Inhibition of prostaglandin synthesis up-regulates cyclooxygenase-2 induced by lipopolysaccharide and peroxisomal proliferators

Primary cultures of fetal hepatocytes expressed cyclooxygenase-2 (COX-2) upon stimulation with bacterial lipopolysaccharide (LPS) or peroxisomal proliferators. This enzyme was active and a good correlation between the mRNA levels, the amount of protein, and the synthesis of prostaglandin E2 was observed. However, when cells were incubated in the presence of indomethacin or the COX-2-specific inhibitor NS398, the amount of COX-2 protein increased 5-fold after activation with LPS and 2-fold after treatment with clofibrate. This up-regulation of COX-2 was not observed at the mRNA level. The mechanism of protein accumulation might involve either a direct stabilization of the enzyme by the inhibitors or the absence of prostaglandins involved in the regulation of its turnover. Among the prostaglandins assayed, only 15-deoxy-Prostaglandin J2 exerted a statistically significant decrease in the COX-2 levels in cells stimulated with LPS or LPS plus NS398. The accumulation of COX-2 in the presence of inhibitors was also observed in peritoneal macrophages treated under identical conditions. These results indicate that COX-2 protein accumulates after enzyme inhibition, and because removal of the inhibitors restored the enzyme activity, suppression of treatment with reversible COX-2 inhibitors may cause a transient overproduction of prostaglandins.     

Selective upregulation of platelet-derived growth factor alpha receptors by transforming growth factor beta in scleroderma fibroblasts

Transforming growth factor beta (TGF-beta), a multifunctional cytokine, is an indirect mitogen for human fibroblasts through platelet-derived growth factor (PDGF), particularly the A ligand-alpha receptor arm of that system. TGF-beta effects on PDGF alpha receptor expression were studied in vitro using ligand binding techniques in three human dermal fibroblast strains: newborn foreskin, adult skin, and scleroderma (systemic sclerosis, SSc). Each cell strain responded differently to TGF-beta. In newborn foreskin fibroblasts, PDGF alpha receptor number decreased in a dose-dependent manner after exposure to low concentrations of TGF-beta (0.1-1 ng/ml). Responses of normal skin fibroblasts were varied, and mean net receptor number was unchanged. Increases in PDGF alpha receptor number by TGF-beta occurred consistently with SSc fibroblasts and low concentrations of TGF-beta (0.1-1 ng/ml) were particularly stimulatory. Increased surface expression of alpha receptor subunit by TGF-beta in SSc fibroblasts correlated with increased new PDGF alpha receptor synthesis as demonstrated by radioimmunoprecipitation analysis of metabolically labeled cells and with increased steady-state levels of corresponding mRNAs. In normal adult skin fibroblasts, TGF-beta had no effect on either synthesis or mRNA expression of alpha receptor subunits. Proliferative responses to PDGF-AA after pretreatment with TGF-beta correlated positively with effects of TGF-beta on expression of alpha receptor subunit. Decreased mitogenic responses to PDGF-AA were observed in foreskin fibroblasts, small changes in responses in adult fibroblasts, and significant increases in SSc fibroblasts. Thus, costimulation with PDGF-AA and TGF-beta selectively enhanced proliferation of fibroblasts with the SSc phenotype. Immunohistochemical examination of SSc and control skin biopsies revealed the presence of PDGF-AA in SSc skin. Data obtained by ligand binding, immunoprecipitation, mRNA, and mitogenic techniques are consistent with the hypothesis that activation of the PDGF-AA ligand/alpha receptor pathway is a characteristic of the SSc fibroblast and may contribute to the expansion of fibroblasts in SSc.     

Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis

Pulmonary fibrosis is the consequence of a variety of diseases with no satisfying treatment option. Therapy-induced fibrosis also limits the efficacy of chemotherapy and radiotherapy in numerous cancers. Here, we studied the potential of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors (RTKIs) to attenuate radiation-induced pulmonary fibrosis. Thoraces of C57BL/6 mice were irradiated (20 Gy), and mice were treated with three distinct PDGF RTKIs (SU9518, SU11657, or Imatinib). Irradiation was found to induce severe lung fibrosis resulting in dramatically reduced mouse survival. Treatment with PDGF RTKIs markedly attenuated the development of pulmonary fibrosis in excellent correlation with clinical, histological, and computed tomography results. Importantly, RTKIs also prolonged the life span of irradiated mice. We found that radiation up-regulated expression of PDGF (A-D) isoforms leading to phosphorylation of PDGF receptor, which was strongly inhibited by RTKIs. Our findings suggest a pivotal role of PDGF signaling in the pathogenesis of pulmonary fibrosis and indicate that inhibition of fibrogenesis, rather than inflammation, is critical to antifibrotic treatment. This study points the way to a potential new approach for treating idiopathic or therapy-related forms of lung fibrosis.     

Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices.

Fibroplasia and angiogenesis are essential components of tissue repair when substantial tissue has been lost at a site of injury. Platelets and monocyte/macrophages accumulate at these sites and release a variety of growth factors that are thought to initiate and sustain the repair. Often the involved tissue contracts, a process that can markedly reduce the amount of fibroplasia and angiogenesis necessary for the reestablishment of organ integrity. Such tissue contraction occurs over hours or days, a much slower time course than the rapid, reversible contraction of muscle tissue. Fibroblasts, which are rich in f-actin bundles, appear to be responsible for wound contraction. However, the signals that stimulate contraction are not known. Using cultured fibroblasts, which are also rich in f-actin bundles, we demonstrate the platelet and monocyte isoforms of platelet-derived growth factor (PDGF; AB and BB) but not PDGF-AA, can stimulate fibroblasts to contract collagen matrix in a time course similar to that of wound contraction. In addition, PDGF appears to be the predominant fibroblast/collagen gel contraction activity released from platelets. Vasoactive agonists known to stimulate smooth and striated muscle contraction do not stimulate fibroblast-driven collagen gel contraction.     

Human platelet-derived growth factor stimulates prostaglandin synthesis by activation and by rapid de novo synthesis of cyclooxygenase.

Human platelet-derived growth factor (PDGF) stimulated prostaglandin (PG) E2 synthesis in the cell cycle of Swiss 3T3 cells at two distinct time intervals, with a first plateau within 10 min and a second plateau within 2-4 h after addition of PDGF. At 4 h, the concentration of PGE2 in PDGF-stimulated cultures exceeded the quiescent control cells by a factor of 10-15. Quiescent cells incubated with up to 16 microM exogenous arachidonic acid (AA) synthesized only small amounts of PGE2. In contrast, 4 h after addition of PDGF, the concentration of PGE2 synthesized from exogenous AA exceeded that in quiescent cultures by a factor of 28. The effect of PDGF stimulation on PG synthesis from exogenous AA could not be explained by growth factor-mediated increase in the cellular free AA pool as shown in experiments using [14C]AA. PDGF also stimulated synthesis of PGI2 (prostacyclin), thromboxane, and PGF2 alpha from exogenous AA. While inhibition of protein synthesis by 10 micrograms/ml cycloheximide had no effect on the early increase in PGE2 synthesis, the second increase was completely prevented. Additionally, cycloheximide treatment at 6 h after PDGF stimulation resulted in rapid decline of PGE2 synthesis from exogenous AA. Quiescent cultures pretreated with 100 microM aspirin and stimulated by PDGF thereafter recovered from cyclooxygenase inhibition within 180 min. Our results suggest that phospholipase activation and resultant AA release is not sufficient to induce the burst of PG synthesis observed in PDGF-stimulated cells. Instead, PDGF stimulates PG synthesis by direct effects on the PG-synthesizing enzyme system, one involving a protein synthesis-independent mechanism and another that requires rapid translation of cyclooxygenase.     

Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells.

In vitro and in vivo studies suggest that liver fat-storing cells (FSC) may play an important role in the development of liver fibrosis. We explored the effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and TGF-beta, and basic fibroblast growth factor (bFGF) on DNA synthesis and growth of rat liver FSC. PDGF, EGF, TGF-alpha, and bFGF induced a dose-dependent increase in DNA synthesis with a peak effect at 24 h. PDGF produced the most striking effect with a maximum 18-fold increase over control. EGF, TGF-alpha, and bFGF elicited a maximum three- to fourfold increase in DNA synthesis. Analysis of growth curves revealed a similar pattern of potency of the growth factors. TGF-beta did not affect DNA synthesis of FSC; however, TGF-beta markedly potentiated the stimulatory effects of both EGF and PDGF. FSC showed high specific binding of 125I-PDGF and Scatchard analysis revealed high affinity receptors with an apparent Kd of 2.3 x 10(-10) M. Our data suggest that PDGF is a key mitogen for FSC and that the coordinate release of other growth factors together with PDGF by inflammatory cells represents a potent potential stimulus for FSC proliferation in conditions of chronic self-perpetuating liver inflammation.     

Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2.

A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.     

Correlation between DNA synthesis and glucosamine incorporation in human diploid fibroblasts stimulated by fibroblast growth factor and fetal calf serum

Summary A 20 and 80% decrease of³H-glucosamine incorporation was observed in resting human fibroblasts stimulated with fibroblast growth factor (FGF) and fetal calf serum (FCS), respectively. The decrease occurred as early as 15 min after stimulation. Inversely, DNA synthesis was increased 1 and 5 fold and cell number was increased 2 and 2.5 fold 72h later.³H-glucosamine was incorporated in cell membrane glycopeptides (GP). Extracted from control or from cells exposed to FGF or FCS, GP have the same molecular weight and identical chemical constitution. The decrease of³H-glucosamine incorporation caused by FGF and FCS appears to be related to the competitive binding between glucosamine-FGF and glucosamine-FCS components to GP, which probably constitute cell membrane receptors. A decline of³H-glucosamine incorporation caused by FCS seems to result from the binding to cell-surface receptors (i.e., GP) of a large number of FCS components. The results suggest a correlation between cell growth rate and the density of growth-promoting molecules received by cells, since a large decrease of³H-glucosamine incorporation corresponds to a high increase of DNA synthesis in cells exposed to FCS, which is a mixture of components with multiple effects.     

Inhibition of mesangial cell proliferation and matrix expansion in glomerulonephritis in the rat by antibody to platelet-derived growth factor

Platelet-derived growth factor (PDGF), a potent mitogen for mesenchymal cells in culture, is expressed in vivo in a variety of inflammatory conditions associated with cell proliferation, including atherosclerosis, wound repair, pulmonary fibrosis, and glomerulonephritis. However, it is not known if PDGF mediates the fibroproliferative responses that characterize these inflammatory disorders. We administered neutralizing anti-PDGF IgG or control IgG to rats with mesangial proliferative nephritis. Inhibition of PDGF resulted in a significant reduction in mesangial cell proliferation, and largely prevented the increased deposition of extracellular matrix associated with the disease. This suggests that PDGF may have a central role in proliferative glomerular disease.     

Glomerulosclerosis induced by in vivo transfection of transforming growth factor-beta or platelet-derived growth factor gene into the rat kidney.

Glomerulosclerosis, a final common lesion of various glomerular diseases, is characterized by mesangial cell proliferation and extracellular matrix (ECM) expansion. TGF-beta and PDGF are known to play a critical role in the regulation of ECM metabolism and mesenchymal cell proliferation, respectively. However, there is little evidence to demonstrate the direct role of each of these growth factors in the pathogenesis of glomerulosclerosis. Using an in vivo transfection technique, we could realize the selective overexpression of single growth factor in the kidney. The introduction of either TGF-beta or PDGF-B gene alone into the kidney induced glomerulosclerosis, although the patterns of action of these growth factors were different; TGF-beta affected ECM accumulation rather than cell proliferation and PDGF affected the latter rather than the former.     

Inhibition of cyclooxygenase-2 by celecoxib reverses tumor-induced wasting

There have been a number of reports suggesting inhibition of prostaglandin production may impact tumor-mediated wasting and levels of associated humoral factors such as hypercalcemia. These reductions were achieved using traditional nonsteroidal anti-inflammatory drugs (NSAIDs), which are often contraindicated in cancer patients. This is especially true during chemotherapeutic regimens due to concerns of bleeding from gastrointestinal and hematopoietic toxicities associated with inhibition of the housekeeping cyclooxygenase enzyme COX-1. Here, we report that celecoxib, one of the new class of selective COX-2 inhibitors, has the potential to reverse tumor-mediated wasting and associated humoral factors such as interleukin (IL)-6 and hypercalcemia in preclinical models of cachexia. Tumor bearing mice in late stage cachexia regained weight within days of the start of celecoxib treatment. Two models were tested. The first was the Colon 26 (Col26) syngeneic murine model that induces high levels of circulating IL-6 and hypercalcemia. The second was the human head and neck 1483 HNSCC xenograft model, which is less inflammatory and produces less prostaglandin than Col26. Despite the observation that no significant impact on tumor growth was observed between vehicle and celecoxib-treated animals over the course of the studies, celecoxib rapidly reversed weight loss in both cachectic models. With the added safety of celecoxib over traditional NSAIDs, these results suggest a possible therapeutic use for celecoxib for treating tumor-mediated wasting.     

