大鼠脑缺血再灌注后糖基化终产物及其受体RAGE、分泌型RAGE的表达

目的:探讨大鼠脑缺血再灌注后血清分泌型RAGE、缺血脑组织糖基化终产物及其受体RAGE的表达。方法:本实验分为假手术组和脑缺血2 h后再灌注12 h、24 h、48 h组,共四个实验组,每组20只健康成年大鼠,雌雄不限。应用单侧大脑中动脉阻断法制作局灶型脑缺血模型,脑缺血2 h后再灌注。采用ELISA法检测血清分泌型RAGE水平,免疫组化法检测缺血脑组织糖基化终产物及其受体的变化,应用原位杂交法检测RAGEm-RNA。结果:与假手术组比较,脑缺血再灌注后大鼠血清分泌型RAGE水平下降。免疫组化结果显示脑组织糖基化终产物及其受体蛋白表达的阳性细胞数量与假手术组比较,呈增高趋势,再灌注24 h时,糖基化终产物及其受体蛋白表达达高峰,再灌注48 h时,表达有所下降。统计学分析提示再灌注24 h组、48 h组分别与假手术组比较,差异显著,具有统计学意义(P<0.05-0.01),而两组之间相比较,无显著差异(P>0.05)。原位杂交法检测脑缺血再灌注48 h组缺血脑组织RAGEmRNA阳性细胞数增加,与假手术组比较,差异具有统计学意义(P<0.01)。结论:脑缺血再灌注后缺血脑组织糖基化终产物及其受体的表达升高,而分泌型RAGE在大鼠脑缺血再灌注后血清中的含量及脑组织内的表达均下降。上述变化可能是脑缺血再灌注损伤的机制之一。     

The Maillard reaction in the human body. The main discoveries and factors that affect glycation

Ever since the discovery of the Maillard reaction in 1912 and the discovery of the interaction between advanced glycation end-products and cellular receptors, impressive progress has been made in the knowledge of nonenzymatic browning of proteins in vivo. This reaction which leads to the accumulation of random damage in extracellular proteins is known to have deleterious effects on biological function, and is associated with aging and complication in chronic diseases. Despite a controlled membrane permeability and a protective regulation of the cells, intracellular proteins are also altered by the Maillard reaction. Two main factors, protein turnover and the concentration of carbonyls, are involved in the rate of formation of the Maillard products. This paper reviews the key milestones of the discovery of the Maillard reaction in vivo, better known as glycation, and the factors which are likely to affect it.     

The effect of clinical and subclinical concentrations of lead on the in vitro aging of human fibroblasts

The effect of 20, 40 and 80 micrograms per 100 ml concentrations of lead on the in vitro senescence of fetal human diploid fibroblasts IMR-90 was determined. The areas and dry mass of the cell, nucleus and nucleolus were measured at early, middle and late passages. There was a decrease in total population doublings as the concentration of lead in the medium was increased. Although there was a decrease in the number of nucleoli per cell with successive doublings, there was no difference between controls and lead-treated cells. There was an increase in nucleolar dry mass as the cells aged and this was most noticeable in the 40- and 80-micrograms groups. There were no noteworthy changes in nuclear and cellular areas and dry mass with respect to lead treatment. The results are discussed and it is concluded that even subclinical concentrations of lead cause an acceleration of cellular aging in vitro.     

