integrins
细胞分化综合征的诊断及治疗
对于出现高白细胞血症的患者，同时启动ATRA/ ATO 及Ara-C可能获益。
治疗：其他
支持治疗：经验性抗生素治疗、利尿、吸氧、呼吸 衰竭时行辅助呼吸。
谢谢！
(1) 毛细血管渗透性增加 (2) 成熟白细胞迁移能力增强 (3) 白细胞integrins表达上调，对血管内皮粘附能力增加
临床表现
典型临床表现：发热、低血压、呼吸困难、体重增 加超过5kg、肌肉骨骼疼痛、全身红疹。
常见症状
呼吸困难
59-95%
水肿
53-81%
无法解释的发热 53-74%
低血压
40%的DS患者中，胸片正常。所以不要非等 到CXR异常才诊断DS。
CT： 弥漫磨玻璃影、胸膜下结节影、胸膜增 厚、胸水及心包积液、片状实变影。
注意肺出血的影像提示。
CXR: BILATERAL INFILTRATES
诊断
DS是一个临床诊断，通常在APL经ARTA或ATO诱导后出现， 包含一组症状及体征。 下列情况出现三种及以上时，应高度怀疑DS并尽快启动 GCS治疗： 发热； 体重增加 ； 呼吸窘迫； 胸片上具片影； 胸水或心包积液； 低血压； 肾功能不全
Sanz MA, et al. Management of acute promyelocytic leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 2009; 113:1875.
细胞分化综合征
DIFFERENTIATION SYNDROME
细胞连接
细胞识别和黏连的分子基础
p200
参与细胞粘着的4类粘着分子: 1 选择蛋白(selectin) 2 免疫球蛋白超家族(IgSF)的某些成员 3 钙粘着蛋白(cadherin) 4 整合蛋白(integrin)家族的某些成员
A 选择素(selectin)
膜整合糖蛋白，能够特异识别并结合从另一个细
Ca2+,Mg2+,and/or Mn2+ are shown as orange spheres.
A model of integrin activation
inactive
The switch in conformation is triggered by the binding of a proteinextracellular matrix(ECM).
III 细胞之间的识别与粘着 P199 Cell-cell recognition and adhesion 细胞识别(Cell recognition)：细胞对同种或异种细胞 、同源或异源细胞及对自己和异己分子的认识和 鉴别。 细胞粘着(Cell adhesion) ：在细胞识别基础上，同类 细胞发生聚集形成细胞团或组织的过程。
Mechanically separated marine sponges reassemble through species-specific homotypic cell adhesion.
a. Two sponges (orange/yellow) growing in the wild. b. After mechanical disruption and mixing of both types of intact sponges, the individual cells were allowed to re-associate for ~30min with gentle stirring.
确定膜蛋白方向的实验程序
第四章细胞质膜与细胞表面一. 教学目标：1掌握质膜的分子模型；2．深刻理解生物膜的分子结构模型及其膜的流动性，并掌握流动镶嵌模型的主要特点；3 掌握细胞连接的方式和特点。
二．重点：流动镶嵌模型结构要点。
三．难点：细胞连接的超微结构。
四．授课方式与教学方法：讲授、讨论、多媒体辅助教学。
五．教学内容:细胞质膜与其他的生物膜一样都是由膜脂和膜蛋白构成的。
膜蛋白又可分为内在蛋白和外在蛋白。
脂双分子层构成了膜的基本结构。
各种不同的膜蛋白及其与膜脂分子的协同作用不仅为细胞的生命活动提供了稳定的内环境，而且还行使着物质转运、信号传递、细胞识别等多种复杂的功能。
第一节细胞质膜与细胞表面特化结构细胞质膜(plasma membrane),又称细胞膜(cell membrane)细胞内膜(intracellular membrane); 生物膜(biomembrane)一、细胞质膜的结构模型二、膜脂三、膜蛋白四、确定膜蛋白方向的实验程序五、生物膜结构特征六、细胞质膜的功能七、膜骨架与细胞表面的特化结构第二节细胞连接细胞连接的功能分类: 封闭连接; 锚定连接; 通讯连接细胞表面的粘连分子一、胞质膜的结构模型细胞膜的化学组成 ; 研究简史; 结构模型; 生物膜结构细胞膜的化学组成●膜脂50%●膜蛋白40%●膜糖类10%左右结构模型◆E.Gorter和F．Grendel（1925）:―蛋白质-脂类-蛋白质‖三夹板质膜结构模型◆J.D.Robertson（1959年）：单位膜模型(unit membrane model)◆S.J.Singer和G.Nicolson（1972）：生物膜的流动镶嵌模型(fluid mosaic model)◆K.Simons et al(1997): 脂筏模型（lipid rafts model）Functional rafts in Cell membranes. Nature 387:569-572生物膜结构◆1.磷脂双分子层是组成生物膜的基本结构成分,尚未发现膜结构中起组织作用的蛋白；◆2.蛋白分子以不同方式镶嵌在脂双层分子中或结合在其表面, 膜蛋白是赋予生物膜功能的主要决定者；◆3.生物膜是磷脂双分子层嵌有蛋白质的二维流体。
炎症的基本病理过程
包 细胞反应 为 白细胞渗出和吞
括
分 噬作用
教材上分为：血管反应和细 胞反应。
1、血流动力学改变 即血管反应、炎性充血。 炎症过程中组织发生损伤后，很快发
生血流动力学变化，即血流量和血管口径 改变。
1）细动脉短暂收缩，持续几秒钟。 （由于神经源性和一些化学介质的机制）。
2）血管扩张和血流加速，先累及细 动脉，随后更多的毛细血管开放，局部血 流加快，造成局部发红发热。（机制是因 神经体液因素的作用，即轴突反射和化学 介质的作用）。
胞连接处伸出伪足，整个白细胞以阿米 巴运动的方式从内皮细胞缝隙中逸出。
嗜中性粒细胞、嗜酸性粒细胞、嗜碱性 粒细胞、单核细胞和各种淋巴细胞都是 以此种方式游出血管。
穿过内皮细胞的白细胞可分泌胶原
酶，降解血管基底膜而进入炎区内（一 个白细胞常需要2～12min才能完全通 过血管壁）。
炎症的不同阶段游出的白细胞的种 类不同。急性炎症的早期6～24h嗜中性 粒细胞首先游出，48h后所见则以单核细 胞在组织内浸润为主，因为嗜中性粒细 胞寿命短，24～48h后崩解消亡，而单 核细胞在组织中寿命长；嗜中性粒细胞 停止游出后，单核细胞可继续游出；炎 症不同阶段所激活的粘附因子和趋化因 子不同，而且嗜中性粒细胞能释放单核 细胞趋化因子，所以引起单核细胞游出。
②渗透压改变
炎区内〔H＋〕增高 盐类解离加强
组织崩解 细胞内K＋释放
由 分解代谢绝亢进 高分子量的糖、
于
蛋白质等分解为许多低分子微粒
炎区内血管通透性增高 血浆蛋白
渗出 炎区内离子增多
（H＋、K＋、PO4-3），分子浓度升高
渗透压升高 吸水性增强 炎性水肿
炎区内渗透压变化和H＋浓度相似， 愈接近炎区中心，渗透压越高。
Src、FAK对E-cadherin和integrin介导的串联以及肿瘤浸润、转移的影响
