EFFECTS OF TGF-β_1 ON THE EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 IN CULTURED HUMAN RENAL INTERSTITIAL FIBROBLASTS

Objective To investigate the effects of transforming growth factor-β1 (TGF-β1 ) on the expression of plasminogen activator inhibitor type 1 (PAI-1 ) mRNA in renal interstitial fibrosis in vitro. Methods Human renal interstitial fibroblasts were isolated and cultured in vitro. The cells wers stimulated by TGF-β1 with different concentration (0 to 10ng/ml ) at different time (0 to 48h). The expression of PAI-1 mRNA was assayed by RT-PCR. Results TGF-β1, had dose-dependent and time-dependent effects on the expression of PAI-1 mRNA in renal interstitial fibroblasts. Conclusion TGF-β1 may partic- ipate in renal fibrosis with inducing the expression of PAI-1 mRNA in renal fibroblasts and affecting the synthesis and degradation of extracellular matrix (ECM).

阿魏酸哌嗪对TGF-β_1诱导肾成纤维细胞表型活化的影响

目的观察阿魏酸哌嗪对转化生长因子-β1(TGF-β1)诱导肾成纤维细胞表型活化的影响,探讨其抗肾间质纤维化的可能机制。方法体外培养正常大鼠肾脏成纤维细胞(NRK-49F),利用MTT观察阿魏酸哌嗪对TGF-β1诱导细胞活力的影响;免疫细胞化学法观察阿魏酸哌嗪对TGF-β1诱导细胞α-平滑肌肌动蛋白(α-SMA)、结缔组织生长因子(CTGF)蛋白表达的影响;实时荧光定量逆转录-聚合酶链反应(realtime RT-PCR)检测阿魏酸哌嗪对TGF-β1诱导细胞α-SMA及CTGFmRNA表达的作用,双抗体夹心ABC-ELISA法检测阿魏酸哌嗪对TGF-β1诱导细胞上清型胶原(Col)、纤维连接蛋白(FN)表达的影响。结果TGF-β1可以显著增加大鼠肾脏成纤维细胞活力,上调细胞α-SMA、CTGF蛋白和mRNA的表达以及Col、FN的表达。与TGF-β1刺激组相比,阿魏酸哌嗪能部分抑制TGF-β1诱导的细胞活力增加、α-SMA、CTGF的表达和细胞外基质(ECM)的合成。结论阿魏酸哌嗪可以在一定程度上拮抗TGF-β1诱导的肾纤维化效应。

Transforming growth factor-β1 involved in urotensin Ⅱ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta

Background Urotensin Ⅱ （UⅡ） is a new vasoconstrictive peptide that may activate the adventitial fibroblasts.Transforming growth factor-β1 （TGF-β1） is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UⅡ-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.Methods Adventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression of α-smooth nuscle actin （α-SM-actin）, the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR （real-time RT-PCR） and Western blotting, respectively.Results UⅡ stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% （P ＜0.01）, than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10-8 mol/L of UⅡ （P ＜0.01）, which was increased by 59.9%,compared with in the control group （P ＜0.01）. The secretion of TGF-β1 induced by UⅡ was significantly blocked by SB-710411 （10-7 mol/L）, a specific antagonist of UⅡ receptor. In addition, both UⅡ （10-8 mol/L） and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UⅡ was inhibited by the specific neutralizing antibody （20 μg/ml） of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 （20 ng/ml）was inhibited by SB-710411 （10-7 mol/L）, the UⅡ receptor antagonist.Conclusion This study suggests that UⅡ could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UⅡ, and TGF-β1 signaling might be one of the important pathways by which UⅡ is involved in vascular fibrosis.

Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

Background Remodeling of the anterior cruciate ligament （ACL） graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site （IRES） sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 （TGFβ1） and vascular endothelial growth factor 165 （VEGF165） genes （named Ad-VEGF165-IRES-TGFβ1）. We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts. Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR. Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers. Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

Transforming growth factor-β1 phage model peptides isolated from a phage display 7-mer peptide library can inhibit the activity of keloid fibroblasts

Background Transforming growth factor-β1 （TGF-β1） is known to have a role in keloid formation through the activation of fibroblasts and the acceleration of collagen deposition. The objective of this current study was to isolate TGF-β1 phage model peptides from a phage display 7-mer peptide library to evaluate their therapeutic effect on inhibiting the activity of keloid fibroblasts.Methods A phage display 7-mer peptide library was screened using monoclonal anti-human TGF-β1 as the target to obtain specific phages containing ectogenous model peptides similar to TGF-β1. Enzyme-linked immunosorbent assay （ELISA） was performed to select monoclonal phages with good binding activity, which underwent DNA sequencing. MTT assay and apoptosis assessment were used to evaluate the biological effects of the phage model peptides on keloid fibroblasts. Immunofluorescence assay was employed to show the binding affinity of the model peptides on phages causing keloid fibroblasts. Quantitative real-time PCR analysis was carried out to detect the expressions of nuclear factor κB （NF-κB） mRNA, connective tissue growth factor （CTGF） mRNA and TGF-β receptor Ⅱ （TβRII） mRNA in keloid fibroblasts.Results Specific phages with good results of ELISA were beneficiated. Four phage model peptides were obtained. The data of MTT showed that TGF-β1 and one phage model peptide （No. 4） could promote keloid fibroblasts proliferation,however, three phage model peptides （No. 1-3） could inhibit keloid fibroblasts proliferation. The results of apoptosis assessment showed that the three phage model peptides could slightly induce the apoptosis in keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the expressions of NF-κB mRNA and CTGF mRNA in the three phage model peptide groups decreased, while the expression of TβRII mRNA slightly increased.Conclusions Three phage model peptides isolated from a phage display 7-mer peptide library can inhibit keloid fibroblasts proliferation and induce the apoptosis in keloid fibroblasts. They can inhibit the activity of keloid fibroblasts by blocking TGF-β1 binding to its receptor and then regulating the expressions of NF-κB, CTGF and TβRII.