Reaction pattern to histamine and codeine in a human intradermal skin test model

BACKGROUND: Skin prick and intradermal skin tests (IDT) are useful tools in evaluating IgE-mediated allergic disorders. In the literature, many variations of the techniques used are described. No general agreement exists on test procedures and reading of test results. OBJECTIVE: To analyse test conditions for IDT to facilitate comparability between different study protocols. METHODS: We tested 24 healthy volunteers with three concentrations of histamine and codeine each on the upper back, lateral upper arm and volar forearm, with/without addition of ethylene diamine tetra-acetic acid. Reading of the resulting weal was performed by taking a digital image of the weal, later outlining the weal perimeter in triplicate and calculating the weal area using the NIH Image J software version 1.3. RESULTS: Weal size was dose dependent for both substances, generally larger on the upper back than on the forearm and upper arm, and larger after codeine than after histamine. Addition of the Ca2+ -chelator ethylene diamine tetra-acetic acid did not significantly affect weal size. Weal size induced by histamine showed better consistency than that induced by codeine. CONCLUSIONS: The results and our technique provide valuable tools for the daily routine as well as for the ability to compare information of intradermal tests from different studies or clinical reports. When assessing skin reactivity, we recommend the use of 1 mg/mL codeine as well as 0.1 mg/mL histamine to reflect aspects of mast-cell releasability and of vascular reactivity. The involvement of local factors influencing the vascular reactivity or differences in opiate receptor density on mast cells surfaces needs to be addressed in future studies.     

Effects of topical DHEA on aging skin: a pilot study

OBJECTIVES: Dehydroepiandrosterone (DHEA) is a steroid hormone involved in physiological aging. When administered by oral route, it has been shown to positively affect skin condition on aged people. The purpose of this pilot study was to observe the in vivo effects on skin aging of topical DHEA (1%). METHODS: The DHEA formulation (1%) or the vehicle was topically applied for 4 months to facial and hand skin, in two groups of 20 post-menopausal women. The efficacy of the treatment was evaluated on the basis of clinical and biophysical signs linked to skin aging. RESULTS: We showed that DHEA treatment increased the rate of sebum, which was perceived rather positively by a menopausal population usually affected with a declining sebum level. Topical DHEA tends to improve skin brightness, to counteract papery appearance of skin and epidermal atrophy, a characteristic feature of hormone-related skin aging. Topical DHEA could also act on skin process related to wrinkles, but this result remains to be confirmed. CONCLUSIONS: This pilot study showed beneficial effects on skin characteristics that are rarely provided by topical treatments. It raised some interesting clues towards the treatment of skin aging.     

Pigmented human skin equivalent—as a model of the mechanisms of control of cell-cell and cell-matrix interactions

The melanin pigment system in human skin is extraordinarly well developed and assures the photoprotection of the skin against harmful solar radiation. Specific cell-cell interactions between one melanocytes and keratinocytes play a fundamental role in the regulation of melanogenesis and melanin pigementation, the two key elements of this system, giving rise to the concept of a structural, functional collaborative ‘epidermal melanin unit,’ Early experiments strongly suggested that melanocyte growth and differentiation are regulated by paracrine factors from keratinocytes and other skin cells. In addition, co-culture studies with keratinocytes has shown that the extracellular matrix acts as a local environmental signal for dendrite formation and melanogenesis. Attempts to reconstruct pigmented human skin in vitro have made great progress over the last decade. The behavior of cells in these pigmented human skin equivalents closely resembles that in vivo, and the cells can still respond to appropriate extrinsic regulatory stimuli such as ultraviolet radiation. Keratinocytes and fibroblasts have been shown to be active partners in the regulation of melanocyte distribution, viability and other differentiation functions, presumably by direct contact and the effects of various soluble paracrine factors. By reproducing cell-cell and cell-matrix interactions, these culture systems provide a promising experimental model for investigating regulation of the skin pigmentary system and the role of photoprotection against harmful solar radiation.     

Collagen biosynthesis in cell culture: comparison of corneal keratocytes and skin fibroblasts. Effect of rhamnose-rich oligo- and polysaccharides

Corneal keratocytes are often confounded with fibroblasts, although their matrix-synthetic phenotype is quite different as shown by the nature and relative amount of the different collagens and proteoglycans-glycosaminoglycans synthesized. In these experiments, we compared the concentration of collagens excreted in the culture medium by human corneal keratocytes and skin fibroblasts at three consecutives passages. Although the keratocytes excreted less collagen at earlier passages, they approached and reached fibroblasts at later passages. This can be taken as an indication of the progressive loss of a specific keratocyte phenotype with increasing passages (in vitro aging). The effect of rhamnose-rich oligo- and polysaccharides on collagen secretion also confirmed the subtle differences between these two cell-types, as well as the difference between saturation density of both cell types at confluence and the proportion of dead cells floating on top of the culture medium. These differences were also attenuated with passage number without disappearing completely. The significance of these findings will be discussed in the light of previous results in our and other laboratories on matrix-secreting phenotypes and aging.     

