原子力显微镜研究UV-B对I型胶原结构的影响

利用原子力显微镜(AFM)成像技术观察胶原蛋白溶液在UV-B照射前后形态的变化,发现UV-B引起胶原纤维交联度的增加,当交联达一定程度后,照射时间的增加对交联度增加的影响不明显。AFM作为一种高分辨的表面分析仪器,为分子生物学领域的研究提供了一种新的手段。是探讨胶原光作用机理直观、有效的方法。     

Ⅰ型胶原对巨噬细胞摄取氧化低密度脂蛋白的作用

为探讨胶原的存在对细胞摄取氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的影响,本研究在体外制成Ⅰ型胶原凝胶和巨噬细胞实验体系。ＬＤＬ经Ｃｕ２＋催化氧化,丙二醛（ＭＤＡ）及乙酰化修饰后,与胶原的结合能力明显增强,但４－羟基壬烯醛（ＨＮＥ）修饰的ＬＤＬ与胶原的结合能力反而不如天然ＬＤＬ。当小鼠腹腔巨噬细胞培养在胶原凝胶上时,其对ｏｘ－ＬＤＬ的摄取明显减少,这时大部分ｏｘ－ＬＤＬ为胶原凝胶所结合。如用细胞松弛素Ｄ（细胞非特异性吞噬抑制剂）处理巨噬细胞,在无胶原存在时,可见细胞对ｏｘ－ＬＤＬ的降解明显减少;而有胶原时,细胞的降解量则无明显变化,其水平与无胶原时的细胞处理组相当。上述结果提示,Ⅰ型胶原的存在可能阻止了巨噬细胞通过非特异性吞噬途径摄取ｏｘ－ＬＤＬ。     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

自由基与软骨基质胶原蛋白类型改变

本文利用ＳＤＳ－聚丙烯酰胺凝胶电泳方法,定量研究了体外培养的软骨细胞和软骨组织基质中Ⅱ型胶原蛋白的含量。结果表明氧自由基（·Ｏ－２和·ＯＨ）和具有自由基性质的物质（黄腐酸,镰刀菌毒素）可使软骨细胞合成,分泌异常的非Ⅱ型的胶原蛋白,同时,硒化合物可明显地抑制此种效应。     

The Influence of Simple Phenols on Collagen Type I Fibrillogenesis in vitro

The influence of phenolic compounds with different numbers of hydroxy groups (phenol, pyrocatechol, resorcinol, and pyrogallol) on the kinetics of in vitro fibrillogenesis of collagen and on fibril structure has been studied. It has been shown that these phenols accelerate fibril formation mainly by shortening the lag phase, presumably facilitating the formation of collagen dimers and their subsequent association to linear aggregates. The accelerating activity of phenols is proportional to the number of hydroxy groups in the molecule. It increases in the series: phenol < resorcinol < pyrogallol. Therefore, the ability of phenols to accelerate fibril formation is likely to stem from the formation of hydrogen bonds with amino-acid residues in collagen chains. The hydrogen bonds may stabilize the structure of the intermediates, facilitating their interaction during fibrillogenesis.     

Accelerating Effect of Soy Peptides Containing Collagen Peptides on Type I and III Collagen Levels in Rat Skin

Two weeks of feeding soy peptides containing 2% collagen peptides increased the levels of type I and III tropocollagen and their mRNAs. In contrast, the diet did not increase the mRNA levels of rat hyaluronan synthases, serine palmitoyltransferase (the rate-limiting enzyme of ceramide synthesis), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (the key enzyme of cholesterol synthesis). These results suggest that feeding of soy peptides with collagen peptides specifically enhanced the tropocollagen level in the skin.     

