IL-13和乙醇促进人肺成纤维细胞胶原的表达

目的:研究乙醇和/或白细胞介素13(IL-13)对人肺成纤维细胞(HFL-1)Ⅰ、Ⅲ型胶原α1链基因(COL1A1、COL3A1)以及Ⅰ型胶原蛋白(CoⅠ)表达的影响,探讨肺纤维化的机制。方法:培养HFL-1,通过实时定量荧光RT-PCR检测乙醇和/或IL-13对HFL-1细胞IL-13受体(IL-13Rα1、IL-13Rα2、IL-4Rα)mRNA、COL1A1 mRNA和COL3A1 mRNA表达的影响,ELISA的方法检测乙醇和/或IL-13对HFL-1分泌CoⅠ的影响。结果:单独低浓度乙醇(25、50、100、200mmol/L)作用HFL-1后,与对照组相比IL-13Rα1 mRNA与IL-4Rα mRNA水平比对照组显著增高(P<0.05),而IL-13Rα2 mRNA水平比对照组显著降低(P<0.05)。单独低浓度乙醇(25、50、100、200 mmol/L)对HFL-1的COL1A1 mR-NA和COL3A1 mRNA表达无影响(P>0.05)。IL-13(10、20、50μg/L)可以促进HFL-1的COL1A1 mRNA和COL3A1 mR-NA的表达(P<0.05),且存在浓度依赖性。乙醇(200 mmol/L)与IL-13(10、20、50μg/L)共同作用刺激HFL-1促进COL1A1 mRNA和COL3A1 mRNA的表达比IL-13(10、20、50μg/L)单独作用强(P<0.05)。IL-13组(10、20、50μg/L)和乙醇(200 mmol/L)与IL-13(10、20、50μg/L)共同刺激组HFL-1均有CoⅠ的分泌,但共同刺激组HFL-1分泌CoⅠ量显著增加(P<0.05)。结论:单独低浓度乙醇(25、50、100、200mmol/L)不影响HFL-1细胞的COL1A1和COL3A1表达,但可以影响HFL-1细胞IL-4Rα、IL-13Rα1和IL-13Rα2的表达,而乙醇与IL-13共同刺激与单独IL-13刺激相比对HFL-1的COL1A1和COL3A1以及CoⅠ的表达有显著的促进作用。     

循环纤维细胞和人胚肺成纤维细胞的比较研究

目的 比较人循环纤维细胞与人胚肺成纤维细胞的形态学特征和合成胶原的能力.方法 体外分离、培养人循环纤维细胞,用酶水解法和天狼星红染色法分别测定培养液中的羟脯氨酸含量和细胞层中的胶原含量,比较其在不同分化阶段与培养的人胚肺成纤维细胞合成胶原的能力,同时用Western blotting分析其分泌胶原的类型.结果 循环纤维细胞在体外分化过程中,形态学特征不断变化,从圆形幼小、多聚集成"细胞岛"的纤维细胞向梭形、透明、边缘多纤毛状突起的成纤维细胞样细胞分化,在分化过程中不断合成胶原,尤其在培养的细胞层中伴有大量胶原沉积,所分泌的胶原以I型和Ⅲ型胶原为主.结论 循环纤维细胞能够在体外分化为成纤维细胞样细胞,与人胚肺成纤维细胞具有相似的胶原合成能力.     

胰蛋白酶与糖皮质激素影响肺成纤维细胞增殖与骨架蛋白的表达

背景：肺成纤维细胞不但是肺组织的结构支持细胞,还能够通过增殖、收缩、趋化及分泌细胞外基质等多种功能在肺组织的炎症损伤-修复过程中发挥关键作用。 目的：观察原代培养的人肺成纤维细胞在胰蛋白酶或糖皮质激素作用下增殖特性与骨架蛋白表达的变化。 方法：采用人肺成纤维细胞体外培养,胰酶处理24 h,终浓度分别为0,0.5,1.0,5,10 mg/L胰酶;地塞米松处理72 h,在有血清培养条件下进行,浓度范围10^-9-10^-6 mol/L。MTT法测定成纤维细胞增殖情况;免疫印迹方法测定细胞波形蛋白和肌动蛋白的表达。 结果与结论：胰蛋白酶在低浓度(0.1-0.5 mg/L)时刺激肺成纤维细胞增殖;而较高浓度(1-10 mg/L)明显抑制细胞生长。地塞米松对肺成纤维细胞增殖作用的影响不明显。胰蛋白酶显著上调肺成纤维细胞α平滑肌肌动蛋白的表达,但对波形蛋白的表达无明显影响;地塞米松(10^-9-10^-6 mol/L)显著抑制波形蛋白的表达。结果表明在慢性阻塞性肺病等支气管肺慢炎症性疾病的发病过程中,胰蛋白酶和糖皮质激素可以通过参与成纤维细胞增殖和骨架蛋白的表达发挥作用。     