血管内皮生长因子基因感染对大鼠创面移植复合皮的影响

目的观察血管内皮生长因子（VEGF）基因感染的复合皮移植大鼠创面后的愈合效果。方法培养自体表皮细胞和异体成纤维细胞，用腺病毒介导的血管内皮生长因子（Ad-VEGF）感染大鼠成纤维细胞。分别构建感染的复合皮（自体表皮细胞＋人脱细胞真皮基质＋Ad-VEGF基因感染的成纤维细胞）和未感染的复合皮（自体表皮细胞＋人脱细胞真皮基质＋未感染的成纤维细胞），移植于大鼠背部皮肤缺损创面。术后2周观察大鼠创面移植皮片存活情况。术后2、4、6周观察大鼠复合皮大体情况，并取创面组织标本进行组织学观察。结果（1）术后2周，感染组复合皮成活面积大于未感染组复合皮成活面积；（2）术后2周，未感染组创面部分结痂。术后6周感染组复合皮表面光滑，有弹性，抗摩擦性强，愈合效果优于未感染组；（3）术后2周感染组皮片内可见较多的毛细血管分布；6周时表皮细胞分化达5-7层，纤维排列致密整齐，毛细血管分布均匀。结论用Ad-VEGF基因感染复合皮，可以刺激新生血管形成，明显提高皮片愈合质量。     

大面积烧伤异体皮移植两种手术方法临床效果观察

目的观察大面积烧伤四肢切削痂后同期完成异体皮移植术与延期完成异体皮移植术两种手术方法的临床效果。方法大面积烧伤8例做为治疗组,四肢切削痂后异体皮延期移植,手术分两次进行,首次只行四肢切削痂术,创面用生物敷料覆盖,3d后去除生物敷料行异体皮移植术;大面积烧伤10例做为对照组,四肢切削痂术与异体皮移植术同期完成。对比观察2组病例手术时间、手术人员、术中切削痂面积、术中输液总量和输注浓缩红细胞数量、术中创面细菌培养结果、术后并发症发生率及植皮成活率、统计手术费用。结果治疗组单次手术时间少于对照组,组间比较差异有统计学意义（P〈0．01）;治疗组手术人员数量少于对照组（P〈0．01）;2组术中切削痂面积比较相近（P〉0．05）;术中输液总量和输注浓缩红细胞数量对照组多于治疗组单次手术的量（P〈0．01）;术中创面菌培养均为无菌生长;术后1周内对照组并发症发生率大于治疗组（P〈0．05）;异体皮移植10d后成活率治疗组大于对照组（P〈0．05）;治疗组单次手术费用少于对照组（P〈0．001）,而治疗组2次手术费用之和大于对照组（P〈0．001）。结论大面积烧伤四肢切削痂后异体皮延期移植,缩短了单次手术时间,减轻了对患者的二次打击,术后并发症发生率低,提高了异体皮的成活率。     

The sour side of neurodegenerative disorders: the effects of protein glycation

Neurodegenerative diseases are associated with the misfolding and deposition of specific proteins, either intra‐ or extracellularly in the nervous system. Although familial mutations play an important role in protein misfolding and aggregation, the majority of cases of neurodegenerative diseases are sporadic, suggesting that other factors must contribute to the onset and progression of these disorders. Post‐translational modifications are known to influence protein structure and function. Some of these modifications might affect proteins in detrimental ways and lead to their misfolding and accumulation. Reducing sugars play important roles in modifying proteins, forming advanced glycation end‐products (AGEs) in a non‐enzymatic process named glycation. Several proteins linked to neurodegenerative diseases, such as amyloid β, tau, prions and transthyretin, were found to be glycated in patients, and this is thought to be associated with increased protein stability through the formation of crosslinks that stabilize protein aggregates. Moreover, glycation may be responsible, via the receptor for AGE (RAGE), for an increase in oxidative stress and inflammation through the formation of reactive oxygen species and the induction of NF‐κB. Therefore, it is essential to unravel the molecular mechanisms underlying protein glycation to understand their role in neurodegeneration. Here, we reviewed the role of protein glycation in the major neurodegenerative disorders and highlight the potential value of protein glycation as a biomarker or target for therapeutic intervention. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mechanical approach to aging and wrinkling of human facial skin based on the multistage buckling theory