蛋白聚糖在维持牙本质胶原空间形貌和水合性能方面的作用

目的:评价牙本质蛋白聚糖对脱矿牙本质胶原纤维形貌和水合性能的影响。方法:新鲜拔除无龋坏人磨牙牙本质酸蚀后分别用胰蛋白酶和硫酸软骨素酶ABC孵育去除牙本质蛋白聚糖和糖胺聚糖侧链,对照组与实验组处理方法相同,但孵育液中不添加酶。然后在牙本质表面不同润湿状态下用场发射扫描电镜和激光共聚焦扫描电镜分别观察牙本质的微观形貌并评价脱矿牙本质的水合性能。结果:硫酸软骨素酶ABC和TRY酶处理改变了牙本质的微观形貌,使胶原纤维间距增大。酶处理、牙本质表面润湿性及两者的交互作用均会显著影响脱矿牙本质的厚度(P0.0001)。结论:牙本质蛋白聚糖和糖胺聚糖侧链在维持牙本质胶原纤维网的空间结构和水合作用方面均发挥着重要作用。蛋白聚糖、胶原纤维-蛋白聚糖以及蛋白聚糖-蛋白聚糖间的的亲水性是影响脱矿牙本质围观形貌和厚度的重要因素。     

Improvement of Shark Type I Collagen with Microbial Transglutaminase in Urea

In the presence of urea, type I collagen could form a gel with crosslinks with microbial transglutaminase (MTGase). Collagen self-assembly was accelerated with the addition of MTGase. The proportion of reconstructed collagen fibrils was raised with the addition of MTGase. MTGase-treated collagen gel remained gelled at high temperatures at which collagen denatured. By treatment with MTGase, collagen could form the gel under impossible condition to collagen self-assembly, and that denaturation temperature was raised.     

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.     

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH₂O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.     

Developmental Stage-dependent Regulation of Prolyl 3-Hydroxylation in Tendon Type I Collagen

3-Hydroxyproline (3-Hyp), which is unique to collagen, is a fairly rare post-translational modification. Recent studies have suggested a function of prolyl 3-hydroxylation in fibril assembly and its relationships with certain disorders, including recessive osteogenesis imperfecta and high myopia. However, no direct evidence for the physiological and pathological roles of 3-Hyp has been presented. In this study, we first estimated the overall alterations in prolyl hydroxylation in collagens purified from skin, bone, and tail tendon of 0.5–18-month-old rats by LC-MS analysis with stable isotope-labeled collagen, which was recently developed as an internal standard for highly accurate collagen analyses. 3-Hyp was found to significantly increase in tendon collagen until 3 months after birth and then remain constant, whereas increased prolyl 3-hydroxylation was not observed in skin and bone collagen. Site-specific analysis further revealed that 3-Hyp was increased in tendon type I collagen in a specific sequence region, including a previously known modification site at Pro⁷⁰⁷ and newly identified sites at Pro⁷¹⁶ and Pro⁷¹⁹, at the early ages. The site-specific alterations in prolyl 3-hydroxylation with aging were also observed in bovine Achilles tendon. We postulate that significant increases in 3-Hyp at the consecutive modification sites are correlated with tissue development in tendon. The present findings suggest that prolyl 3-hydroxylation incrementally regulates collagen fibril diameter in tendon.     

Proteolytic Cleavage of Type I Collagen Generates an Autoantigen in Autoimmune Uveitis

This study was initiated to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats by melanin-associated antigen (MAA; 22-kDa fragment of type I collagen α2 chain) derived from rat iris and ciliary body (CB), to localize MAA within the eye, and to investigate the possible mechanism of MAA generation in vivo. The EAAU model replicates idiopathic human anterior uveitis. Lewis rats sensitized to rat MAA developed anterior uveitis, and EAAU induced by rat MAA can be adoptively transferred to naive syngenic rats by MAA-primed T cells. Animals immunized with rat MAA developed cellular immunity to the antigen. MAA was detected only in the iris and CB of the eye. Iris and CB were the major source of matrix metalloproteinase-1 (MMP-1) in the naive eye, and ocular expression of MMP-1 was up-regulated, whereas expression of tissue inhibitor of metalloproteinase 1 decreased before the onset of EAAU. These results demonstrated that EAAU can be induced by autologous MAA. Uveitogenic antigen is present only in the iris and CB of the eye, and the imbalance between MMP-1 and tissue inhibitor of metalloproteinase 1 may play a role in the generation of MAA in vivo. Collectively, the evidence presented here suggests that MAA is an autoantigen in EAAU. These observations may extend to idiopathic human anterior uveitis and facilitate the development of antigen-specific therapy.     

