细胞外信号调节激酶及应激活化蛋白激酶通路蛋白在瘢痕疙瘩成纤维细胞中的表达

目的通过比较瘢痕疙瘩及正常皮肤中细胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、应激活化蛋白激酶(c-Jun amino-terminal kinase,JNK)信号通路的表达,探讨其在瘢痕疙瘩形成中的作用。方法取四川大学华西医院烧伤整形科收治的26例患者皮肤组织,其中行瘢痕疙瘩切除患者(实验组)16例,瘢痕疙瘩形成时间8个月～10年;胸部6例,耳垂4例,会阴部2例,肩部3例,腹部1例;均经病理检查确诊为瘢痕疙瘩。10例整形手术患者自愿捐赠的正常皮肤作为对照组,腹部4例,大腿3例,肩部2例,背部1例。取标本采用Envision二步法行免疫组织化学染色,观察磷酸化及非磷酸化JNK和ERK表达情况,并采用Image Pro Plus 4.5图像分析系统测定积分吸光度(IA)值,观察阳性染色强度。结果免疫组织化学染色观察显示,对照组正常皮肤成纤维细胞中未见明显的磷酸化及非磷酸化ERK、JNK阳性表达;而实验组主要在成纤维细胞中表达,阳性颗粒主要位于细胞质和细胞核内。实验组磷酸化ERK及JNK的IA值明显高于对照组(P<0.05),非磷酸化ERK及JNK两组间差异无统计学意义(P>0.05)。结论磷酸化JNK、ERK信号通路蛋白在瘢痕疙瘩中异常高表达,提示该通路可能与瘢痕疙瘩形成密切相关。     

Ticlopidine inhibits activation of mitogen-activated protein kinase by platelet-derived growth factor in cultured rat renal mesangial cells

Background We previously found that ticlopidine inhibits the proliferation of cultured rat mesangial cells that is induced by fetal bovine serum. This study was designed to examine the effects of ticlopidine on platelet-derived growth factor (PDGF)-induced DNA synthesis and mitogen-activated protein (MAP) kinase activation in such mesangial cells to clarify the mechanism of the antiproliferative action. Methods Glomerular mesangial cells were isolated from rat kidneys, and cells were incubated with various combinations of ticlopidine, PDGF, epidermal growth factor, phorbol 12-acetate 13-myristate (PMA), cilostazol (a phosphodiesterase inhibitor), and H-89 (a cAMP-dependent protein kinase A [PKA] inhibitor). A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and cell count involving the trypan blue exclusion test were performed for determination of cell viability. DNA synthesis and MAP kinase activity were assessed by means of a tritiated thymidine ([³H]thymidine) incorporation assay and the Biotrak^TM MAP kinase assay system, respectively. Results Ticlopidine (1 μmol/L) significantly inhibited both PDGF-induced DNA synthesis and MAP kinase activation. Also, 1 μmol/L ticlopidine substantially blocked PMA-induced MAP kinase activation. Pretreatment with H-89 did not abolish the ability of ticlopidine to inhibit PDGF-induced MAP kinase activation, while H-89 pretreatment significantly reserved the inhibitory action of cilostazol on PDGF-induced MAP kinase activation. Conclusion These results suggest that ticlopidine might inhibit PDGF-induced DNA synthesis after MAP kinase activation by intercepting the signal transduction from c-Raf-1 to MAP kinase, independent of the cAMP-PKA pathway.     

反义结缔组织生长因子对人瘢痕疙瘩成纤维细胞的作用

目的：研究结缔组织生长因子（CTGF）反义寡核苷酸（ASODN）对人瘢痕疙瘩成纤维细胞CTGFmRNA的表达和细胞增殖及胶原合成的影响,探讨瘢痕疙瘩的基因治疗。方法：体外分离、培养人正常皮肤成纤维细胞（NSF）和瘢痕疙瘩成纤维细胞（KF）,以脂质体介导方法将CTGFASODN转染KF中,用逆转录-聚合酶链式反应（RT-PCR）方法检测细胞中CTGFmRNA的表达;采用MTT方法测细胞增殖3;H-脯氨酸掺入法检测细胞的胶原合成量。结果：CTGFmRNA在NSF中几乎无表达,在KF中表达增高,CTGFASODN可以抑制其增殖和胶原合成（P〈0.05）。结论：CTGFASODN能够抑制成纤维细胞CTGFmRNA表达和细胞增殖及胶原合成,表明阻断CTGF可能是延缓瘢痕纤维化的有效手段。     

MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖中的作用

目的 探讨MEK/ERK信号通路在吡咯喹啉醌促雪旺细胞增殖过程中的作用. 方法 体外培养雪旺细胞,S-100免疫荧光鉴定;Western blot检测MEK下游因子ERK1/2磷酸化激活形式(p-ERK1/2)的表达;MEK抑制剂(PD98059)阻断该通路后检测p-ERK1/2的表达;MTT法检测经PD98059阻断MEK通路后雪旺细胞的增殖情况. 结果 吡咯喹啉醌可激活雪旺细胞内MEK/ERK信号通路,在加入吡咯喹啉醌1 h后p-ERK1/2表达最高;吡咯喹啉醌在1～500 nmol/L范围内可使p-ERK1/2表达增加,1 000 nmol/L时与对照组比较差异无统计学意义,10 000 nmol/L时则表现为抑制作用(P＜0.05);经PD98059阻断MEK通路后p-ERK1/2的上调效应消失(P＜0.05).而且加入PD98059阻断MEK通路后吡咯喹啉醌对雪旺细胞的促增殖效果减弱. 结论 吡咯喹啉醌可激活雪旺细胞MEK/ERK信号通路,且该通路在吡咯喹啉醌促雪旺细胞增殖过程中发挥作用.     

CTGF反义寡核苷酸对人瘢痕疙瘩成纤维细胞的作用

目的探讨结缔组织生长因子在人瘢痕疙瘩发病机制中的作用。方法在脂质体介导下,将异硫氰酸荧光素标记的结缔组织生长因子反义寡核苷酸转染至体外培养的人瘢痕疙瘩成纤维细胞中,通过MTT比色实验检测细胞增殖活力,通过流式细胞仪检测细胞凋亡,通过H3脯氨酸掺入法检测细胞胶原合成。结果转染后和对照组相比,反义寡核苷酸能够抑制细胞增殖及胶原合成,并促进细胞凋亡(P<001)。结论在体外,结缔组织生长因子反义寡核苷酸具有抗人瘢痕疙瘩纤维化的作用;结缔组织生长因子在人瘢痕疙瘩纤维化进程中具有促进作用。     

脂质体介导血小板衍化生长因子-B基因转染成纤维细胞的表达

目的用脂质体介导血小板衍化生长因子(PDGF)-B真核表达质粒转染成纤维细胞，使PDGF-B基因在成纤维细胞中特异表达。方法构建PDGF—B真核表达质粒，用脂质体LipofectAMINE介导转染成纤维细胞，G418筛选阳性克隆。逆转录-聚合酶链反应(RT-PCR)和细胞爬片免疫组织化学染色检测PDGF-B基因在成纤维细胞中的表达。结果RT—PCR检测结果显示，转染组PDGF-BmRNA的表达量较未转染组明显增加。免疫组织化学染色结果显示转染组成纤维细胞胞浆中有大量棕黄色阳性颗粒。结论脂质体介导PDGF-B基因成功转染成纤维细胞并表达。为PDGF基因治疗奠定基础。     

Differential expression of platelet-derived growth factor receptors in porcine fibroblasts cultured from skin and granulation tissue

This study investigated the biological response of fibroblasts cultured from uninjured skin and granulation tissue from different stages of healing wounds to the three isoforms of platelet-derived growth factor. Fibroblasts were derived by explant culture from the skin or the granulation tissue that formed within open mesh nylon Schilling-Hunt chambers (postoperative days 10, 20, 30, and 50) which had been implanted subcutaneously in the backs of domestic pigs. Cells were cultured under identical conditions in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Mitogenic activity was measured with (3)H-thymidine incorporation into DNA. Fibroblasts from normal skin responded equally well to all of the platelet-derived growth factor isoforms in the mitogenic assays. All of the wound fibroblasts exhibited a decreased response to platelet-derived growth factor compared with those from skin. Granulation tissue fibroblasts responded to platelet-derived growth factor BB, less to platelet-derived growth factor AB, and poorly to platelet-derived growth factor AA. These results correlated with a significantly decreased growth rate of fibroblasts in culture from both 30- and 50-day postsurgical wound tissue compared with normal skin. Western blot studies of cell membrane extracts showed that wound fibroblasts contained less than 20% as many platelet-derived growth factor-alpha receptors as found in fibroblasts cultured from normal skin. No significant difference in the amount of platelet-derived growth factor-beta receptor was detected. The decreases in platelet-derived growth factor-alpha receptors are sufficient to account for the diminished response of the wound fibroblasts to all platelet-derived growth factor isoforms and the differential loss of responsiveness to platelet-derived growth factor AA. These results show that fibroblasts derived from granulation tissue of pig skin wounds exhibit a decreased growth response to platelet-derived growth factor and a decreased growth rate in culture media as compared with fibroblasts derived from uninjured skin. How these differences may relate to the physiologic characteristics of normal and healing-impaired wounds is considered.     