A wrinkle formation mechanism with cutaneous aging is addressed through a mechanical calculation of linear buckling. Skin is divided into five mechanically distinct layers in this study. In general, the outer layer is stiffer than the inner layer, so buckling occurs in the outer layer against the uniform compression caused by muscle contraction. This buckling damages the skin and affects the formation of permanent wrinkles. We propose a multistage buckling theory for evaluation of the wrinkle property, namely, the specific wrinkle size and critical strain in three stages. The specific wrinkle size is derived as the wavelength of the minimum-buckling mode for infinite-length skin. A sensitivity analysis is carried out to investigate the effect of age-related changes of the mechanical parameters on the wrinkle property. We employ some aging hypotheses and prepare two sets of mechanical parameters, one for young and one for aged skin. The numerical results show that the buckling mode switch from Stage I to Stage II is the main reason why pronounced wrinkles suddenly appear in aged skin.     

The effects of human skin fibroblast monolayers on human sperm motility and mouse zygote development.

A new system for co-culture in human in-vitro fertilization (IVF), using human skin fibroblasts, is described and tested pre-clinically. The first test involved the development of 1-cell mouse embryos which exhibit the 2-cell developmental block in vitro. Passage through this block (pb1-ratio) was determined by the ratio of compacted morula stages on day 4 of incubation. For nine human skin cell lines tested (fetal, neonatal and adult), the pb1-ratio was approximately 0.45 (0.07 in culture medium alone; P less than 0.0005). At the compacted morula stage, a second developmental block was observed. The ratio of passing this block (pb2-ratio) was 0.70 +/- 0.09 on skin fibroblasts obtained from fetal or neonatal tissue. On fibroblasts from adult patients the pb2-ratio was 0.30 +/- 0.04 (P less than 0.0005). The second test examined the influence of skin fibroblasts from fetal or neonatal tissue on human sperm motility. After 24 h of incubation, all skin cell lines had a positive influence (P less than 0.01) on the percentage motility compared to culture medium alone. The curvilinear velocity was not significantly increased. From the results we conclude that (i) human skin fibroblasts (especially from fetal tissue) have a positive influence on the development of mouse embryos in vitro, (ii) there is a positive influence of human skin fibroblasts on the percentage motility of human spermatozoa, and (iii) a clinical trial of co-culture with human skin fibroblasts can be justified.     

糖基化终产物(AGEs)的病理意义及其抑制性药物开发原理的研究进展

糖基化终产物(AGEs)是体内蛋白质与还原糖在无酶条件下发生反应后的产物。AGEs与糖尿病及其并发症、血管疾病、衰老以及肾脏疾病等密切相关。本文从AGEs的产生过程、生理作用的分子机制、病理意义以及采用药物干预糖基化终产物的形成和生物学效应等方面进行了综述。     

Comparison of the effects of desloratadine and levocetirizine on histamine-induced wheal,flare and itch in human skin

