正常人成纤维细胞老化过程中活性氧水平的变化

目的 研究正常人成纤维细胞在老化过程中细胞活性氧水平的变化。方法 取常规体外培养的原代人成纤维细胞,按不同传代数（PD22－24和PD50－52）分为两组,代表老化过程中两个阶段（早期和中－晚期）,用流式细胞仪检测罗丹明123显示细胞活性氧水平,同时用β-半乳糖苷酶组化染色,形态观察衰老细胞,用群体倍增时间和流式细胞周期分析显示细胞增殖情况。结果 中－晚期细胞中衰老细胞增多,活性氧水平增高,未出现增殖阻滞。结论 正常人成纤维细胞在老化过程中细胞活性氧水平呈升高趋势。     

Prednicarbate Versus Conventional Topical Glucocorticoids: Pharmacodynamic Characterization In Vitro

Purpose. Pharmacodynamic characterization of topical glucocorticoids as prednicarbate (PC), its metabolites prednisolone 17-ethylcarbonate (PEC) and prednisolone (PD), betamethasone 17-valerate (BMV), beta-methasone (BM) and desoximetasone (DM) by evaluating their effects on epidermal and dermal cells. Synopsis of pharmacokinetic and pharmacodynamic studies, possibly explaining the improved benefit-risk ratio of prednicarbate. Methods. Isolated foreskin keratinocytes were used to investigate the influence on epidermal inflammatory processes, dermal fibroblasts of the same origin to study antiproliferative activities of glucocorticoids. Interleukins were measured by ELISA-assay, the influence on II-l-production also on mRNA-level by RNAse protection assay. Proliferation was assessed by ³H thymidine incorporation and biodegradation by HPLC/UV-absorption. Cell viability was controlled by MTT assay. Results. In keratinocytes, inflammation was induced by TNF, resulting in an increased II- l synthesis. This cytokine was particularly suppressed by PC and BMV, whereas PEC, PD, DM and BM were less potent (p 0.05). Since, however, the double ester PC is rapidly degraded in keratinocytes, a RNAse-protection assay of II-1 mRNA was performed allowing short incubation times and thus minimizing biodegradation effects. In agreement with the previous experiment, the antiinflammatory potency of native PC was confirmed. In fibroblasts, II-l and II-6 synthesis indicate proliferation and inflammation respectively. Whereas PC inhibited II- l and II-6 production in fibroblasts to a minor extent only, it was strongly reduced by the conventional glucocorticoids and PEC (p 0.05). The minor unwanted effect of PC on fibroblasts was also reflected by its low influence on cell proliferation as assayed by ³H thymindine incorporation. More pronounced antiproliferative features were observed with BM, PEC and espectially BMV. Conclusions. Correlating antiphlogistic effects in keratinocytes (suppression of II-l) with antiproliferative effects in fibroblasts (suppression of II-l and II-6), the improved benefit–risk ratio of PC compared to conventional glucocorticoids does not result only from distinct drug metabolism in the skin but also from a specific influence on the cytokine network.     

DNA repair synthesis following irradiation with 254-nm and 312-nm ultraviolet light is not diminished in fibroblasts from patients with dysplastic nevus syndrome

The DNA excision repair capacity of 23 primary fibroblast lines from patients with dysplastic nevus syndrome was investigated and DNA repair synthesis (unscheduled DNA synthesis) was determined after UV exposure. Seventeen fibroblast lines from normal donors served as controls. The dose/response experiments included up to ten dose levels and two wavelength ranges: UV-C (using a low-pressure mercury lamp emitting predominantly 254-nm light) and UV-B (artificial sunlamp radiation centering around 312-nm light). For each dose level, silver grains over fibroblast nuclei were counted by visual inspection. Twelve cell lines were also evaluated for both UV wavelength ranges using a new semi-automatic image analyzing system. This system included components for rapid sequential identification of both fibroblast nuclei and silver grains sited above them. Silver grains over 100 nuclei were determined for each UV dose level. Dose/response curves were established and analyzed by linear regression. As a quantitative term for assessing DNA excision repair capacity of a cell line we calculated the linear increase (G ₀) in the number of grains per nucleus, when the UV dose was multiplied by the factor e (i.e. 2.72). The sensitivity of grain detection and resolution ofoverlapping grains was approximately threefold better in visual than in automatic counting, especially when there were more than 70 grains over nuclei. The time recired for visual conting, however, was tenfold that of automatic counting. The varianceweighted meanG ₀ ^v,w of all fibroblast lines from patients with dysplastic nevus syndrome was found to be 79.1 (±1.8-grains/nucleus, that of fibroblast lines from normal donors was 74.2 (±1.7) grains/nucleus. This difference revealed a slightly better repair capability for cell lines from patients but was at the borderline of detection and, therefore, should not be overinterpreted. From the experimental accuracy achieved by determination of the varianceweighted means of the two groups, we would have been able to detect a difference of 7 and more grains [> 2 x (_normal + _patients)]. The variance-weighted meanG _o ^v,w of all fibroblast lines from patients with dysplastic nevus syndrome was found to be 76.4 (±1.4) grains/nucleus, whereas that of fibroblast lines from normal donors was only 66.6 (±1.8) grains/nucleus. This difference was statistically significant and, contrary to expectation, revealed better, not worse post-UV DNA repair capability in cell lines from patients that in those from normal donors. From the experimental accuracy achieved by determination of the variance-weighted means of the two groups, we would have been able to detect a difference of 6.4 or more grains [> 2 x (_normal + _patients)]. Variation between cell lines belonging to the same group was expressed by the standard deviation. On average, the standard deviation was in the range 18.2–21.1 grains/nucleus. This variation did not reflect experimental inaccuracy but different responses of individual cell lines to UV irradiation. On the basis of our data, we consider the hypothesis that patients with dysplastic nevus syndrome are prone to melanoma development because of a general defect in post-UV DNA repair to be improbable.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 136     

