expressed in cytoplasm in control group, while it was expressed in both cytoplasm and nucleus in H2O2 group. Compared with H2O2 group, NF-κB P65 was mainly expressed in cytoplasm. (3) Compared with control group, the expressions of FN, COL1, P-NF-κB P65 proteins and IL-1β gene were significantly increased in TGF-β2 group (P < 0.05). Compared with TGF-β2 group, the indicators mentioned above were significantly decreased in TGF-β2+RSV group (P<0.05). Conclusion H2O2 and TGF-β2 can upregulate the expression of FN, COL1, P-NF-κB P65 proteins and IL-1βgene in HTMCs, which may be involved in the development and progression of glaucoma. RSV can inhibit the influence of H2O2 and TGF-β2 in HTMCs and exert a protective effect on glaucoma.
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Objective To observe the expression of heat shock protein 47 (HSP47) and transforming growth factor-β2 (TGF-β2) in vitreous specimens and epiretinal membranes of patients with proliferative vitreoretinopathy diseases.Methods Vitreous specimens and epiretinal membranes were obtained from 48 patients (48 eyes) with proliferative vitreoretinopathy (PVR) and 50 patients (50 eyes) with proliferative diabetic retinopathy (PDR).Vitreous specimens and internal limiting membranes were collected from 20 patients (20 eyes) with idiopathic macular hole (IMH) as control group.The expression of HSP47 and TGF-β2 in the vitreous specimens was evaluated using enzyme linked immunosorbent assay.The expression of HSP47,TGF-β2,types Ⅰ and Ⅲ collagen in epiretinal membrane and internal limiting membrane specimens were observed for immunohistochemical staining method.The correlation between the positive expression of HSP47 and TGF-β2,types Ⅰ and Ⅲ collagen in epiretinal membrane specimens of patients with PVR and PDR were analyzed.Results The expression of HSP47 in vitreous specimens of patients with PVR,PDR and IMH were (212.35±23.32),(231.30±26.79),(171.06±28.91) pg/ml,respectively.The expression of TGF β2 in vitreous specimens of patients with PVR,PDR and IMH were (1919.96 ± 318.55),(1939.39 ± 177.57),(1194.61 ± 234.20) pg/ml,respectively.The expression of HSP47,TGF-β2 in the vitreous specimens of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=12.952,34.532;P＜0.01).The epiretinal membrane of patients with PVR and PDR showed markedly increased expression of HSP47,TGF β2,types Ⅰ and Ⅲ collagen in the cytoplasm and extracellular matrix.The expression of HSP47 and type Ⅲ collagen was negative and the expression of TGF-β2 was weakly positive and the expression of types Ⅰ collagen was positive in internal limiting membrane of patients with IMH.The expression of HSP47,TGF β2,types Ⅰ and Ⅲ collagen in the epiretinal membrane of patients with PVR and PDR were significantly increased compared with patients with IMH and the difference was statistically significant (F=13.469,18.752,12.875,20.358;P＜0.01).The expression of HSP47 was positively correlated with thepositive expression of TGF-~,types Ⅰ and Ⅲ collagen in epiretinal membrane specimens of patients withPVR (r=0.475,0.556,0.468;P＜0.05) and PDR (r=0.484,0.589,0.512;P＜0.05).Conclusions This study showed increased consistent expression of HSP47 and TGF-β2 in vitreous and epiretinal membrane specimens of patients with PVR and PDR.Both HSP47 and TGF-β2 were expressed in the cytoplasm and extracellular matrix.HSP47 and TGF-β2 may be involved in the pathological process of PDR and PVR by promoting collagen synthesis.
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Objective To predict the binding sites of transforming growth factor-βreceptor Ⅱ (TβRⅡ ) ectodomain and the aptamer S58 specifically targeted TβRⅡ ,and to confirm the structure stability of the aptamer S 58 in vitro .Methods We created three-dimensional structure by utilizing ssDNA aptamer sequences ,the crystal structure of the TβRⅡ was searched by protein data bank database .According to the results of the molecular docking experiments on aptamer S 58 and TβRⅡ ectodomain ,we sheared the aptamer sequences ,then verified its affinity respectively by biosensor technology and Western blot .Results Binding sites of aptamers S58 and TRβⅡ ectodomain included site Ⅰ(T4 ,T5 ,G6 ,C7) ,site Ⅱ(G13 ,A14 ,T15 ,C16 ,G17 ,C18 ) ,site Ⅲ (T31 ,G32 , T33 ,C34) and site Ⅳ(G40 ,A41 ,T42 ,T43 ,T44 ,G45 ,G46) .We validated the high affinity between aptamer S58 and TRβⅡ ectodo-main .The expression of α-smooth muscle actin(α-SMA) protein in the human tenon′s capsule fibroblasts was descended obviously after the experiment of the aptamer S58 in comparing with the control of DMEM (P< 0 .05) .But the new ssDNA by shear the aptamer ssDNA S58 according to the results were poor than aptamers S58 .Conclusion The aptamer S58 targeted TβRⅡ was high-ly specific with a certain stability ,any changing of structure will reduce the affinity of TβRⅡ .Computer-aided molecular docking technology has become an important means of an exploratory intermolecular interaction ,and can provides a good theoretical basis on medical research .
