尖顶羊肚菌胞外多糖提取物对皮肤成纤维细胞增殖和衰老的影响

不同浓度尖顶羊肚菌胞外多糖提取物作用人皮肤成纤维细胞（human skin fibroblasts,HSF）,检测对HSF细胞形态、细胞增殖、衰老相关β-半乳糖苷酶活性、羟脯氨酸含量的影响,探究尖顶羊肚菌胞外多糖提取物对HSF增殖和衰老的影响。结果显示,125μg/mL尖顶羊肚菌胞外多糖提取物使HSF细胞活力增加了25.2%,羟脯氨酸含量增加了12.1%,β-半乳糖苷酶活性降低了48.1%。说明适宜浓度尖顶羊肚菌胞外多糖提取物具有促进HSF细胞增殖、胶原蛋白合成,延缓细胞衰老的作用。     

Proteome analysis of dermal fibroblasts cultured in vitro from human healthy subjects of different ages

Aging is a complex multifactorial process still far from being completely understood. The aim of the present study was to compare the proteome of in vitro cultured dermal fibroblasts from healthy subjects of different ages (i.e. 15 +/- 2, 41 +/- 4 and 82 +/- 3 years old). Proteins of the cell layer were separated by two-dimensional electrophoresis and protein identification was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry; moreover, synthetic gels were qualitatively and quantitatively analyzed by Melanie 3 software. Our study did not reveal any protein typical of any one age group. On the other hand, we observed 38 proteins exhibiting more than three-fold reproducible variations with aging, some (45%) being reduced such as F-actin capping protein alpha1, proteasome subunit alpha type 3, heat shock protein 27, ubiquitin carboxyl-terminal hydrolase isozyme L1, mitochondrial thioredoxin-dependent peroxide reductase, cathepsin B, glutathione S-transferase P, cyclophilin A and calgizzarin. In contrast, T-complex protein 1, probable protein disulfide isomerase ER60, phosphoglycerate kinase 1, Ran-specific GTPase-activating protein, proteasome subunit alpha type 5, triosephosphate isomerase and superoxide dismutase (Mn) increased with age. Furthermore, annexin 1, elongation factor 1beta, proteasome activator complex subunit 1, phosphoglycerate mutase, superoxide dismutase (Cu-Zn) and cofilin, exhibited the highest levels in adult cells; whereas, septin 2 homolog, RNA-binding protein regulatory subunit and ATP synthase D chain revealed the lowest values in adults. The present investigation, underlining the complexity of the aging process, highlights the role of synthetic and degradative pathways in modulating the whole cell machinery and emphasizes that metabolic impairment with age could depend partly on different expression of a number of genes and leading to an imbalance among functional proteins.     

Effects of cell density and cell proliferation on acid cholesterol esterase and cathepsin activity of cultured human skin fibroblasts

We tested the effects of fibroblast cell density and proliferation on the activities of acid cholesterol esterase and cathepsins, the lysosomal enzymes which degrade low-density lipoprotein. Rates of cell proliferation were increased by: (1) fibroblast conditioned medium, (2) increasing the time since subculture from 3 to 7 days, and (3) decreasing the plating density of cells. Cathepsin activity was consistently decreased as cellular proliferation was increased by these various methods. Changes in acid cholesterol esterase activity were more variable. For example, acid cholesterol esterase activity was consistently a positive function of cell density only at densities under 3 micrograms protein/cm2, while cathepsin activity increased up to densities of 16 micrograms protein/cm2. However, the activities of both enzymes were lower at cell densities of under 3 micrograms cell protein/cm2 compared to confluent cultures. Sparse fibroblast cultures may provide a unique model system to study low-density lipoprotein metabolism since, at low cell density, LDL receptor activity is high while lysosomal activity is low, making it possible that lysosomal degradation could become the rate-limiting step in the process of LDL degradation rather than receptor-mediated internalization of the lipoprotein. This might then allow an accumulation of lipoprotein-derived cholesteryl esters in the cell. Such a model could be relevant to the propensity of arterial cells to become foam cells during atherogenesis.     