Expressions of TGF-β2, bFGF and ICAM-1 in lens epithelial cells of complicated cataract with silicone oil tamponade

AIM： To investigate the expression differences of transforming growth factor-β2（TGF-β2）, basic fibroblast growth factor（b FGF） and intercellular cell-adhesion molecule-1（ICAM-1） in lens epithelial cells（LECs） of complicated cataract with silicone oil tamponade and agerelated cataract. METHODS： Totally 150 eyes of 150 patients（aged 35 to 77y） were investigated, including 75 patients with complicated cataract after silicone oil tamponade and 75 patients with age-related cataract. The central piece of anterior capsules was collected during cataract surgery. TGF-β2, b FGF and ICAM-1 were detected in the 60 specimens of the two groups by immunohistochemistry. The expression levels of the three kinds of messenger ribonucleic acid（m RNA） were determined by real-time quantitative reverse transcriptionpolymerase chain reaction in the 90 specimens of the two groups.RESULTS： TGF-β2 was detected in the cytomembrane and cytoplasm of the LECs and b FGF was detected in the nucleus. ICAM-1 was positive in the cytomembrane of the LECs and the distribution of positive cells was uneven. The m RNA genes expression of the TGF-β2, b FGF and ICAM-1 was significant differences between the two groups and markedly increased in complicated cataract group（P〈0.05）.CONCLUSION： The up-regulated TGF-β2, b FGF and ICAM-1 maybe associate with the occurrence and development of complicated cataract with silicone oil tamponade.

Different Regulation Effects of Gekko Sulfated Glycopeptide in Breast and Liver Cancer Cell Lines

Background:Our previous work demonstrated that Gekko sulfated glycopeptide extracted from the Chinese gecko Shou gong(Gekko swinhonis Guenther)could inhibit tumor growth by regulating basic fibroblast growth factor.However,basic fibroblast growth factor has opposing effects on growth in breast and liver cancers.Direct effects and mechanisms of Gekko sulfated glycopeptide on tumor growth remain unclear and are ripe for further exploration.Methods:Differential regulation by Gekko sulfated glycopeptide and bFGF were studied in established human breast cancer MCF-7 cells and hepatocarcinoma HepG2 cells.Cell proliferation was evaluated with a Trypan blue exclusion assay.Cell cycle phases were measured by flow cytometry.Basic fibroblast growth factor,transforming growth factor-β1,and p21WAF1/CIP1 mRNA and protein expression levels were detected by real-time PCR(mRNA)and ELISA(protein).Changes in phosphorylated extracellular signal-regulated kinase levels were analyzed by Western blot.Results:Data indicated that Gekko sulfated glycopeptide inhibited the proliferation of HepG2 cells(P<0.001)and also blocked basic fibroblast growth factor-stimulated proliferation of these cells(P=0.001).Gekko sulfated glycopeptide was shown to increase transforming growth factor-β1 and p21WAF1/CiP1 expression(P<0.01)and partially compensate for reductions therein induced by basic fibroblast growth factor.Conversely,in MCF-7 cells,Gekko sulfated glycopeptide alone had no observable effect on transforming growth factor-β1 and p21WAF1/CiP1 expression.Gekko sulfated glycopeptide did,however,enhance basic fibroblast growth factor-induced inhibition of cell proliferation and transforming growth factor-β1 and p21WAF1/CiP1 expression in the MCF-7 cells(P=0.001,P<0.01,P<0.01,respectively).Gekko sulfated glycopeptide was shown to suppress basic fibroblast growth factor secretion in both HepG2 and MCF-7 cell lines(P<0.05 and P<0.01,respectively)and inhibited extracellular signal-regulated kinase 1/2 phosphorylation facilitated by basic fibroblast growth factor.Gekko sulfated glycopeptide alone decreased phosphorylated extracellular signal-regulated kinase in HepG2 cells but did not visibly affect phosphorylated extracellular signal-regulated kinase levels in MCF-7 cells.Conclusions:Gekko sulfated glycopeptide,a basic fibroblast growth factor inhibitor,differentially regulates growth in different cancer cell lines,and these differences may be determined by the opposing effects of basic fibroblast growth factor on transforming growth factor-β1 and p21WAF1/CiP1 levels in breast and liver cancer cells.

Effect and Mechanism of Ganoderma lucidum Polysaccharides on Human Fibroblasts and Skin Wound Healing in Mice

Objective: To investigate the effects of Ganoderma lucidum polysaccharides(GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. Methods: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide(MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type Ⅰ(CICP) and transforming growth factor-β1(TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. Results: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts(P<0.05 or P<0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group(P<0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group(P<0.05 or P<0.01). Conclusion: A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.