新型激光光敏根管消毒剂在牙本质中渗透性的研究

目的：观察新型激光光敏根管消毒剂、甲苯胺蓝和亚甲基蓝在牙本质中的渗透效果,评价三种光敏剂在牙本质中的渗透性。方法：收集新鲜拔除的离体单根管牙90颗,根管预备后随机分为三组。每组30颗。A组为新型激光光敏根管消毒剂组;B组为甲苯胺蓝组;c组为亚甲基蓝组。A、B、C三组实验根管内分别用浸有饱和的新型激光光敏根管消毒剂、甲苯胺蓝和亚甲基蓝的棉捻在根管内停留60秒。沿牙体长轴颊舌纵向劈开,倒置荧光显微镜下观察并拍照,用Photoshop8．01软件测量三种光敏剂渗透入牙本质的平均深度。结果：新型激光光敏根管消毒剂在牙本质内的渗透平均深度为553．25μm,甲苯胺蓝在牙本质内的渗透平均深度为350．75μm,亚甲基蓝在牙本质内的渗透平均深度为168．25μm。结论：新型激光光敏根管消毒剂在牙本质渗透性明显优于甲苯胺蓝和亚甲基蓝,具有良好的牙本质渗透性。     

新型激光光敏根管消毒剂在牙本质中渗透性的研究

目的:观察新型激光光敏根管消毒剂、甲苯胺蓝和亚甲基蓝在牙本质中的渗透效果,评价三种光敏剂在牙本质中的渗透性。方法:收集新鲜拔除的离体单根管牙90颗,根管预备后随机分为三组。每组30颗。A组为新型激光光敏根管消毒剂组;B组为甲苯胺蓝组;C组为亚甲基蓝组。A、B、C三组实验根管内分别用浸有饱和的新型激光光敏根管消毒剂、甲苯胺蓝和亚甲基蓝的棉捻在根管内停留60秒。沿牙体长轴颊舌纵向劈开,倒置荧光显微镜下观察并拍照,用Photoshop8.01软件测量三种光敏剂渗透入牙本质的平均深度。结果:新型激光光敏根管消毒剂在牙本质内的渗透平均深度为553.25μm,甲苯胺蓝在牙本质内的渗透平均深度为350.75μm,亚甲基蓝在牙本质内的渗透平均深度为168.25μm。结论:新型激光光敏根管消毒剂在牙本质渗透性明显优于甲苯胺蓝和亚甲基蓝,具有良好的牙本质渗透性。     

Recruitment of Type I Collagen Producing Cells from the Microvasculaturein Vitro

We have previously suggested that microvascular pericytes can differentiate into fibroblast-like, type I collagen-producing cells during excessive dermal scarringin vivo(Sundberg, C., Ivarsson, M., Gerdin, B., and Rubin, K.,Lab. Invest.74, 454–468, 1996). Here we have investigated to what extent pericytes derived from microvessels of full-term human placenta exhibited this capacityin vitro.Vascular fragments of human term placenta were isolated by enzymatic digestion and separation in Percoll. Their microvascular origin was ascertained by confocal microscopy using antibodies specific for endothelial cells (PAL-E) and pericytes (high-molecular-weight–melanoma-associated antigen). When vascular fragments were culturedin vitro,large cells with irregular edges migrated out from the fragments. After 4–6 days in culture, these cells started to proliferate and reached near confluence after approximately 8 days. The cultures were not overgrown by clones of cells with a high proliferative capacity, as demonstrated by cell membrane fluorescence staining and Ki67 expression. Expression of PAL-E, high-molecular-weight–melanoma-associated antigen, smooth muscle α-actin, desmin, and collagen synthesis (prolyl-4-hydroxylase and type I procollagen, as well as collagen pro-α1(I) mRNA) were followed during a culture period of 8 days. The cells were PAL-E negative but expressed high-molecular-weight–melanoma-associated antigen, smooth muscle α-actin, and desmin. Based on morphology and expression of the various markers, the outgrowing cells were identified as pericytes. With time in culture the cells decreased their expression of all these markers and increased their expression of prolyl-4-hydroxylase, type I procollagen, and collagen pro-α1(I) mRNA. Metabolic labeling and SDS–PAGE analysis of labeled proteins revealed that type I collagen was the major collagen species synthesized in the cultures. Our results support the hypotheses that pericytes can leave the vasculature and differentiate into collagen-producing cells and that cultured “fibroblasts” are derived from pericytes.     