Src、FAK对E-cadherin和integrin介导的串联以及肿瘤浸润、转移的影响周俭珊;黄海燕【摘要】钙黏蛋白(E-cadherin)和整合素(Integrin)在协调控制细胞基本的生理和病理过程中扮演着重要的角色,包括形态发生、组织分化、伤口愈合、免疫监视、炎症反应、肿瘤进展和转移等.然而,目前调节钙黏蛋白和整合素之间通信的根本性分子机制仍然不是很清楚.尽管大量的证据支持两种黏附受体家族间存在有精细调控的串联,而且这种串联可以影响他们的表达、翻转、定位和/或功能,并可根据细胞内外的环境背景来增强或抑制黏附连接,然而这些重要的现象中涉及到的分子和分子调控机制目前还不完全清楚.最近越来越多的证据表明,非受体酪氨酸激酶Src和FAK与整合素和钙黏蛋白调控的细胞间黏附和信号转导的过程密切相关,本文主要是综述Src及FAK在串联中的重要作用,及探讨这种串联对肿瘤细胞的集体迁移、浸润和转移的潜力的影响.【期刊名称】《海南医学》【年(卷),期】2016(027)001【总页数】4页(P93-96)【关键词】钙黏蛋白;整合素;串联;Src;FAK【作者】周俭珊;黄海燕【作者单位】三峡大学医学院,湖北襄阳 443000;三峡大学医学院,湖北襄阳443000【正文语种】中文【中图分类】R73-37所谓分子串联是指信号通路间的通信，在细胞生物学中起着核心作用，使细胞能够连接到相邻细胞或者较远的分子功能组件，来产生协同或拮抗效应，最终产生生物学效果[1]。
细胞间最重要的串联事件是连接到整合素和钙黏蛋白家族的黏附分子受体的信号网络。
钙黏蛋白(E-cadherins)和整合素(Integrins)是在上皮中分别介导细胞和细胞间、细胞和胞外基质间黏附的主要分子。
已有研究证实，这些分子参与了如细胞迁移、增殖、分化，生存和基因表达等重要生物过程的调节。
大量的体内和体外实验都证明了在细胞黏附和移动过程中E-cadherins和Integrins两者介导的连接存在着串联，且这种串联可以调控肿瘤细胞的可塑性，在肿瘤细胞的局部浸润和远处转移中发挥了重要作用[2-3]。
integrin review 整合素受体信号通路综述
Rheumatol IntDOI 10.1007/s00296-014-3137-5Role of integrins and their ligands in osteoarthritic cartilageJian Tian · Fang‑Jie Zhang · Guang‑Hua LeiReceived: 25 May 2014 / Accepted: 17 September 2014 © Springer-Verlag Berlin Heidelberg 2014[1]. Radiographic evidence of OA occurs in the majority of people by 65 years of age, and among them about 80 % in people who aged over 75 years [2]. However, the pathogen-esis of this disease is not fully elucidated.Cartilage damage is one of the major pathological changes in OA. Articular cartilage is an avascular, a neu-ral, alymphatic, and viscoelastic connective tissue that functions autonomously to bear loads and provide almost friction-free movement of diarthrodial joints [3]. Chondro-cytes, the only cell population of adult articular cartilage, are strongly involved in maintaining the dynamic equi-librium between synthesis and degradation of the extra-cellular matrix (ECM) [4]. Collagens represent the major structural components of the articular cartilage. Cartilage is made up of two main ECM macromolecules: type II collagen and aggrecan, a large aggregating proteoglycan [5, 6]. Cartilage destruction is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for the cartilage collagen break-down, whereas enzymes from disintegrin and metallopro-teinase domain with thrombospondin motifs (ADAMTS) family mediate cartilage aggrecan loss [7]. Activation of biochemical pathways involves the production of proin-flammatory cytokines, inflammation, degradation of the ECM by MMPs and ADAMTS, and cessation of ECM syn-thesis via dedifferentiation and apoptosis of chondrocytes [8, 9]. Therefore, the ECM is a vital cellular environment, and interactions between the cell and ECM are important in regulating many biological processes, which include cell growth, differentiation, and survival [10, 11].Cell–matrix interactions control cell function and behav-ior by signal transduction through a variety of cell sur-face receptors. The integrins are the major family of ECM receptors, which can transmit information from the matrix to the cell. Integrin binding of ECM ligands results in theAbstract Osteoarthritis (OA) is a degenerative disease, which is characterized by articular cartilage destruction, and mainly affects the older people. The extracellular matrix (ECM) provides a vital cellular environment, and interactions between the cell and ECM are important in reg-ulating many biological processes, including cell growth, differentiation, and survival. However, the pathogenesis of this disease is not fully elucidated, and it cannot be cured totally. Integrins are one of the major receptors in chondro-cytes. A number of studies confirmed that the chondrocytes express several integrins including α5β1, αV β3, αV