Adrenomedullin inhibits connective tissue growth factor expression, extracellular signal-regulated kinase activation and renal fibrosis

Systemic administration of the potent vasodilating peptide adrenomedullin reduces cardiac and renal fibrosis in hypertensive animals. Here, we investigated the effects of kidney-specific adrenomedullin gene delivery in normotensive rats after unilateral ureteral obstruction, an established model of renal tubulointerstitial fibrosis. Overexpression of exogenous adrenomedullin in the renal interstitium following ureteral obstruction significantly prevented fibrosis and proliferation of tubular and interstitial cells. In this model, there is upregulation of connective tissue growth factor (CTGF) mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation, and adrenomedullin overexpression suppressed both of these activities without altering the blood pressure. In NRK-49F renal fibroblasts, adrenomedullin reduced transforming growth factor-beta-induced CTGF and fibronectin mRNA upregulation through the cyclic AMP/protein kinase A signaling pathway, and suppressed ERK phosphorylation and cell proliferation. In the kidneys with an obstructed ureter, adrenomedullin receptor gene expression was upregulated along with cyclic AMP production in kidney slices. The latter effect was partially blocked by a neutralizing antibody to adrenomedullin, indicating that an endogenous peptide-receptor system was activated. Our results show that overexpression of exogenous adrenomedullin in the ureteral-obstructed kidney prevents tubulointerstitial fibrosis and cell proliferation through the cyclic AMP-mediated decrease of CTGF induction and ERK phosphorylation.     

Plasma levels of platelet-derived growth factor BB and transforming growth factor in patients with failed hip prostheses

Background The role of growth factors in prosthesis loosening is unclear. We evaluated the levels of plate-let-derived growth factor BB (PDGF-BB), transforming growth factors β1 (TGF-β1) and β2 (TGF-β2), both before and after activation, in patients with aseptic loosening of their hip prosthesis.

Patients and methods 26 patients with loosened hip implants were compared with 21 patients who had stable hip prostheses, and 28 patients undergoing primary hip replacement. The plasma levels of the growth factors were analyzed by enzyme immunoassay. TGF-β1 and TGF-β2 were determined both before and after activation.

Results Patients with aseptic loosening had significantly lower PDGF-BB levels than patients undergoing primary hip replacement, and significantly lower TGF-β2 levels than patients with a stable implant. Patients with stable prostheses had significantly higher TGF-β1 and TGF-β2 levels than patients undergoing primary hip replacement.

Interpretation It is possible that the prosthetic implant itself causes a local increase in PDGF-BB, TGF-β1 and TGF-β2, released by osteoblasts and other cells in the microenvironment. The plasma PDGF-BB measured does not correspond to local release, which is probably due to local consumption or degradation. The consumption of PDGF-BB is low in stable implants, and TGF-β1 and TGF-β2 levels increase during bone formation. In loosening, PDGF-BB consumption is higher and causes a significant reduction in plasma levels as compared to presurgery. The formation of poor-quality bone may be related to the scarce increase in TGF-β1 and TGF-β2. In conclusion, compared with patients with a stable implant, a reduction in bone-forming growth factors appears to occur in individuals with aseptic loosening.     

Nicotine reorganizes cytoskeleton of vascular endothelial cell through platelet-derived growth factor BB

BACKGROUND: Cigarette smoking has been directly linked to atherosclerosis formation and vascular graft failures but the role of nicotine in these processes is not yet completely understood. We investigated the release of platelet-derived growth factor BB (PDGF BB) by the bovine aortic endothelial cell (EC) after nicotine administration at concentrations similar to those found in plasma of active and passive smokers and the role of PDGF BB, autocrinally released, in EC cytoskeletal modification. METHODS: EC were stimulated in a serum-free medium for 72 h with (-)-nicotine (from 6 x 10(-4) to 6 x 10(-8) M). The release of PDGF BB was assessed by inhibition antibody-binding assay and confirmed by Western blotting. Mitogenic activity of nicotine on EC was also determined. The EC cytoskeleton was studied with specific antibodies anti-alpha-actin fibers and anti-vimentin and the modification induced by PDGF BB was assessed by blocking PDGF BB activity with specific antibodies. RESULTS: The greatest PDGF BB release was noted at a (-)-nicotine concentration of 6 x 10(-6) M (P < 0.001). The addition of antibody anti-PDGF BB to EC exposed to (-)-nicotine decreased tritiated thymidine uptake by 20% (P < 0.001). EC exposed to (-)-nicotine concentrations of 6 x 10(-6) and 6 x 10(-8) M had a significant alteration in the expression of alpha-actin fibers and vimentin as compared with control. Administration of the antibody anti-PDGF BB in the culture medium reversed cytoskeletal alteration. CONCLUSIONS: Nicotine enhanced the release of PDGF BB by EC which in turn caused an alteration in cytoskeletal organization.     