绝经后骨质疏松患者血清I型胶原氨基末端、I型胶原羧基末端及骨钙素的表达变化及临床意义

摘要 目的：探讨绝经后骨质疏松患者血清I型胶原氨基末端（NTx）、I型胶原羧基末端（CTX）及骨钙素（BGP）的表达变化及临床意义。方法：选取我院2017年8月-2022年8月收治的60例绝经后骨质疏松患者作为研究对象,将其分为观察组,另选取同期来我院体检的60名绝经后健康志愿者作为对照组。对比两组患者NTx、CTX、BGP表达水平,并建立受试者特征工作（ROC）曲线分析NTx、CTX、BGP对绝经后骨质疏松的诊断效能。3个月后对所有患者进行门诊复查随访,将症状明显明显减轻,X线检查明显改善,骨密度值明显增加的35例绝经后骨质疏松患者分为预后良好组,将其余25例未达到上述标准的患者分为预后不良组,对比两组患者临床一般情况,并应用Logistic回归分析NTx、CTX、BGP对绝经后骨质疏松的预后预测价值。结果：两组受检者NTx、CTX、BGP表达水平对比差异显著,观察组NTx、CTX高于对照组,BGP低于对照组（P＜0.05）; NTx、CTX、BGP三者联合对绝经后骨质疏松的诊断效能优于单一检测（P＜0.05）;预后良好组与预后不良组患者年龄、BMI、合并基础疾病、病程、Ca表达水平对比无明显差异（P＞0.05）,预后良好组与预后不良组患者病情严重程度、骨密度T值、雌二醇、血清NTx、CTX、BGP表达水平对比差异显著（P＜0.05）;logistic回归分析结果表明：CTX、BGP为绝经后骨质疏松的预后不良的独立影响因素（P＜0.05）。结论：绝经后骨质疏松患者血清I型胶原氨基末端、I型胶原羧基末端表达水平高于非骨质疏松群体,骨钙素低于非骨质疏松群体,三者联合可提升绝经后骨质疏松的诊断效能。另外,CTX、BGP作为绝经后骨质疏松的预后不良的独立影响因素,CTX水平越高、BGP水平越低可能预示患者预后不良,因此临床需针对此类患者及时改良治疗措施,提升其预后水平。     

Factors in Bovine Colostrum that enhance the Migration of Human Fibroblasts in Type I Collagen Gels

Bovine colostrum has an activity that increases the migration of WI38 fibroblasts. We evaluated the motility of fibroblasts by their ability to contract collagen gels. Part of the activity was absorbed by anion-exchange chromatography at pH 6.4, and eluted by 0.2-0.3 M sodium chloride. The activity was separated into many fractions corresponding to 20-150 kDa by gel filtration chromatography under acidic conditions. The major peak of the activity coincided with 50-70 kDa.     

Effects of Desiccation and Sediment Type on Early Regeneration of Plant Fragments of Three Species of Aquatic Macrophytes

We tested the effects of desiccation and type of sediment (clay or sand) on the regeneration of fragments of the apical shoots of three species of Hydrocharitaceae: Egeria najas, E. densa and Hydrilla verticillata. The shoots were left to dry on clay or sand from zero to four days, and were then returned to aquaria containing water. To approximate natural conditions, the aquaria containing clay had higher turbidity and nutrient (P and N) concentrations than did the aquaria with sand. All species dried faster on sand substrate, which led to lower regeneration (in terms of dry weight, length, sprout and root formation) in this treatment. H. verticillata fragments elongated faster than the other species, but E. najas was the most successful species (in terms of increase in dry weight) in the sand treatment. Our results indicate that the exotic H. verticillata has a competitive advantage, at least in its early stages of regeneration, over the other two, native species, especially in more eutrophic and turbid habitats; whereas E. najas fragments have a competitive advantage in less‐turbid, oligotrophic and sand‐dominated sites. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin producing beta cells focus on soluble molecules whereas the impact of cell-matrix interactions has been mainly unattended. In this study almost 500 different extracellular matrix protein combinations were screened to systemically identify extracellular matrix proteins that influence differentiation of human embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed by time lapse studies compared to cells on the other tested substrates. Global gene expression analysis showed that cells differentiated on collagen I were largely similar to cells on fibronectin after completed differentiation. Collectively, the data suggest that collagen I induces a more rapid and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation.