低剂量长波紫外线反复照射诱导培养成纤维细胞表达基质金属蛋白酶实验研究

目的：探讨长波紫外线引起皮肤光老化、皱纹形成的机制。方法：以7.2J/cm2长波紫外线单次或多次照射培养真皮成纤维细胞,光镜、电镜观察细胞的形态学变化,原位杂交和免疫组化的方法检测基质金属蛋白酶中的间质胶原酶（MMP-1）、间质溶解素-1（MMP-3）mRNA及其共同的组织抑制因子（TIMP-1）蛋白在各实验组成纤维细胞的表达并进行定量分析。结果：在长波紫外线累积剂量达57.6J/cm2后,培养真皮成纤维细胞开始出现具有细胞衰老特征性的形态改变,但仅经两次7.2J/cm2 UVA照射,培养成纤维细胞就出现MMP-1、MMP-3 mRNA的表达并随累积照射剂量增加逐渐增强,但各照射组的TIMP-1蛋白均仅在照射后48h内呈一过性轻度表达。结论：皮肤光老化皱纹形成可能与长波紫外线照射引起真皮成纤维细胞基质金属蛋白酶及其组织抑制剂表达失衡密切相关。     

Keratinocytes stimulate fibroblast hyaluronan synthesis through the release of stratifin: A possible role in the suppression of scar tissue formation

Scar tissue formation during healing of extensive skin wounds may be related to delayed reepithelialization, while elevated levels of hyaluronan may suppress scar tissue formation. This study investigates the expression of hyaluronan by human skin fibroblasts in response to keratinocyte‐conditioned medium (KCM), and attempts to identify any active factors within the conditioned medium. Serum‐free KCM was assessed for its ability to stimulate the incorporation of ³H‐glucosamine into fibroblast glycosaminoglycans, and hyaluronan synthesis. Conditioned medium was concentrated with an ultrafiltration membrane with a 30 kDa cutoff. Stratifin was assessed for its ability to stimulate hyaluronan synthesis and its role in KCM. KCM stimulated fibroblast glycosaminoglycan synthesis up to a 3.3‐fold increase and a 6.5‐fold increase in hyaluronan synthesis compared with serum‐free controls. Preliminary characterization of the active factors showed that they are retained by a >30 kDa ultrafiltration membrane, and are protease sensitive and heat resistant. Emmprin and stratifin were shown to stimulate fibroblast hyaluronan synthesis, and the hyaluronan‐stimulating activity of KCM was removed following stratifin depletion and to a lesser extent by emmprin depletion. Keratinocytes release soluble factors, including stratifin, that stimulate fibroblast hyaluronan synthesis, and this stimulation of fibroblast hyaluronan may contribute to the suppression of scar tissue formation.     

Effect of heparin on production of transforming growth factor (TGF)-beta1 and TGF-beta1 mRNA expression by human normal skin and hyperplastic scar fibroblasts

Heparin affects both dermal fibroblast proliferation and collagen and may mediate these effects by altering the levels of transforming growth factor-beta1 (TGF-beta1) production and TGF-beta1 mRNA expression as a wound healing modulator. The purpose of this study is to probe the effect of heparin on TGF-beta1 and TGF-beta1 mRNA production by human normal skin and hyperplastic scar fibroblasts. This research investigates the effect of heparin on TGF-beta1 and TGF-beta1 mRNA production by human normal skin and hyperplastic scar fibroblasts with exposure to 0 microg/mL, 100 microg/mL, 300 microg/mL, or 600 microg/mL heparin for 24, 48, 72, or 96 hours in a serum-free in vitro model. Levels of TGF-beta1 in the supernatants and TGF-beta1 mRNA expression of fibroblasts were determined by enzyme-linked immunosorbent assay (ELISA) and real time RT-PCR, respectively. Heparin (300 microg/mL and 600 microg/mL) stimulated TGF-beta1 production by normal skin (26% to 83%) and hyperplastic scar fibroblasts (63% to 85%), with statistical significance (P < 0.05) at various time points. Heparin (300 microg/mL and 600 microg/mL) also stimulated TGF-beta1 mRNA expression by normal skin (12% to 53%) and hyperplastic scar fibroblasts (33% to 52%), with statistical significance (P < 0.05) at various time points. These effects of heparin on normal skin and hyperplastic scar fibroblasts may have implications for hyperplastic scar formation and wound healing in vivo.     