LncRNA-ATB抑制人瘢痕疙瘩成纤维细胞的增殖和胶原合成

目的 探讨lncRNA-ATB对人瘢痕疙瘩成纤维细胞(KFB)增殖和胶原合成的影响及其可能的分子机制。方法 体外分离培养人KFB和正常成纤维细胞(NFB),将KFB分为NC组、si-NC组、si-lncRNA-ATB组、si-lncRNA-ATB+TNF-α组;RT-qPCR检测NFB和KFB中lncRNA-ATB表达水平;MTT检测细胞活力;流式细胞仪检测细胞周期;Western blot检测cyclinD1、胶原合成和NF-κB信号通路相关蛋白的表达。结果 与NFB比较,KFB细胞中lncRNA-ATB表达水平显著升高(P<0.05);敲减lncRNA-ATB表达降低了KFB细胞活力、cyclinD1表达水平以及S期细胞百分比,升高了G₀/G₁期细胞百分比(P<0.05);降低了胶原蛋白Col-Ⅰ和Col-Ⅲ的表达水平(P<0.05);降低了NF-κB信号通路相关蛋白p-p65和p-IκBα的表达水平(P<0.05);NF-κB活化激活剂TNF-α减轻敲减lncRNA-ATB表达对KFB细胞增殖和胶原合成的抑制作用...     

羟基喜树碱与人病理性瘢痕成纤维细胞的增殖

背景:课题组前期实验表明病理性瘢痕成纤维细胞中拓扑异构酶Ⅰ存在高表达,实验以此设想作为拓扑异构酶Ⅰ抑制剂的羟基喜树碱能否抑制体外培养的人病理性瘢痕成纤维细胞的增殖?目的:探讨羟基喜树碱对人病理性瘢痕成纤维细胞增殖的影响。方法:体外培养人病理性瘢痕成纤维细胞。选取羟基喜树碱2～1000μg/L共10个药物质量浓度梯度,分别选取给药后24,48和72h时间点,采用MTT法检测羟基喜树碱对成纤维细胞生长的抑制作用。结果与结论:低质量浓度(2～8μg/L)下羟基喜树碱对成纤维细胞的增殖有抑制作用,质量浓度8～125μg/L时出现平台期,质量浓度125～500μg/L时抑制作用进一步增强,说明羟基喜树碱对成纤维细胞的增殖的抑制作用具有明显的剂量依赖性。药物作用质量浓度和成纤维细胞抑制率呈正相关(r=0.87,P0.05)。随着作用时间延长,细胞抑制率无明显增加,药物作用24,48和72h后,半数抑制率(IC50)分别为233,176及103μg/L。因此实验推论羟基喜树碱能够抑制体外培养的人病理性瘢痕成纤维细胞的增殖。     

类胰蛋白酶和环孢素体外促人胚肾成纤维细胞的增殖

自 1976年报道环孢素A(cyclosporinA ,CsA)可明显改善器官移植的成功率以来 ,现已广泛应用于器官移植及自身免疫病的治疗 ,尤其在防止早期的移植排斥反应、延长移植物的存活期方面 ,已作为临床的首选药物。但随着应用时间的延长 ,发现其存在着不可忽视的毒副作用 (如慢性肾毒性     

人巨细胞病毒感染人胚肺成纤维细胞时NF-κB的表达及意义

研究表明人巨细胞病毒（HCMV）感染能激活核转录因子NF-κB，该因子在病毒完成复制和生活周期中起重要作用。人胚肺成纤维细胞（human embryonic lung fibroblasts，RELY）是HCMV的容许细胞，HCMV可在该细胞内活化复制。     

人肝癌细胞与人胚肺成纤维细胞定位共育培养时bFGF的表达

人肝癌细胞与人胚肺成纤维细胞定位共育培养时ｂＦＧＦ的表达张华＊高福云＊赵天德＊魏育林郭崇洁（首都医科大学组胚教研室＊中日友好临床医学研究所）碱性成纤维细胞生长因子（ｂＦＧＦ）是一种多功能的生长因子，在许多正常细胞和肿瘤细胞中均有表达，它通过自分泌及旁...     