Objective: A previous study showed the inhibitory effects of loratadine on histamine-induced wheal, flare and itch in human skin to be very variable between individuals. It was hypothesised that this variability may have been due to differences in the rates of metabolism of loratadine to its active form, desloratadine. This double blind, crossover study examined the effects of desloratadine in 12 healthy volunteers. Levocetirizine was used as a comparator. Methods: Desloratadine (5 mg), levocetirizine (5 mg) or placebo was taken orally 4 h before an intradermal injection of histamine (20 l, 100 M) or vehicle control into the forearm skin. Flare areas were assessed by scanning laser Doppler imaging before and at 30 s intervals for a period of 9 min. Wheal areas were measured by planimetry at 10 min. Itch was scored every 30 s for 5 min using a visual analogue scale. Results: Following placebo administration, the mean (± SEM) wheal area at 10 min was 79.3 ± 6.9 mm², mean flare area for the first 5 min following challenge 26.6 ± 2.7 cm², and itch score for the same period 48.5 ± 7.6%. The effects of desloratadine were variable between individuals, mean reductions in the wheal and flare areas being 17% (P = 0.033) and 12% (P = 0.036). Desloratadine did not reduce itch significantly. Levocetirizine was more consistent in its effects, mean reductions in wheal, flare and itch being 51%, 67% 78% respectively (all P < 0.001). Conclusions: A single dose of 5 mg levocetirizine produced more consistent and greater inhibitory effects on histamine-induced wheal, flare and itch than did 5 mg desloratadine. The difference is suggested to reflect the basic pharmacokinetics of the two drugs.Received 21 May 2003; returned for revision 29 May 2003; accepted by M. Parnham 5 June 2003     

The concentration-dependent effect of anethole on collagen,MMP-2 and GAG in human skin fibroblast cultures

PurposeIn aging skin and some skin disorders, components of skin extracellular matrix (ECM) are disturbed and therefore research to find skin drugs is important. Evaluation of anethole impact on collagen, GAGs and MMP-2 in human skin fibroblasts was the aim of this study.Materials and methodsFor collagen assay the Sircol dye, 5-[³H]proline and real time-PCR were used. MMP-2 activity was detected by zymography. GAG concentration was determined using 1,9-dimethylmethylene blue (DMMB). Cell viability was assayed with MTT.ResultsIn cells treated with 1 and 10 μM anethole, a significant increase in collagen synthesis was demonstrated. In contrast, collagen synthesis was significantly decreased in cells exposed to 100 μM anethole. Similar alterations were found in collagen type I expression. The concentration of collagen secreted into the medium was higher only in cells exposed to 1 μM anethole, while it was lower under the influence of higher compound concentrations. It may be due to the lack of pro-MMP-2 activation at 1 μM and a significant increase in the level of MMP-2 at 10 and 100 μM anethole. GAG concentration was reduced under the influence of 100 μM anethole, whereas anethole at lower concentrations revealed the ability to prevent H₂O₂-induced GAG increase. No significant cytotoxicity of anethole to fibroblasts was noted.ConclusionsOur findings demonstrate the concentration-dependent action of anethole on the crucial components of ECM in cultured skin fibroblasts, which may be somewhat beneficial and may possibly be developed towards a therapeutic use in some skin disorders.     

Comparison of the effects of levocetirizine and loratadine on histamine-induced wheal, flare, and itch in human skin

BACKGROUND: This randomized, double-blind, crossover study compared the effects of the R-enantiomer of cetirizine, levocetirizine, with those of loratadine on the wheal, flare, and itch response to histamine in human skin. METHODS: Levocetirizine (5 mg), loratadine (10 mg), or placebo was taken orally 4 h before the intradermal injection of histamine (20 microl, 100 microM) or the control vehicle into the forearm skin of healthy volunteers. Flare areas were assessed by scanning laser Doppler imaging before and at 30-s intervals for a period of 9 min. Wheal areas were measured by planimetry at 10 min. Itch was scored every 30 s with a visual analogue scale. RESULTS: After placebo administration, the mean peak flare area was 23.01+/-1.94 cm(2), the wheal area 248+/-27 mm(2), and the cumulative itch score 28.8+/-4.6% (mean+/-SEM). Levocetirizine reduced the flare, wheal, and itch by 60%, 68%, and 91%, respectively (all P<0.001, Student's t-test for paired data). The effects of loratadine were variable and not statistically significant. CONCLUSION: Levocetirizine (5 mg) is a potent inhibitor of the effects of histamine in human skin with an efficacy that exceeded that of loratadine (10 mg) when single doses of the drugs were administered 4 h before the test.     