The responses of extrinsic fibroblasts infiltrating the devitalised patellar tendon to IL-1beta are different from those of normal tendon fibroblasts

In order to clarify the role of cytokines in the remodelling of the grafted tendon for ligament reconstruction we compared the responses to interleukin (IL)-1beta, platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta1 of extrinsic fibroblasts infiltrating the frozen-thawed patellar tendon in rats with that of the normal tendon fibroblasts, in regard to the gene expression of matrix metalloproteinase (MMP)-13, using Northern blot analysis. We also examined, immunohistologically, the local expression of IL-1beta, PDGF-BB, and TGF-beta1 in fibroblasts infiltrating the frozen-thawed patellar tendon. Northern blot analysis showed that fibroblasts derived from the patellar tendon six weeks after the freeze-thaw procedure in situ showed less response to IL-1beta than normal tendon fibroblasts with respect to MMP-13 mRNA gene expression. The immunohistological findings revealed that IL-1beta was over-expressed in extrinsic fibroblasts which infiltrated the patellar tendon two and six weeks after the freeze-thaw procedure in situ, but neither PDGF-BB nor TGF-beta1 was over-expressed in these extrinsic fibroblasts. Our findings indicated that IL-1beta had a close relationship to matrix remodelling of the grafted tendon for ligament reconstruction, in addition to the commencement of inflammation during the tissue-healing process.     

截短型转化生长因子-βⅡ型受体在皮肤成纤维细胞的过度表达

目的　研究截短型转化生长因子 -β(TGF-β) 型受体在皮肤成纤维细胞的过度表达对 TGF-β1 自分泌的抑制作用。　方法　组织块法体外培养正常皮肤成纤维细胞 ,加入重组人 (rh) TGF- β1 (5ng/ ml)或含截短型 TGF- β 型受体基因的重组腺病毒 (50 pfu/ cell) ,并用 Northern印迹观察其对 TGF-β1 的基因调控。　结果　 rh TGF-β1 能上调TGF-β1 和 I型前胶原的基因表达。截短型 TGF-β 型受体的过度表达能减少细胞 TGF-β1 的基因表达。　结论　截短型TGF- β 型受体的过度表达可以通过阻断 TGF- β1 的信号转导来抑制皮肤成纤维细胞 TGF- β1 的自分泌。为研究瘢痕的基因治疗提供了新的策略     

肾上腺素对增生性瘢痕成纤维细胞TGF-β1、bFGF及Ⅰ型前胶原表达的影响

目的实验研究肾上腺素对体外培养人皮肤成纤维细胞中Ⅰ型前胶原、TGF—β1及bFGF表达的影响。方法实验采用RT-PCR检测。肾上腺素作用24h后bFGF、TGF-β1、人Ⅰ型前胶原的mRNA表达差异，并以ELISA检测bFGF、TGF-β1在肾上腺素作用24h后的蛋白表达差异。结果与对照组比较，肾上腺素上调bFGF mRNA及蛋白的表达和下调TGFβ1 mRNA及蛋白的表达；但在增生性瘢痕成纤维细胞中，0．20μmol／L的。肾卜腺素可显著下调人Ⅰ型前胶原mRNA水平的表达，而在正常皮肤成纤维细胞中，0．05、0．1和0．2μmol／L的。肾上腺索均可下调人Ⅰ型前胶原mRNA水平的表达，且其差异有统计学意义。结论在增生性瘢痕成纤维细胞生长融合后早期（24h），肾上腺素可促进成纤维细胞bFGF的mRNA和蛋白的合成，降低TGF—β1的mRNA和蛋白的生成，降低Ⅰ型前胶原mRNA的表达。     

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.     

Expression of TGF-beta and fibrogenic genes in transplant recipients with tacrolimus and cyclosporine nephrotoxicity