β5, α6β1, α1β1, α2β1, α10β1, and α3β1, and some integrins ligands might act as the OA progression biomarkers. This review focuses on the functional role of integrins and their extracellular ligands in OA progression, especially OA car-tilage. Clear understanding of the role of integrins and their ligands in OA cartilage may have impact on future develop-ment of successful therapeutic approaches to OA.Keywords Chondrocyte · Integrin · Fibronectin · Tenascin C · Osteopontin · Osteoarthritis · CartilageIntroductionOsteoarthritis (OA) is a degenerative disease and is char-acterized by articular cartilage destruction along with changes occurring in other joint components including bone, menisci, synovium, ligaments, capsule, and musclesRheumatologyINTERNATIONALJ. Tian · F.-J. Zhang · G.-H. Lei (*)Department of Orthopaedics, Xiangya Hospital, Central South University, No. 87 Xiangya Road, Changsha 410008, Hunan, Chinae-mail: gh.lei9640@; lgh9640@Rheumatol Intformation of signaling complexes, which play a key role in the regulation of cell survival, adhesion, proliferation, dif-ferentiation, and matrix remodeling [11, 12]. To develop new and successful approaches for the treatment for OA, it is essential to elucidate the role of integrins and their ligands in the pathogenesis of OA. In this study, we have reviewed the role of integrins and their ligands on the OA cartilage, consequently which contributes to OA progression. Integrins structure and functionThe first integrin was identified almost 30 years ago; “integrin” was named for this protein complex because of its role as an integral membrane complex involved in the transmembrane association between the ECM and the cytoskeleton [13]. The first integrin of which cDNA was sequenced encodes a polypeptide of 89 kD, with the pres-ence of a large N-terminal extracellular domain, a single transmembrane segment, and a small C-terminal cytoplas-mic domain. The extracellular domain contains a threefold repeat of a novel 40 residue cysteine-rich segment, and the cytoplasmic domain contains a tyrosine residue that is a potential site for phosphorylation by tyrosine kinases [13]. So far, it is well known as a family of heterodimeric trans-membrane receptors consisting of an α and a β subunit, which each have a large ectodomain, a single transmem-brane domain, and a generally short cytoplasmic tail. All of the different 18 α and 8 β subunits are known in humans, which can be combined to 24 different integrin receptors [14, 15]. Multiple α subunits can combine with single βsubunits (and vice versa), giving rise to “combinatorial” ligand specificity, as shown in Fig. 1.The 24 known integrin heterodimers can be classified as arginine–glycine–aspartate (RGD)-binding, the α4 family, leukocyte adhesion integrins, laminin-binding, and I-domain collagen-binding, as shown in the Table 1. All of these integ-rins can be further segregated into two groups, either contain-ing or the other lacking an extra von Willebrand factor type A domain (known as αA or αI in integrins) in their α subunits. The I-domain subunits contain α1, α2, α10, α11, αL, αM, αX, αD, and αE, and non-I-domain subunits are α3, α4, α5, α6, α7, α8, α9, αV, and α IIb, as shown in Fig. 1. In I-domain integ-rins, the I-domains play a central role in ligand binding and intercellular adhesion, whereas in integrins, which lack the αI domain, the binding site in the integrin “head” is formed by structural