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Background Researches demonstrated that dendritic cells(DCs) are uniformly immature in the central cornea but mature in the peripheral region of cornea.So an important question is which factor impact the maturation of DCs,especially in terms of corneal transplant rejection and the known roles of DCs in the development and persistence of some corneal diseases.Objective This study aimed to examine whether corneal stroma cells (CSCs) inhibit DCs maturation through secreting transforming growth factor beta 2 (TGF-β2) and prostaglandin E2 (PGE2).Methods DCs,T cells and CSCs were isolated and cultured from clean BALB/c and C57BL/6 mice.The level of PGE2 and TGF-β2in CSCs culture supernatant and the fresh RPMI 1640 medium were then analyzed by enzyme linked immunosorbent assay (ELISA).During the DCs maturation stage,the neutralizing TGF-β2 antibody and the EP2 receptor antagonist AH6809 were added in the CSCs culture supernatant respectively.According to the different treatment,cultured cells were assigned to different groups as follows:control group,CSCs culture supernatant group,AH6809 group,TGF-β2 antibody group,AH6809 +TGF-β2 antibody group.Subsequently,the cellular surface markers for DCs,including CD11c,CD80,CD86,and MHC- Ⅱ,were analyzed by flow cytometry.The capability of stimulating the proliferation of T lymphocytes was evaluated by allogeneic mixed lymphocyte reactions,and the function of endocytosis was assessed by fluorescein isothiocyanate-dextran(FITC) uptake.Results The data of ELISA showed a higher concentration of TGF-β2 and PGE2 in murine CSCs culture supernatant than in the fresh RPMI 1640 medium.Compared with the CSCs culture supernatant group,the expression of CD80,CD86,and MHC- Ⅱ was up-regulated ( P ＜ 0.05 ),the expression of dextran was down-regulated ( P ＜ 0.05 ),and the stimulate index was increased( P＜ 0.05 ) in the TGF-β2 antibody group; the expression of CD86,and MHC-Ⅱ was up-regulated (P＜0.05),the expression of dextran was down-regulated ( F =13.740,P =0.006 ),and the stimulate index was increased(P＜0.05) in the AH6809 group;the expression of MHC-Ⅱ was up-regulated and the stimulate index was increased with statistical difference in interaction(P＜0.05 ) in the AH6809+TGF-β2 antibody group.Compared with the control group,the expression of CD80 and CD86,and the stimulate index was still lower(P＜0.05 ).Conclusions TGF-β2 and PGE2 contribute to the inhibitory effects on DCs maturation mediated by murine CSCs in vitro and further have additive effect on the immunosuppression of DCs.
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Laser photocoagulation is commonly used for the treatment of retinal neo-vascularization in ischemic retinal diseases. Transforming growth factor-beta2 is known to be produced by RPE cells after laser photocoagulation and to be related to both regression of neovascularization and acceleration of preretinal fibrosis. In this study, we investgated the safe extent of laser photocoagulation not promoting the fibrosis by measuring intravitreal TGF-beta2 released following treatment. In gray rabbit eyes, trans pars plana vitrectomies and laser photocoagulations[1/8, 1/4, 1/2 of the fundus]were performed. The each eyeball was enucleated and TGF-beta2 concentration was measured at 12, 24, 72 hours after laser photocoagulation. And fibrotic activity was observed by the subconjunctival injection of fibrocyte and TGF-beta2 [0.1, 1, 10ng/milliliter]. The results were as follows:1. When TGF-beta2 was injected subconjunctivally along with fibrocytes, fibrocytic activity was observed found at the concentration of 1 or 10ng/milliliter of TGF-beta2. 2.The intravitreal concentration of TGF-beta2 after laser photocoagulation to the 1/8, 1/4, 1/2 of fundus area were 0.05, 0.1, 0.2ng/milliliter respectively. From the above results, we suggest to treat limit the area of laser photo-coagulation to 1/8 of fundus area in order to prevent the retinal fibrosis.
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Acceleration , Fibrosis , Light Coagulation , Retinal Diseases , Retinaldehyde , Transforming Growth Factor beta2 , VitrectomyABSTRACT
To explain a part of the machanism of regression of new vessels after laser photocoagulation in retinal vascular occlusive diseases causing neovascularization, particularly for diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, and age-related macular degene- ration, we performed this study. Photocoagulation was done with argon green laser on the right superofrontal retina of the 15 eyes of white rats. On the first, third, and sixth day after laser photocoagulation, both eyes of 5 rats were enucleated and stained with polyclonal anti-transforming growth factor-beta2(Anti-TGF-beta 2) and anti-vascular endothelial growth factor(Anti-VEGF) antibody by immuno peroxidase technique. TGF-beta2 and VEGF were not expressed in retina of normal control eyes. After laser photocoagulation, degree of expression of TGF-beta2 and VEGF increased in 24 hours, only in and adjacent to the photocoagulated area. Thereafter, degree of expression of both factor decreased, especially that of VEGF decreased much more than that of TGF-beta2. To elucidate the exact mechanism, more qualitative and quantitative analysis will be necessary.