BACKGROUND: Long-term treatment with cyclosporine (CsA) or tacrolimus (Tac) results in chronic nephrotoxicity. Transforming growth factor-beta (TGF-beta) and other pro-fibrogenic molecules have been known to contribute to this side effect. A comparison of intrarenal expression of TGF-beta and other fibrogenic genes in biopsies from patients with either CsA or Tac nephrotoxicity have not been documented. This study compared the expression of TGF-beta, collagen, fibronectin, metalloproteinases (MMP-2, -9), tissue inhibitors of metalloproteinases (TIMP-2) and osteopontin in renal biopsies obtained from renal transplant recipients treated with either CsA or Tac as primary immunosuppressive agents. METHODS: Using RT-PCR, intrarenal expression of TGF-beta, collagen, fibronectin, MMP-2, MMP-9 and TIMP-2 were studied in renal biopsies from patients with histological diagnosis of CsA or Tac nephrotoxicity and acute rejection. TGF-beta protein expression was studied by staining section of biopsies with anti-TGF-beta antibody. RESULTS: Intrarenal expression of TGF-beta, collagen, fibronectin, MMP-2, TIMP-2, and osteopontin were significantly increased in patients treated with Tac nephrotoxicity compared with CsA nephrotoxicity. The intrarenal mRNA expression of these genes was higher in patients diagnosed with Tac/CsA nephrotoxicity compared to acute rejection. CONCLUSIONS: This study compares the intrarenal expression of TGF-beta and profibrogenic genes in renal transplant recipients treated with Tac and CsA. The results show that patients diagnosed with Tac nephrotoxicity exhibit increased expression of profibrogenic genes compared to CsA nephrotoxicity.     

Paclitaxel interrupts TGF-beta1 signaling between gallbladder epithelial cells and myofibroblasts

BACKGROUND: The cellular and molecular mechanisms of fibrogenesis in the extrahepatic biliary epithelium are not known. Transforming growth factor-beta1 (TGF-beta1) is a cytokine implicated in signaling pathways that mediate collagen formation. An observation that paclitaxel (PT), applied topically into the rat common bile duct, inhibited stricture formation led us to hypothesize that PT's effects might be due to interruption of TGF-beta1 signaling between biliary epithelial cells and subepithelial myofibroblasts. MATERIALS AND METHODS: We tested this hypothesis using an in vitro cell-culture model in which murine gallbladder epithelial cells (GBEC) are cultured separately or cocultured with human gallbladder myofibroblasts (GBMF). RESULTS: Exposure to Escherichia coli lipopolysaccharide (LPS) enhanced TGF-beta1 mRNA expression and stimulated TGF-beta1 protein secretion into both apical and basolateral compartments in GBEC. This effect was more prominent with basolateral secretion and was also more pronounced in the coculture system. In GBMF, collagen I mRNA expression and protein secretion were stimulated by treatment with LPS or TGF-beta1. GBMF also expressed TGF-beta1 mRNA, whose levels were enhanced by exposure to either LPS or exogenous TGF-beta1. PT inhibited LPS-induced TGF-beta1 mRNA expression and protein secretion in GBEC in both culture systems. Tumor necrosis factor-alpha mRNA expression and protein secretion were not affected by PT in GBEC, demonstrating that the effects were specific for TGF-beta1. PT also inhibited LPS- and TGF-beta1-induced collagen I mRNA expression and protein secretion in GBMF. CONCLUSIONS: These findings support a model in which GBEC communicate with subepithelial GBMF via TGF-beta1, leading to collagen deposition and fibrosis, and in which GBMF possess autocrine mechanisms involving TGF-beta1 that could regulate collagen production. PT inhibits these fibrogenic pathways.     

Relaxin down-regulates renal fibroblast function and promotes matrix remodelling in vitro.

BACKGROUND: Renal fibroblasts are important effector cells in tubulointerstitial fibrosis, with experimental antifibrotic strategies focusing on the functional down-regulation of these cells. Several experimental models of fibrosis have provided evidence for the effectiveness of the polypeptide hormone relaxin as a potential antifibrotic agent. This study was conducted to further elucidate the antifibrotic mechanisms of relaxin on renal fibroblasts in vitro. METHODS: Rat cortical fibroblasts were obtained from outgrowth culture of renal tissue isolated from kidneys 3 days post-unilateral ureteric obstruction and constituted 100% of cells studied. A relaxin radio-receptor assay was used to establish binding of relaxin to renal fibroblasts in vitro. Functional studies then examined the effects of H2 relaxin (0, 1, 10 and 100 ng/ml) on fibroblast kinetics, expression of alpha-smooth muscle actin (alpha-SMA), total collagen synthesis, collagenase production and collagen-I lattice contraction. CTGF mRNA expression was also measured by northern analysis. RESULTS: H2 relaxin bound with high affinity to rat renal fibroblasts, but receptor numbers were low. Consistent with its previously reported bimodal effect, transforming growth factor (TGF-beta 1) reduced fibroblast proliferation, an effect abrogated by H2 relaxin. Fibroblasts exposed to H2 relaxin (100 ng/ml) for 24 h demonstrated decreased immunostaining for alpha-SMA and reduced alpha-SMA protein expression compared with controls. There was a trend for a relaxin-mediated reduction in total collagen synthesis and alpha 1(I) mRNA expression with large dose-related increases in collagenase protein expression being observed. TGF-beta 1-stimulated collagen-I lattice contraction was significantly inhibited following co-incubation with 100 ng/ml relaxin. Incremental doses of H2 relaxin had no significant effect on CTGF mRNA expression. CONCLUSIONS: The findings of this study suggest that the antifibrotic effects of relaxin involve down-regulation of fibroblast activity, increase in collagenase synthesis and restructuring of collagen-I lattices, which are consistent with its known physiological role of matrix remodelling. Although there appears to be an interaction between TGF-beta 1 and H2 relaxin, this does not appear to involve a reduction in CTGF mRNA expression.     

Glucan stimulates human dermal fibroblast collagen biosynthesis through a nuclear factor-1 dependent mechanism

Glucan, an immunomodulator, has been reported to increase collagen deposition and tensile strength in experimental models of wound repair. Previous data suggest that glucan modulates wound healing via an indirect mechanism in which macrophages are stimulated to release growth factors and cytokines. However, recent data have shown the presence of glucan receptors on normal human dermal fibroblasts, suggesting that glucans may be able to directly stimulate fibroblast collagen biosynthesis. To test this hypothesis, we examined the effect of glucan on collagen biosynthesis in normal human dermal fibroblasts. We assessed nuclear factor-1 (NF-1) activation, procollagen mRNA expression, collagen biosynthesis, and whether there was a causal link between glucan treatment, NF-1 activation, and collagen expression. Glucan (1 microg/ml) increased NF-1 binding activity by 46% (8 hours), 64% (24 hours), 215% (36 hours), and 119% (48 hours) in cultured normal human dermal fibroblasts. Alpha 1(I) and alpha1 (III) procollagen mRNA were increased in glucan-treated normal human dermal fibroblasts when compared with the untreated fibroblasts. Collagen synthesis was increased at 24 hours and 48 hours following glucan treatment of normal human dermal fibroblasts. Down-regulation of NF-1 by pentifylline inhibited glucan-induced procollagen mRNA expression. These data indicate that glucan can directly stimulate human fibroblast collagen biosynthesis through an NF-1-dependent mechanism.     

beta2-microglobulin induces MMP-1 but not TIMP-1 expression in human synovial fibroblasts

BACKGROUND: beta2-Microglobulin (beta2m) amyloidosis is a destructive articular disease that causes significant morbidity in patients undergoing hemodialysis. The amyloid deposits contain beta2m, some of which is altered with advanced glycation end products (AGE-beta2m). The deposits are located principally in joint structures, with adjacent degradation of cartilage and bone. We hypothesized that one of the mechanisms by which beta2m induces joint destruction is to induce the release of matrix metalloproteinase-1 (MMP-1), but not tissue inhibitor of metalloproteinase-1 (TIMP-1), from synovial fibroblasts. METHODS: To test this hypothesis and determine the role of AGE-beta2m, we incubated human osteoarthritic synovial fibroblasts in the presence and absence of beta2m and AGE-beta2m and measured the release of interstitial collagenase (MMP-1) and/or TIMP-1 by enzyme-linked immunosorbent assay and Northern blot analysis. RESULTS: beta2m and AGE-beta2m at 10 and 25 microg/mL induced the release of MMP-1 from human osteoarthritic synovial fibroblasts at 24 hours. In contrast, there was no increased release of TIMP-1, leading to an increase in the MMP-1/TIMP-1 ratio indicative of uncontrolled collagenolysis. A similar dose response was observed at 48 hours, except that AGE-beta2m had no effect over control cultures. MMP-1 mRNA expression by Northern blot analysis paralleled these findings. The source of the fibroblasts did not alter the results. Finally, we demonstrated that doxycycline, a treatment for arthritis, can inhibit the release of MMP-1 from synovial fibroblasts incubated with beta2m. CONCLUSION: beta2m, at physiologically relevant concentrations, induces the release of MMP-1 without concomitant release of TIMP-1 from human synovial fibroblasts, leading to uncontrolled collagenolysis. The alteration of beta2m with AGE did not alter this effect at 24 hours, but blocked the effect at 48 hours. These findings may account for the tissue destruction seen in beta2m amyloidosis.