contributions of both the α and β chains [16].Although the 24 heterodimers can be defined into different groups, different heterodimers can also be expressed on a sin-gle cell and each can interact with multiple intracellular sign-aling cascades. Depending on the cellular microenvironment, the biological effect of ligating or activating an integrin can vary dramatically [15, 17]. The regulation of integrin activ-ity is complex. Integrin affinities for their cognate extracellu-lar ligands, such as fibronectin, fibrinogen, and collagen, are regulated by cellular signaling, resulting in integrin activation through “inside–out” signaling [15, 18] leading to conforma-tional changes that result in increased affinity for extracellu-lar ligands [18]. Inside–out signaling controls the adhesion strength and enables sufficiently strong interactions between integrins and ECM proteins to allow integrins to transmit the forces required for cell migration and ECM remodeling and assembly [18]. Integrins have no intrinsic enzymatic activity but, following binding to extracellular ligands, they become activated, can cluster on the cell surface, and undergo con-formational changes that propagate across the membraneFig. 1 Integrins superfamily. All 18 different α and 8 dif-ferent β subunits are known in humans, which can combine to24 different integrin receptorsRheumatol Int(“outside–in”) to activate cytoplasmic kinase- and cytoskele-tal-signaling cascades. These in turn control cell attachment, movement, growth and differentiation, and survival [15, 17]. Therefore, integrin activation can increase ligand binding, resulting in outside–in signaling. Converse ligand binding can generate signals that cause inside–out signaling [18].Expression of integrins in chondrocytesPrevious studies confirmed that the chondrocytes express several integrins including α5β1, αV β3, αV β5, α6β1, α1β1,α2β1, and α10β1 [18–23], while α3β1 was expressed by occasional cells only [24]. The expression level of above-mentioned integrins was in different percentages and in dif-ferent zones. Fetal chondrocytes strongly expressed β1 and β5 chains [24, 25]. Chondrocytes from osteoarthritic car-tilage expressed high levels of β1 integrin and all of the α chains. The α1 was the most frequently expressed α chain, followed by α3, α5, α2, αv. Integrin expression decreased from the least to the most damaged zone of articular car-tilage, and cell cycle analysis showed that proliferating chondrocytes (S phase) were prevalent in the latter zone. The expression of β2, β3, β2, and β5 is usually very lowTable 1 24 human integrin heterodimers and their ligands ADAMs a disintegrin and metalloproteinases, ICAM intercellular adhesion molecules, VCAM vascular adhesion molecules, TGF β LAP trans-forming growth factor β latency-associated peptide, MadCaM mucosal address in cell adhesion molecule, VEGF vascular endothelial growth factorHuman integrins Ligands Cellular and tissue distributionRGD -binding α5β1FibronectinChondrocytes , endothelial cellsα8β1Fibronectin, vitronectin, tenascin C, osteopontin, nefronectin Smooth muscle cells αV β1Fibronectin, vitronectin Smooth muscle cells, fibroblasts, osteoclasts, tumor cells αV β3Fibrinogen, fibronectin, vitronectin, tenascin C, osteopontin, bone sialoprotein, MMP-2Smooth muscle cells, fibroblasts, osteoclasts, tumor cells, Chondrocytes, endothelial cells, platelets, epithelial cells,leukocytesαV β5Vitronectin Smooth