Effect of cholesterol and growth factors on the proliferation of cultured human skin fibroblasts

A cholesterol-deficient growth medium for human skin fibroblasts was prepared by adding to Eagle's Minimum Essential Medium a bovine serum treated with ultracentrifugation to remove bulk lipoproteins followed by silicic acid adsorption to remove residual lipoproteins and cholesterol. Cell growth was slow, but the daily cell doublings could be increased by 76% by including 7.5 micrograms purified cholesterol/ml in the medium. Cell growth in cholesterol-deficient culture medium could be increased to that seen with medium containing 15% untreated fetal bovine serum by the inclusion of the following growth factors: epidermal growth factor (EGF), cortisol, non-essential amino acids, insulin, transferrin and selenium. Cholesterol increased the proliferation of these rapidly-growing cultures by 19%. No effect of cholesterol was observed in transformed L-cell mouse fibroblasts.     

Growth of human skin fibroblasts in dialyzed fetal bovine serum

Summary Human diploid fibroblast cultures plated at or below a density of 2×10³ cells per cm² grew very slowly or not at all in MEM supplemented with 10% fetal bovine serum that had been dialyzed for 24 hr. Adding serine (0.2 mM) or pyruvate (1.0 mM) to MEM and 10% dialyzed serum restored growth to the level observed with 10% nondialyzed serum. Serine and pyruvate also were able to overcome partially the growth arrest induced by a reduced serum concentration (1 or 2%). Human fibroblast cultures grew very well in 100% fetal bovine serum that had been dialyzed against MEM. For cells grown in dialyzed serum, the final number increased with increasing serum concentration, in contrast to the well established toxic effects of high concentrations of nondialyzed serum. This research was supported by NIH Grants CA15207 and HD03110.     

Proliferation dynamics in cultured skin fibroblasts from Down syndrome subjects

With a view to better understanding the role of oxidant/antioxidant variables in proliferation dynamics of somatic cells, we explored the relationships among superoxide dismutase (SOD) activity, glutathione peroxidase (Gpx) activity, reactive oxygen intermediates (ROI), and indices of cellular proliferation and senescence in cultured fibroblasts from Down syndrome and normal donors. We found that Down syndrome cells had a significantly slower proliferative rate, but attain replicative senescence at similar population doubling (PD) as control cells. Irrespective of donor origin, the number of PD until replicative senescence was positively correlated with Gpx activity (r = 0.784, P = 0.007). In addition, the presence of exogenous catalase in the growth medium significantly extended the number of PD until replicative senescence (P = 0.011). The loss of telomere repeats per PD was not different between Down syndrome cells and controls. However, SOD activity was inversely correlated with the loss of telomere repeats per PD. Collectively, these findings suggest that replicative senescence ultimately relates to mechanisms downstream to SOD (i.e., Gpx and catalase) and confirmed previous observations about inverse relationships between SOD activity and telomere repeat loss per cellular replication.     

Intracellular activity and secretion of various lysosomal glycosidases in cultured human skin fibroblasts

The intracellular activities of four lysosomal glycosidases (alpha-L-fucosidase, beta-D-hexosaminidase, beta-D-galactosidase and beta-D-glucuronidase) in human skin fibroblasts cultured in a medium with 0.1% serum increased in a greater degree than that in a medium with 10% serum. Only two glycosidases (alpha-L-fucosidase and beta-D-hexosaminidase) were secreted by fibroblasts in the culture medium. The extracellular activity of alpha-L-fucosidase and beta-D-hexosaminidase was equivalent to 80 and 25% of their intracellular activity in serum-sufficient fibroblasts and 40 and 15%--in serum-restricted fibroblasts. These results suggest that the observer phenomena are controlled by the levels of autophagy, endocytosis and membrane recycling.     

Effects of human and ovine pancreatic secretory trypsin inhibitors on the proliferation of normal human fibroblasts

Human pancreatic secretory trypsin inhibitor inhibited cell-surface proteolytic activity in human fibroblasts. In the range of concentrations which caused proteinase inhibition, fibroblast proliferation was also inhibited by this reagent and by the ovine equivalent. At lower concentrations, there was some evidence for a mitogenic effect, and this was confirmed by obvious stimulation of DNA synthesis at these concentrations. Human alpha 1-proteinase inhibitor, previously demonstrated to be an inhibitor of fibroblast proliferation, was also mitogenic at concentrations lower than those which inhibited proteolytic activity and cell proliferation. Human pancreatic secretory trypsin inhibitor and epidermal growth factor apparently work through independent mechanisms, since their mitogenic effects are additive.     

Generation of insulin-secreting islet-like clusters from human skin fibroblasts

Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future.     