低氧对人胚肺成纤维细胞转化生长因子β1表达的影响

应用核酸原位杂交及增强化学发光免疫斑点法,观察常氧（Ｎ）、低氧（Ｈ）、常氧和低氧猪肺动脉内皮细胞条 件培养液（ＮＥＣＣＭ和 ＨＥＣＣＭ）对人胚肺成纤维细胞转化生长因子β_１（ＴＧＦβ_１） ｍＲＮＡ和蛋白表达的影响。结果表 明：Ｈ组ＴＧＦβ_１ｍＲＮＡ表达为Ｎ组的１．３６倍（Ｐ＜０．０１）,同时在其培养上清液中,ＴＧＦβ_１蛋白含量也明显升高为Ｎ 组的１．８倍。ＨＥＣＣＭ组ＴＧＦβ_１ｍＲＮＡ和蛋白表达较ＮＥＣＣＭ组均下降,ｍＲＮＡ为ＮＥＣＣＭ组的９０％,Ｐ＜０．０１。蛋 白含量为后者的６２．２％。提示：（１）低氧促进入胚肺成纤维细胞合成和分泌ＴＧＦβ_１。（２）在低氧条件下,肺动脉内皮 细胞可能合成某种因子,抑制成纤维细胞ＴＧＦβ_１基因转录和蛋白表达。     

Extracellular glutathione suppresses human lung fibroblast proliferation

Alveolar epithelial lining fluid glutathione (GSH) is markedly decreased in patients with idiopathic pulmonary fibrosis (IPF). Because patients with IPF have exaggerated numbers of fibroblasts in their lower respiratory tract, we hypothesized that GSH can suppress lung fibroblast proliferation. To verify this hypothesis, we examined the ability of GSH to suppress human lung fibroblast (ATCC; HFL-1) proliferation in vitro in the presence of either IPF bronchoalveolar lavage fluid (BAL) or calf serum (CS). Both CS at a concentration of 10% and IPF BAL markedly increased fibroblast proliferation when compared to cells grown without CS or IPF BAL (10% CS = 93 +/- 4%, P less than 0.001; IPF BAL = 47 +/- 4%, P less than 0.001). In the presence of physiologic concentrations of GSH (0 to 500 microM), both CS- and IPF BAL-mediated fibroblast proliferation were markedly reduced, with 500 microM GSH inducing complete inhibition. Interestingly, glutathione disulfide (GSSH) and S-methylglutathione did not suppress proliferation, whereas various sulfhydryl-containing molecules (cysteine, N-acetylcysteine, 2-mercaptoethanol, and low concentrations of dithiothreitol) induced an inhibition of fibroblast proliferation similar to that observed with GSH. Most of the suppressive effect of GSH was mediated at the cell level since incubation of fibroblasts with 500 microM GSH for 1 h completely blocked the ability of the cells to subsequently proliferate in the presence of untreated 10% CS. Treatment of CS with 500 microM GSH for 1 h followed by removal of GSH by molecular sieve chromatography had no detectable effect on fibroblast proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