Effect of phosphatidylserine on free radical susceptibility in human diploid fibroblasts

Summary We studied the effect of phosphatidylserine (PdtSER) on oxygen metabolite toxicity in skin fibroblast cell lines from apparently normal subjects. Fibroblast damage was produced by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by xanthineoxidase (Xo). In order to quantify cell damage, we measured lactate dehydrogenase (LDH) activity in culture medium and cell viability in fibroblast cultures, with and without preincubation for 4 days with PdtSER 13 M, after Xo incubation. We found a significant increase of LDH activity in culture medium of cells without preincubation with PdtSER. No significant increase of LDH activity was observed in the same cell lines after preincubation with PdtSER.     

复方鳖甲软肝片对心肌成纤维细胞增殖影响的研究

目的　观察复方鳖甲软肝片对心肌成纤维细胞 (CFs)增殖的影响。方法　采用培养的新生大鼠CFs,以MTT作为反映细胞增殖的指标 ,流式细胞仪分析细胞周期。结果　复方鳖甲软肝片能明显抑制CFs增殖 ,并将细胞周期阻抑在G1期 ,强度与氯沙坦相近。结论　复方鳖甲软肝片具有防治心肌纤维化的作用。     

经IFN-γ诱导的人IL-2基因修饰的人原代成纤维细胞体外细胞因子表达及某些免疫学特性的研究

成纤维细胞作为基因治疗载体细胞是肿瘤基因治疗的一项重要途径,但以往的研究仅仅将成纤维细胞作为载体而忽略了其自身的免疫学功能的发挥。本文采用本室构建的逆转录病毒载体将IL-2基因转入人原代成纤维细胞,再用IFN-7诱导。结果表明,IFN-7诱导后,IL-2基因修饰的成纤维细胞MHC-Ⅰ、MHC-Ⅱ、CD40等分子表达具有一定程度的增高,并且表达IL-2、IL-1、IL-6等,由于这些免疫分子及细胞因子的表达与肿瘤抗原的递呈、效应细胞的激活密切相关,提示这种经IFN-7诱导后的IL-2基因修饰的成纤维细胞可能作为抗原递呈细胞而参与肿瘤抗原的递呈及效应细胞的激活。     

牛磺酸抑制新生大鼠心肌重塑的体外研究

目的 通过牛磺酸对新生大鼠心肌成纤维细胞增殖和胶原合成及对I／Ⅲ型胶原比值的影响，从细胞水平探讨其在体外抑制心肌重塑的作用。方法 在培养的心肌成纤维细胞中加入不同浓度的牛磺酸，用羟脯氨酸法测定胶原量，MTT比色法检测细胞增殖情况，SDS-PAGE电泳测胶原I／Ⅲ型比值。结果 100mmol／L、200mmol／L牛磺酸用药24h和48h及50mmol／L用药72h后能明显抑制心肌成纤维细胞的增殖及胶原的合成，I／Ⅲ型胶原比值明显下降。结论 牛磺酸能通过抑制心肌成纤维细胞增殖和胶原合成，降低I／Ⅲ型胶原比值起到抗心肌重塑的作用。     

MyoD基因诱导大鼠心脏成纤维细胞分化为成肌细胞的实验研究

目的：探讨MyoD基因诱导大鼠心脏成纤维细胞分化为成肌细胞的可能性。方法：将MyoD基因重组腺病毒载体转染大鼠心脏成纤维细胞，观察转染后大鼠心脏成纤维细胞形态的变化；用RT-PCR和Western blot方法检测MyoD和肌细胞生成素的表达；免疫组化检测骨骼肌肌球蛋白和结蛋白的表达。结果：MyoD基因转染后的大鼠心脏成纤维细胞形态和排列方式发生明显变化；RT-PCR可检测出转染后细胞表达MyoD；Western blot结果表明转染后细胞表达MyoD和肌细胞生成素；免疫组化检测骨骼肌肌球蛋白和结蛋白表达均为阳性。结论：MyoD基因可诱导体外培养的大鼠心脏成纤维细胞分化为成肌细胞，为进一步研究MyoD对心肌损伤的修复作用奠定了基础。