Angiotensin II further enhances type IV collagen production stimulated by platelet-derived growth factor and fibroblast growth factor-2 in cultured human mesangial cells

SUMMARY: Angiotensin II (Ang II) is considered to play a role in the development of glomerulosclerosis, which is characterized by excessive accumulation of mesangial matrix after mesangial cell proliferation. We have reported that platelet-derived growth factor (PDGF) and fibroblast growth factor-2 (FGF-2) stimulate type IV collagen production by cultured human mesangial cells (HMC). Although Ang II is well known to have a mitogenic effect in various kinds of cells, its role in the production of extracellular matrix is still undetermined. This study was designed to examine the effect of Ang II and its interaction with PDGF and FGF-2 in type IV collagen production by HMC. Cultured HMC were incubated with Ang II with or without PDGF or FGF-2 for 72 h and type IV collagen, fibronectin and laminin in the cell supernatants were measured. Ang II (10⁻⁶–10⁻⁸) itself did not change the production of type IV collagen, fibronectin, laminin and transforming growth factor-β. PDGF and FGF-2 enhanced type IV production, although they did not stimulate the production of fibronectin and laminin. Ang II further increased the stimulating effect of PDGF and FGF-2 in type IV collagen production in a dose-dependent manner. This effect of Ang II was completely blocked by Ang II type I receptor antagonist (Losartan). These data imply that Ang II is a potent stimulator of type IV collagen production by HMC in the presence of growth factors.     

Accelerated diabetic wound healing using cultured dermal fibroblasts retrovirally transduced with the platelet-derived growth factor B gene

The treatment of diabetic wounds is a considerable clinical challenge. In this study, mouse dermal fibroblasts retrovirally transduced with the human platelet-derived growth factor B (PDGF-B) gene were used to treat diabetic mouse wounds. The PDGF-B gene was obtained from human umbilical vein endothelial cells, cloned into retroviral vectors, and introduced into diabetic mouse C57B1/ks-db/db dermal fibroblasts. In vitro results demonstrated production of PDGF-B protein by these transduced cells at steady-state levels of 1000 ng PDGF-B/10(6) cells/24 hours, and expression of PDGF-B mRNA. These cells were seeded onto polyglycolic acid scaffold matrices and used to treat diabetic mouse 20-mm x 20-mm full-thickness excisional dorsal skin wounds. Measurement of the residual epithelial gap at 21 days showed significantly accelerated healing (P < 0.05) of wounds treated with PDGF-transduced cells (epithelial gap 10.46 +/- 1.20 mm) compared with untreated wounds (14.66 +/- 0.591 mm), wounds treated with polyglycolic acid alone (14.80 +/- 0.575 mm), or wounds treated with negative control LNCX-transduced cells (13.76 +/- 0.831 mm). Immunohistochemical staining showed intense staining for PDGF in wounds treated with PDGF-B-transduced cells. This study demonstrates the promising potential for gene therapy in diabetic wound healing.     

细胞外信号调节蛋白激酶在前列腺上皮内瘤组织中的表达及意义

目的 探讨细胞外信号调节蛋白激酶（ERK）在前列腺上皮内瘤（PIN）组织中表达的意义。方法 应用免疫组织化学SP法检测12例PIN、11例前列腺癌（PCa)及16例BPH组织标本中ERK的表达。结果 12例PIN标本均有表达,其中6例为高表达;11例PCa组织10例为阳性,但多为弱表达;16例BPH组织12例为阳性,表达也较弱。PIN组织中ERK表达与PCa组和BPH相比ERK在PIN组织中呈高表达,与早期前列腺癌的发生有密切关系。     

Evidence for mitogen-associated protein kinase activation and transduction of mitogenic signals by platelet-derived growth factor in human meningioma cells