丝裂原活化蛋白激酶介导人胚肺成纤维细胞增生的观察

目的探讨丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎａｃｔｉｖｉｔｅｄｐｒｏｔｅｉｎｋｉｎａｓｅ，ＭＡＰＫ）在６０Ｃｏγ射线照射后人胚肺成纤维细胞（ｈｕｍａｎｅｍｂｒｙｏｌｕｎｇｆｉｂｒｏｂｌａｓｔ，ＨＥＬＦ）增生中的作用及其与血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ，ＡⅡ）的关系。方法对培养的ＨＥＬＦ进行１～５戈瑞（Ｇｙ）的６０Ｃｏγ－射线照射，用酶标方法检测细胞增生，用免疫细胞化学结合图像分析检测ＡⅡ、ＭＡＰＫ和Ⅰ型前胶原合成以及硝普钠抑制ＡⅡ的作用。结果１～５Ｇｙ照射能促进细胞增生及Ⅰ型前胶原合成，受照射细胞ＡⅡ和ＭＡＰＫ合成增加，外源性ＡⅡ能促进细胞增生而硝普钠则能抑制ＡⅡ的作用。结论６０Ｃｏγ－射线照射促进ＨＥＬＦ增生是一种链锁反应过程，ＡⅡ和ＭＡＰＫ介导了这一反应，ＭＡＰＫ在细胞增生信号传导中可能起着限制性阀门的作用。     

Muscarinic receptors mediate stimulation of human lung fibroblast proliferation

Airway remodeling is a structural alteration associated with chronic inflammatory and obstructive airway diseases, wherein fibroblasts are crucially involved. The present study investigates whether lung fibroblast proliferation is influenced by muscarinic mechanisms. For this purpose, expression of muscarinic receptors in MRC-5 human lung fibroblasts was characterized by semiquantitative RT-PCR, and the effects of muscarinic agonists and antagonists on ((3)H)-thymidine incorporation as a measure of proliferative activity were studied under different culture conditions. MRC-5 fibroblasts express mRNA encoding different subtypes of muscarinic receptors (M(2) > M(3) > M(4), traces for M(5) and no M(1)). Expression of M(2) and M(3) receptors was confirmed at the protein level by immunoblot analysis. Under different culture conditions, carbachol (up to 10 microM) or oxotremorine (10 microM) stimulated ((3)H)-thymidine incorporation, with maximum increases between about 40 and 100%. The stimulatory effect of 10 microM carbachol was prevented by pretreatment with pertussis toxin and antagonized in a concentration-dependent manner by the muscarinic receptor antagonists tiotropium, AQ-RA 741, AF-DX 384, 4-diphenylacetoxy-N-methylpiperidine methoiodide, himbacine, p-fluorohexahydrosiladifenidol, and pirenzepine, with concentrations producing 50% inhibition of 14 pM, 24, 64, 127, 187, 452 nM, and 1.5 microM, respectively. Primary human lung fibroblasts were also found to express mRNA for muscarinic receptors (M(2) > M(1) > M(3), traces for M(4) and no M(5)), and showed a pertussis toxin-sensitive proliferative response to muscarinic receptor stimulation. In conclusion, proliferation of human lung fibroblasts can be stimulated by activation of muscarinic receptors with a pharmacologic profile correlating best to M(2) receptors.     

Expression of the cytoskeleton linker protein ezrin in human cancers

Expression of the metastasis-associated protein, ezrin, in over 5,000 human cancers and normal tissues was analyzed using tissue microarray immunohistochemistry. Ezrin staining was compared between cancers and their corresponding normal tissues, between cancers of epithelial and mesenchymal origin, in the context of the putative inhibitor protein, merlin, and against clinicopathological data available for breast, lung, prostate cancers and sarcomas. Ezrin was found in most cancers and normal tissues at varying levels of intensity. In general ezrin was expressed at higher levels in sarcomas than in carcinomas. By normalizing the expression of ezrin in each cancer using ezrin expression found in the corresponding normal tissue, significant associations between ezrin were found in advancing histological grade in sarcomas (P = 0.02) and poor outcome in breast cancer (P = 0.025). Clinicopathologic associations were not changed by simultaneous assessment of ezrin and merlin in each patient sample for the cancer types examined. These data support a role for ezrin in the biology of human cancers and the need for additional studies in breast cancer and sarcoma patients that may validate ezrin as a marker of cancer progression and as a potential target for cancer therapy. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

巨细胞病毒感染对人胚肺成纤维细胞增殖及凋亡的影响

目的探讨人巨细胞病毒（human cytomegalovirus,HCMV）感染对宿主细胞增殖及凋亡的影响。方法用HCMV AD169感染人胚肺成纤维细胞（human embryonic lung fibroblast cells,HEL）,倒置显微镜观察不同感染时间细胞病变,采用MTT法检测HCMV感染对HEL细胞增殖的抑制作用,同时采用流式细胞术检测HCMV感染细胞对照组细胞凋亡指数。结果在HCMV感染48h内,细胞增殖及凋亡指数与细胞对照组无明显差异。自HCMV感染72h后,细胞增殖明显受抑制,凋亡指数明显增高,细胞病变逐渐加重。结论在HCMV感染早期,对宿主细胞的增殖及凋亡影响不明显,在感染后期,HCMV通过抑制宿主细胞增殖,加重细胞病变,促进感染细胞的凋亡而发挥致病作用。     