muscle cells, fibroblasts, osteoclasts, Chondrocytes,platelets, leukocytes, epithelial cellsαV β6Fibronectin, TGF-β LAP Epithelial cells, carcinoma cells αV β8Vitronectin Melanoma, kidney, brian, ovary, uterus, placenta αIIb β3Fibrinogen Fibronectin, vitronectinPlatelets The α4 family α4β1Fibronectin, VCAMLeukocytes, endothelial cells,α4β7Fibronectin, VCAM, MadCaMLeukocytes,α9β1Tenascin C, osteopontin, ADAMs, factor XIII, VCAM, VEGF-C, VEGF-DEndothelial cells, keratinocytesLeukocyte adhesion integrins αD β2ICAM, VCAM Leukocytes αM β2ICAM, VCAM, iC3b, factor X, fibrinogen Leukocytes αL β2ICAM Leukocytes αX β2Fibrinogen, plasminogen, heparin, iC3b Leukocytes αE β7E-cadherin Leukocytes,Laminin -binding α3β1Laminins (collagens)Keratinocytesα6β1Laminins, ADAMs Endothelial cells, Chondrocytes α6β4Laminins Endothelial cellsα7β1LamininsDifferentiated muscle cells I -domain collagen -binding α1β1Collagens, semaphorin7A, (laminins)Endothelial cells, Chondrocytesα2β1Collagens, tenascin C, (laminins)Keratinocytes, endothelial cells, Chondrocytes, platelets α10β1Collagens Chondrocytesα11β1CollagensMesenchymal non-muscle cellsRheumatol Int[25]. With immunohistochemical methods using monoclo-nal and polyclonal antibodies, the integrin pattern in joint cartilage from rats corresponded largely to integrin expres-sion described for human cartilage tissue: β1, α1, α3, and αv subunits and the α5β1 and αvβ3 heterodimers were con-sistently expressed [26]. Moreover, an inverse correlation was demonstrated between the severity of the anatomical changes found in the zones and the phenotypic/metabolic changes in the cells. These results, together with the well-known inside–out signaling function of the adhesion mol-ecules, highlight the key role of matrix interactions in the pathogenesis of the anatomic changes in OA cartilages [22, 27]. Expression of integrins on chondrocytes is correlated with the degree of cartilage damage in human OA [22].All of the α5β1, αVβ3, and αVβ5 contain the RGD-binding domain; α6β1 and α3β1 contain the laminin-binding domain, while α1β1, α2β1, and α10β1 contain the I-domain collagen-binding motif. The α5β1 integrin serves as the primary chondrocyte fibronectin (FN) receptor [28], while αV-containing integrins bind to vitronectin [29] and osteopontin (OPN) [30], and may serve as alternative FN receptors. αVβ3 integrin binds to tenascin C [31]. All of the α1β1, α2β1, and α10β1 integrins can serve as receptors for collagens [32–34], and α6β1 and α3β1 integrins could bind to certain cartilage extracellular matrix proteins such as laminin [35–37]. The aforementioned integrins and their corresponding ligands all played the important roles in OA pathologic changes.Important roles of integrins in OA cartilageIntegrins mediate cells adhesionThe cartilage surface defect is a common change in OA. The initial adhesion of transplanted chondrocytes to sur-rounding host cartilage may be important in the repair of articular defects [38]. Adhesion may set position for cells to secrete molecules that fill the defect and integrate repair tissue with host tissue, while chondrocytes are known to become increasingly adherent to cartilage with time. It is well known that Annexins (mainly A5), CD44, and integ-rins are the important molecules involved in chondrocyte adhesion with ECM.In vitro experiments, under the conditions in which chondrocytes were cultured in high-density monolayer, released with