OBJECT: Coexpression of platelet-derived growth factor (PDGF)-BB and activated PDGF-beta receptor in meningioma cells indicates that this cytokine may act as an autocrine or paracrine stimulant of meningioma growth. The intracellular events transducing signals from PDGF-beta receptor tyrosine kinases are unknown. In this study the authors evaluated whether or not mitogen-activated protein kinases (MAPKs) are expressed in meningiomas, regulate their growth, and transduce mitogenic signals of PDGF-BB. METHODS: Ten human meningioma tumors as well as cells cultured from two normal leptomeninges and 10 additional human meningiomas were evaluated using Western blot analysis to determine the presence of MAPK and phosphorylated (activated) MAPK. The effects of PD098059, a selective inhibitor of MAPK phosphorylation/activation, on proliferation of meningioma cells stimulated with 10% fetal bovine serum was also evaluated. Last, the authors evaluated whether PDGF-BB stimulation of meningioma cells was associated with activation of MAPK. Western blots of lysates from meningiomas and from cultured leptomeningeal and meningioma cells demonstrated MAPK and phosphorylated MAPK. Treatment with PD098059 produced a 52 to 84% (x = 69.8) loss in [3H]thymidine incorporation, which was associated with a partial or complete loss of phosphorylated MAPK after 3 days of treatment. The PDGF-BB produced a significant increase in [3H]thymidine incorporation and phosphorylation of MAPK at 1 and 3 days. Coadministration of PD098059 completely blocked PDGF-BB's stimulation of [3H]thymidine incorporation and cell proliferation concomitant with reduced MAPK phosphorylation. CONCLUSIONS: The findings indicate that MAPK is constitutively expressed in leptomeningeal and meningioma cells and transduces mitogenic signals of PDGF, contributing to the growth of human meningiomas.     

ERK/MAPK通路参与肝癌产生多药耐药的胞内信号传导

目的探讨微环境诱导肝癌产生多药耐药的胞内信号传导途径。方法分别使HepG2细胞在缺氧、低糖环境下生长或稳定整合HBX基因,运用Western蛋白印迹法检测这些细胞内ERK／MAPK的活性。用ERK／MAPK特异性阻断剂U0126处理这些细胞后,用Western蛋白印迹法检测缺氧诱导因子-1α（HIF—1α）和多药耐药相关蛋白的表达变化,逆转录聚合酶链反应和免疫细胞化学技术分别检测HIF-1α在mRNA水平表达量和部位的改变。结果不同环境下生长的HepG2细胞中,磷酸肜非磷酸化ERK／MAPK比例均有不同程度的增高。用U0126处理12h后,这些细胞中HIF-1α和多药耐药相关蛋白的表达下降,且HIF-1α表达由胞核向胞质转位,其mRNA水平无显著变化。结论ERK／MAPK信号通路是微环境诱导肝癌产生多药耐药的重要胞内信号传导途径。     

Effect of dexamethasone on cultured human tenocytes and its reversibility by platelet-derived growth factor

BACKGROUND: Many cases of tendon rupture after glucocorticoid injections have been reported in the literature. Despite previous studies on the histological and biomechanical changes in tendons after glucocorticoid injections, the role of glucocorticoid in causing tendon rupture still remains controversial. The objective of this study was to determine whether glucocorticoid has deleterious effects on the cellular metabolism and collagen production of cultured human tenocytes and the reversibility of these effects by platelet-derived growth factor-BB (PDGFBB). METHODS: Primary cultures of human tenocytes obtained from explants of healthy patellar tendon, harvested during anterior cruciate ligament reconstructions, were performed. The effects on cell viability, cell proliferation, and induction of apoptosis were measured by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, 5-bromo-deoxyuridine incorporation, and DNA fragmentation assay, respectively. The effect on collagen synthesis was measured by (3) H-proline incorporation assay. RESULTS: The number of viable cells was decreased, in a dose-dependent manner, by the administration of 10 (-9) to 10 (-4) -M dexamethasone. This dose range also suppressed cell proliferation. No apoptotic effect was detected. Treatment with 10 (-6) -M dexamethasone significantly reduced the amount of collagen synthesis. Co-incubation with 10 ng/mL of PDGFBB significantly reversed the effects caused by 10 (-6) -M dexamethasone. CONCLUSIONS: Dexamethasone significantly decreased cell viability, suppressed cell proliferation, and reduced collagen synthesis in cultured human tenocytes. The effects were reversed by the simultaneous administration of PDGFBB.