肝素促肺成纤维细胞增殖的实验研究

目的：探讨肝素对肺成纤维细胞增殖的作用。方法：应用细胞计数、＾３Ｈ－ＴｄＲ掺入和增殖细胞枋抗原（ＰＣＮＡ）细胞免疫组化等方法，检测肝素对培养的Ｗｉｓｔａｒ乳鼠的肺成纤维细胞（ＬＦｂ）的作用。结果：肝素可促进ＬＦｂ增殖且呈量效关系，以０．１ｍｇ·Ｌ＾－１作用最强，其次为１ｍｇ·Ｌ＾－１，大于１０ｍｇ·Ｌ＾－１则表现为抑制效应。肝素可协同ＴＮＦ促ＬＦｂ增殖。结论：低浓度肝素可能直接或与其他因子协同促进     

Eosinophil- and neutrophil-mediated injury of human lung fibroblast cells

The effect of culturing purified rat peritoneal neutrophils and eosinophils with the human foetal lung fibroblast cell line, MRC-5, was studied. Target cell damage was measured by the failure of the MRC-5 cells to form confluent monolayers during a 6-day incubation period. Neutrophils were more effective at inhibiting cell growth than were eosinophils with greater than 50% inhibition being recorded at initial effector: target cell ratios of 5:1 using neutrophils and 40:1 using eosinophils. Following stimulation with phorbol myristate acetate (PMA), one quarter the number of eosinophils was required to give 50% inhibition of cell growth. The addition of either catalase or superoxide dismutase partly reduced the activity of PMA-stimulated eosinophils, although only with catalase did this reach significance. PMA-stimulated neutrophils did not show any enhanced activity against the MRC-5 cells. Addition of the bacterial analogue f-Met-Leu-Phe (10(-7)M) was unable to increase the cytotoxic activity of eosinophils. Disrupted eosinophil suspensions were as effective as intact cells at inhibiting the growth of target cells whereas some loss in effectiveness was seen with disrupted neutrophils. Membrane-free supernatants from both eosinophils and neutrophils were inactive. The results suggest that although eosinophils and neutrophils are both capable of damaging lung fibroblast cells in culture, the latter is more effective, eosinophils appearing to require an additional stimulation in vitro. Possible mechanisms of cytotoxicity are discussed.     

Thrombin enhances lung fibroblast proliferation in bleomycin-induced pulmonary fibrosis.

To clarify the role of thrombin in fibroblast growth and the development of pulmonary fibrosis in bleomycin-induced interstitial lung disease, we examined the relationship of thrombin activity to fibroblast growth-stimulating activity (FGA) in bronchoalveolar lavage (BAL) fluid from bleomycin-treated rats. Male Wistar rats were given a single intratracheal injection of bleomycin, BAL was performed 2, 6, and 15 days later, and the BAL fluid was assayed for thrombin activity and FGA. Higher FGA than the control value was detected in the BAL fluid from rats on day 6 after bleomycin administration. In bleomycin-treated rats, thrombin activity in the BAL fluid was significantly elevated on day 2 and maximal on day 6. The FGA of the BAL fluid from bleomycin-treated rats on day 6 was significantly decreased by its treatment with various thrombin inhibitors, such as alpha 1-protease inhibitor, antithrombin III, hirudin, and MD-805. In our assay, purified rat thrombin also showed FGA in vitro, and its FGA was inhibited by the same concentrations of these thrombin inhibitors as those inhibiting the activity in the BAL fluid. On ammonium sulfate fractionation, most of the thrombin activity was recovered in the fraction of 35 to 50% saturation in which most of the FGA was detected. These results suggest that the FGA of the BAL fluid from bleomycin-treated rats was at least partly due to thrombin is responsible, at least in part, for fibroblast growth and pulmonary fibrosis in bleomycin-induced interstitial lung disease.