Inhibition of Na+ influx and DNA synthesis in human fibroblasts and neuroblastoma-glioma hybrid cells by amiloride analogs

Identification of a Na+ influx inhibitor that is significantly more potent than amiloride and devoid of the nonspecific effects of amiloride would be of great value in determining the validity of the hypothesis that mitogen-stimulated Na+ influx acts as a signal for induction of cell proliferation. In this study, we evaluated a number of amiloride analogs for potency of Na+ influx inhibition in human fibroblasts (HSWP). One analog, benzamil, was found to exhibit a 60-fold enhanced potency relative to amiloride. We also assessed the relative efficacies with which amiloride and benzamil inhibit Na+ influx and DNA synthesis in HSWP cells and neuroblastoma-glioma hybrid cells (NG108-15). Concentrations of benzamil required for 50% inhibition (ID50) of Na+ influx and DNA synthesis of HSWP cells are in excellent agreement (15 and 18 microM, respectively), an observation which, on the surface, is supportive of the hypothesis in question. Benzamil also inhibits Na+ influx of NG108-15 cells with an ID50 comparable to that for HSWP cells (18 microM) and suppresses DNA synthesis with a slightly higher ID50 (38 microM). Although the benzamil concentrations needed to inhibit cell growth and Na+ influx are in reasonable agreement, caution should be exercised in interpreting the effects of benzamil on cell growth with respect to the role of Na+ influx as we also observed that an analog of benzamil with a reduced ability to inhibit Na+ influx gave inhibition of DNA synthesis at concentrations which do not inhibit Na+ influx.     

血小板源生长因子受体在瘢痕疙瘩形成中的作用

目的:观察血小板源生长因子受体α和β在瘢痕疙瘩中的表达,并与正常皮肤比较。方法:实验于2005-04/10在解放军第二军医大学长海医院中心实验室进行。切取12例瘢痕疙瘩和6例正常皮肤标本,先经原代培养为成纤维细胞,取3～6代细胞分别应用免疫细胞化学和实时定量聚合酶链反应技术,检测瘢痕疙瘩成纤维细胞和正常皮肤成纤维细胞中血小板源生长因子受体α和血小板源生长因子受体β的蛋白和mRNA表达。结果:①免疫细胞化学结果:与正常皮肤相比,瘢痕疙瘩中的血小板源生长因子受体α和血小板源生长因子受体β的染色都增强,但血小板源生长因子受体α的染色增强尤其明显;图像分析定量统计显示瘢痕疙瘩中血小板源生长因子受体α的染色阳性指数显著高于正常皮肤(2.76±0.52,0.74±0.17,P<0.01),而血小板源生长因子受体β的染色阳性指数与正常皮肤比较差异不显著(0.95±0.202,0.76±0.17,P=0.07)。②实时定量聚合酶链反应结果:瘢痕疙瘩成纤维细胞中血小板源生长因子受体α的每百万看家基因含量显著高于正常皮肤(21.73±6.51,14.41±3.37,P=0.02),而血小板源生长因子受体β的每百万看家基因含量与正常皮肤比较差异不显著(P=0.06)。结论:在瘢痕疙瘩形成过程中,血小板源生长因子受体α的蛋白和mRNA表达都显著升高,可能是其病因机制之一。     

血小板源生长因子受体在瘢痕疙瘩形成中的作用

目的：观察血小板源生长因子受体α和β在瘢痕疙瘩中的表达，并与正常皮肤比较。 方法：实验于2005-04／10在解放军第二军医大学长海医院中心实验室进行。切取12例瘢痕疙瘩和6例正常皮肤标本，先经原代培养为成纤维细胞，取3～6代细胞分别应用免疫细胞化学和实时定量聚合酶链反应技术，检测瘢痕疙瘩成纤维细胞和正常皮肤成纤维细胞中血小板源生长因子受体α和血小板源生长因子受体β的蛋白和mRNA表达。 结果：①免疫细胞化学结果：与正常皮肤相比，瘢痕疙瘩中的血小板源生长因子受体α和血小板源生长因子受体β的染色都增强，但血小板源生长因子受体α的染色增强尤其明显；图像分析定量统计显示瘢痕疙瘩中血小板源生长因子受体α的染色阳性指数显著高于正常皮肤（2.76&;#177;0．52，0．74&;#177;0．17，P〈0．01），而血小板源生长因子受体β的染色阳性指数与正常皮肤比较差异不显著（0．95&;#177;0．202，0．76&;#177;0．17，P=0．07）。②实时定量聚合酶链反应结果：瘢痕疙瘩成纤维细胞中血小板源生长因子受体α的每百万看家基因含量显著高于正常皮肤（21．73&;#177;6．51，14．41&;#177;3．37，P=0．02），而血小板源生长因子受体β的每百万看家基因含量与正常皮肤比较差异不显著（P=0．06）。 结论：在瘢痕疙瘩形成过程中，血小板源生长因子受体α的蛋白和mRNA表达都显著升高，可能是其病因机制之一。     

Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha

The immunoregulatory effects of transforming growth factor beta (TGF-beta) and recombinant murine tumor necrosis factor alpha (rMuTNF-alpha) on CTL generation and activity were examined. The results demonstrate that TGF-beta, in a dose-dependent manner, inhibited CTL generation but not CTL activity. The inhibitory effects were detected only when TGF-beta was added within the first 48 h of the MLC. Little activity was seen when it was added thereafter, including the addition of TGF-beta to the cytotoxicity assay. The production of TNF-alpha, which occurs during early phases of the MLC and which is inhibited in the presence of TGF-beta, appears to have an important regulatory role, as altering the levels of TNF-alpha in an MLC can significantly influence CTL development. The inhibitory effects of TGF-beta on the MLC can be significantly reversed by the addition of rMuTNF-alpha to the cultures. These results demonstrate that TGF-beta can inhibit MLC and subsequent CTL generation at early stages of the reaction, and such inhibition may involve the suppression of TNF-alpha production.     

Tissue localization of beta receptors for platelet-derived growth factor and platelet-derived growth factor B chain during wound repair in humans.

The expression and localization of PDGF beta receptors and PDGF-AB/BB in human healing wounds was evaluated by immunohistochemical techniques and in situ hybridization. Expression of PDGF beta receptor protein and PDGF-AB/BB were analyzed in wound margin biopsies using the PDGFR-B2 and PDGF 007 antibodies. PDGF beta receptor expression was minor in normal skin. An increased expression of PDGF beta receptor protein was prominent in vessels in the proliferating tissue zone in wounds as early as 1 d after surgery and was apparent < or = 4 wk after surgery. There was also a concordant increase in PDGF beta receptor mRNA detected by in situ hybridization. PDGF-AB/BB was present in healing wounds as well as in normal skin. In normal skin, expression of PDGF-AB/BB was confined to peripheral nerve fibers and to solitary cells of the epidermis and of the superficial dermis. In wounds, infiltrating mononuclear cells also stained for PDGF-AB/BB. To identify cell types expressing PDGF AB/BB and PDGF beta receptors, respectively, we performed double immunofluorescence stainings. PDGF beta receptors were expressed by vascular smooth muscle cells and cells in capillary walls; the receptor protein could not be detected in neurofilament containing structures, T lymphocytes, or CD68 expressing macrophages. PDGF-AB/BB colocalized with neurofilaments, it was present in Langerhans cells of the epidermis and in HLA-DR positive cells located in the epidermal/dermal junction area. Of the macrophages infiltrating the wound, 43 +/- 18% stained positively for PDGF AB/BB. Since PDGF-AB/BB and PDGF beta receptors are expressed in the healing wound, two essential prerequisites for a role of PDGF in wound healing are fulfilled.     