Comparative studies on the secretion and activation of MMPs in two reconstructed human skin models using HaCaT- and HaCaT-ras-transfected cell lines

Matrix metalloproteinases play an important role in tissue regeneration, wound healing and tumor invasion. Our previous studies have shown a higher motility of HaCaT-ras-transfected cells compared with HaCaT or normal human keratinocytes (NHK) in correlation with a higher secretion of MMP-2 (72 kDa) or MMP-9 (92 kDa), according to the medium used for cell cultures. Presently, the expression and activity of MMPs were investigated in two reconstructed skin models, using a dead de-epidermized dermis (DED) or a dermal substitute including living fibroblasts. In all experiments, MMP-9 was essentially secreted by NHK and to a greater extent by HaCaT cells. Its active form (86 kDa) was only detected in both reconstructed skin models according to keratinocyte differentiation. MMP-2 was mainly secreted by living fibroblasts included in the dermal substitute skin model. In this case, its activation was up-regulated when HaCaT cell lines were seeded onto the dermal substitute according to their culture at air/liquid interface as shown for MMP-9. The collagenase MMP-1 and stromelysin-1 (MMP-3), susceptible to activate pro-MMP-2 and -9, respectively, were detected in their inactive form by ELISA. MMP-1 was expressed in both models but MMP-3 required the presence of living fibroblasts. Their activities were not detected using specific fluorogenic substrates. In the skin equivalent model using HaCaT, the extensive secretion and activation of MMP-2 and MMP-9 could explain the defect observed in basal membrane reconstruction, suggesting a direct interaction of HaCaT with fibroblasts.     

糖尿病大鼠血管糖基化终产物含量与其受体的ICAM—1表达的关系

探讨糖尿病大鼠血管组织糖基化终产物（AGEs)含量与其受体（RAGE）和细胞间粘附因子－1（ICAM－1）表达的关系。复制糖尿病大鼠模型,采用荧光法、RT－PCR及原位杂交方法检测主动脉及心肌组织的AGEs含量以及RAGE和ICAM－1基因的表达。发现糖尿病大鼠主动脉和心肌组织AGEs含量升高（P＜0．01）;RAGE和ICAM－1基因表达增强（P＜0．05－0．05）;AGEs含量与RAGE及ICAM－1呈明显正相关（P＜0．01）;氨基胍治疗可缓解上述指标的变化。提示 AGEs可诱导RAGE和ICAM－1的表达。推测AGEs-RAGE相互作用是引起糖尿病血管内皮细胞功能紊乱和损伤的关键环节。     

The budding yeast, Saccharomyces cerevisiae, as a model for aging research: a critical review

In this review we discuss the yeast as a paradigm for the study of aging. The budding yeast Saccharomyces cerevisiae, which can proliferate in both haploid and diploid states, has been used extensively in aging research. The budding yeast divides asymmetrically to form a ‘mother’ cell and a bud. Two major approaches, ‘budding life span’ and ‘stationary phase’ have been used to determine ‘senescence’ and ‘life span’ in yeast. Discrepancies observed in metabolic behavior and longevity between cells studied by these two systems raise questions of how ‘life span’ in yeast is defined and measured. Added to this variability in experimental approach and results is the variety of yeast strains with different genetic make up used as ‘wild type’ and experimental organisms. Another problematic genetic point in the published studies on yeast is the use of both diploid and haploid strains. We discuss the inherent, advantageous attributes that make the yeast an attractive choice for modern biological research as well as certain pitfalls in the choice of this model for the study of aging. The significance of the purported roles of the Sir2 gene, histone deacetylases, gene silencing, rDNA circles and stress genes in determination of yeast ‘life span’ and aging is evaluated. The relationship between cultivation conditions and longevity are assessed. Discrepancies between the yeast and mammalian systems with regard to aging are pointed out. We discuss unresolved problems concerning the suitability of the budding yeast for the study of basic aging phenomena.