8,12;8,20-diepoxy-8,14-secopregnane glycosides from roots of Asclepias tuberosa and their effect on proliferation of human skin fibroblasts

A pregnane glycoside fraction from the roots of Asclepias tuberosa L. caused normal human skin fibroblasts to proliferate. This fraction contained 21 pregnane glycosides whose structures were established using NMR spectroscopic analysis and chemical evidence. The aglycones of most of these compounds were identified as 8,12;8,20-diepoxy-8,14-secopregnanes, such as tuberogenin or 5,6-didehydrotuberogenin, the same aglycones as constituents of the aerial parts of this plant. Some of these compounds also caused proliferation of skin fibroblasts.     

Proteoglycans synthesized by gingival fibroblasts derived from human donors of different ages

The proteoglycans synthesized by fibroblasts derived from human donors of ages ranging from 12 years to 68 years have been studied. In addition, the in vitro proliferation rates of the various cell strains were studied and demonstrated that increasing donor age correlated with a decrease in proliferative activity. The incorporation of [35S]-sulfate into proteoglycans decreased with increasing donor age with cells from the oldest donor demonstrating a 50% reduction compared with cells from the youngest donor. Analysis on Sepharose CL-4B of isolated [35S]-labeled proteoglycans for molecular size distribution revealed few differences between the cell-layer-associated proteoglycans of all cell strains studied. However, analysis of the medium-associated [35S]-labeled proteoglycans demonstrated an increase in the amount of small molecular size proteoglycans with increasing age. More specific analysis of the glycosaminoglycan composition revealed an increase in heparan sulfate from 52% to 73% in the cell-layer-associated proteoglycans of cells from the youngest and oldest donors, respectively. Accompanying this increase was a relative decrease in dermatan and chondroitin sulfate content from 24% to 13% and 25% to 16%, respectively, with increasing donor age. Additionally, the degree of N-sulfation of cell layer heparan sulfate increased with age. Heparan sulfate levels increased in the medium as well with increasing age, with a concomitant decrease in chondroitin sulfate. The quantity of medium-derived dermatan sulfate remained relatively evenly distributed throughout the various ages studied. The various differences noted are considered to reflect the general metabolic changes associated with aging. In particular the increase in heparan sulfate content with age is considered to be related to the decreased proliferative activity of the fibroblasts with increasing age.     

阿魏酸钠对增生性瘢痕成纤维细胞增殖及胶原合成的影响

探讨阿魏酸钠(SF)对增生性瘢痕成纤维细胞(HSFb)增殖及胶原合成的影响.体外培养HSFb,MTT法计算SF的LC50及最佳药物时间后,分为空白对照组、SF干预组(高、中、低浓度分别为0.3、0.03、0.003 mg/mL),培养72 h后,在倒置显微镜和透射电镜下观察HSFb微观形态学变化;MTT法、Wester...     

Interleukin-6 receptor superantagonist Sant7 inhibits TGF-beta-induced proliferation of human lung fibroblasts

Abstract.  Objectives : Both interleukin-6 (IL-6) and transforming growth factor-β (TGF-β) are crucially involved in fibrotic events that characterize interstitial lung diseases (ILD). Therefore, the aim of this study was to investigate in primary cultures of normal and fibrotic human lung fibroblasts (HLF), exposed to either IL-6 or TGF-β1, the effects on phosphorylation of mitogen-activated protein kinases (MAPK) and cell growth of IL-6 signalling inhibition, performed by the IL-6 receptor superantagonist Sant7. Materials and methods : MAPK phosphorylation was detected by Western blotting, HLF viability and proliferation were evaluated using the trypan blue staining and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Results : Sant7, at a concentration of 1 µg/mL, was capable of significantly inhibiting HLF proliferation and MAPK phosphorylation induced by cell exposure to IL-6 (100 ng/mL) or TGF-β1 (10 ng/mL), whose actions were more evident in fibrotic cells. Conclusions : These findings suggest that, in HLFs derived from patients with ILDs, the proliferative mechanisms activated by TGF-β1 are at least in part mediated by an increased release of IL-6, leading to phosphorylation-dependent MAPK activation. Such preliminary findings may thus open new therapeutic perspectives for fibrogenic ILDs, based on inhibition of signal transduction pathways stimulated by the IL-6 receptor.     

Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture

Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.