表皮生长因子诱导糖尿病难愈性创面成纤维细胞增殖的对照观察

目的：探讨表皮生长因子对糖尿病难愈性创面成纤维细胞增殖的影响。方法：选择2004-01／2005-12广西医科大学第一附属医院烧伤整形康复中心住院糖尿病足患者5例（糖尿病组）。平均年龄72岁；病程14年，经严格内科及换药治疗创面超过8周仍不愈合。另选同期健康志愿者7人（对照组），平均年龄74岁。实验在广西医科大学实验中心完成。将糖尿病难愈性创面及对正常对照组成纤维细胞于Dulbecco＇s改良的Eagle＇s培养基中培养，待细胞培养成对数生长期，分别加入浓度为0．1，0．25，0．5，0．75，1，5，10μg／L的表皮生长因子于Dulbecco＇s改良的Eagle＇s培养基中培养24h，与未加入表皮生长因子的成纤维细胞（空白对照组）进行比较，分别改用加有不同浓度表皮生长因子的培养液继续培养，以四甲基偶氮唑盐法测定成纤维细胞增殖活性，各孔细胞在490nm处的光吸收值（A值）越大表示细胞增殖越强。结果：在不同浓度的表皮生长因子刺激下，两组成纤维细胞的增殖活动均较未加入表皮生长因子的空白对照组有明显提高，差异具有显著性意义（P〈0．05）；糖尿病组最大吸光值为0．35&;#177;0．13，表皮生长因子刺激的最适浓度为0．5μg／L，对照组最大吸光值为0．58&;#177;0．07，表皮生长因子刺激的最适浓度为0．25μg／L。两组表皮生长因子刺激的最适浓度及成纤维细胞最大增殖程度差异有显著性意义（P=0．026）。结论：表皮生长因子能促进糖尿病难愈性创面成纤维细胞的增殖．但是糖尿病难愈性创面成纤维细胞对于表皮生长因子的增殖应答被削弱。     

表皮生长因子诱导糖尿病难愈性创面成纤维细胞增殖的对照观察

目的:探讨表皮生长因子对糖尿病难愈性创面成纤维细胞增殖的影响。方法:选择2004-01/2005-12广西医科大学第一附属医院烧伤整形康复中心住院糖尿病足患者5例(糖尿病组)。平均年龄72岁;病程14年,经严格内科及换药治疗创面超过8周仍不愈合。另选同期健康志愿者7人(对照组),平均年龄74岁。实验在广西医科大学实验中心完成。将糖尿病难愈性创面及对正常对照组成纤维细胞于Dulbecco’s改良的Eagle’s培养基中培养,待细胞培养成对数生长期,分别加入浓度为0.1,0.25,0.5,0.75,1,5,10μg/L的表皮生长因子于Dulbecco’s改良的Eagle’s培养基中培养24h,与未加入表皮生长因子的成纤维细胞(空白对照组)进行比较,分别改用加有不同浓度表皮生长因子的培养液继续培养,以四甲基偶氮唑盐法测定成纤维细胞增殖活性,各孔细胞在490nm处的光吸收值(A值)越大表示细胞增殖越强。结果:在不同浓度的表皮生长因子刺激下,两组成纤维细胞的增殖活动均较未加入表皮生长因子的空白对照组有明显提高,差异具有显著性意义(P<0.05);糖尿病组最大吸光值为0.35±0.13,表皮生长因子刺激的最适浓度为0.5μg/L,对照组最大吸光值为0.58±0.07,表皮生长因子刺激的最适浓度为0.25μg/L。两组表皮生长因子刺激的最适浓度及成纤维细胞最大增殖程度差异有显著性意义(P=0.026)。结论:表皮生长因子能促进糖尿病难愈性创面成纤维细胞的增殖,但是糖尿病难愈性创面成纤维细胞对于表皮生长因子的增殖应答被削弱。     

Inhibition of the growth of Rickettsia prowazekii in cultured fibroblasts by lymphokines

The effect of lymphokine treatment of mouse and human fibroblast cell lines on the growth of Rickettsia prowazekii within the fibroblasts was studied. Treatment of mouse L929 cells with concanavalin A- or antigen- induced mouse lymphokines both before and after infection with R. prowazekii led to clearance of the rickettsiae from a substantial proportion of the cells and suppression of rickettsial growth in those cells which remained infected. Similar but less dramatic anti- rickettsial effects were observed in L929 cells treated with mouse lymphokines either only before or after infection with rickettsiae. Mouse lymphokine treatment of L929 cells had similar anti-rickettsial effects on the avirulent E strain and the virulent Breinl strain of R. prowazekii. Addition of cycloheximide or emetine to L929 cells at the same time as the lymphokines markedly suppressed the inhibition of rickettsial growth by the lymphokines. Mouse lymphokine treatment inhibited rickettsial survival and growth in mouse 3T3-A31 cells as well as in mouse L929 cells, but had no effect on rickettsial survival and growth in human foreskin fibroblasts. Conversely, concanavalin A- induced human lymphokines inhibited rickettsial survival and growth in human foreskin fibroblasts but had no effect on rickettsial survival and growth in mouse L929 cells. The rickettsia inhibitory activity in concanavalin A-induced mouse lymphokines was destroyed by heating the lymphokines at 80 degrees C for 10 min or by holding the lymphokines at pH 2 for 24 h but was retained after heating at 56 degrees C for 30 min.