trypsin, and allowed to recover in suspen-sion for 2 h at 37 °C, β1-integrins appear to mediate chon-drocyte adhesion to a cut cartilage surface. Delineation of the mechanisms of adhesion may have clinical impli-cations by allowing cell manipulations or matrix treat-ments to enhance chondrocyte adhesion and retention at a defect site [39]. Under the culture and seeding conditions in high-density or low-density monolayer, β1, α5β1, and αVβ5 integrins mediate human chondrocyte adhesion to cartilage [19]. These chondrocyte integrins have a potential role in the initial adhesion and retention of chondrocytes at a cartilage defect site following clinical procedures of chondrocyte transplantation [19]. In an experiment about the chondrocytes attached to hyaline or calcified carti-lage and bone, freshly isolated (primary) or passaged (P1) chondrocytes were seeded on the top of bone plugs having either a surface composed of mid-deep zone hyaline carti-lage or calcified cartilage or bone only. Both primary and passaged chondrocytes attached efficiently to all of the three surfaces (over 88 % of seeded cells). The chondro-cytes showed a punctate distribution of β1-integrin and vin-culin, which are colocalized with actin, suggesting that the cells formed focal adhesions. Blocking either β1-integrin or αVβ5 integrin partially inhibited (between 27–48 and 26–37 %, respectively) attachment of both primary and pas-saged chondrocytes to all surfaces. Blocking αVβ3 had no effect on adhesion [38].Besides cell adhesion, integrins also mediate chondro-cytes adhesion to their extracellular ligands. Cell adhesion assays revealed that both α1β1 and α2β1 can serve as chon-drocyte adhesion receptors for types II and VI collagen. In cell lines expressing both integrins, α1β1 was the preferen-tial receptor for type VI collagen, while α2β1 was the pref-erential receptor for type II collagen [23]. Thus, α1β1 and α2β1 integrins play the roles to mediate chondrocyte adhe-sion to types II and VI collagen, respectively [23]. α1β1 also mediates chondrocyte adhesion to type VI collagen [40]. Integrins also mediate attachment of chondrocytes to fibronectin and matrix Gla protein (MGP) [41].Integrins in chondrocytes mechanotransductionIn OA, mechanical forces play an important role in tissue homeostasis and remodeling [42]. Chondrocytes are poten-tially exposed to a variety of different mechanical forces including stretch, shear, or compressive forces in vivo [42]. Matrix synthesis and chondrocyte proliferation are up-regulated by the physiological levels of mechanical forces [43]. It is well know that integrins as mechanoreceptors regulate the cellular response to both changes in the ECM and mechanical stresses that chondrocytes are subjected to [44–46]. Integrin activity is important in the early cel-lular responses to mechanical stimulation, regulating the activation of a number of intracellular cascades that induce changes in gene expression and tissue remodeling. In nor-mal human articular chondrocytes, integrin activation, con-sequent to mechanical stimulation in vitro, results in tyros-ine phosphorylation of regulatory proteins and subsequent secretion of autocrine and paracrine acting soluble media-tors including substance P and interleukin 4 [47]. NormalRheumatol Intchondrocytes in monolayer exposed to 0.33-Hz mechanical stimulation for 20 min resulted in increased GAG synthesis that was blocked by the presence of antibodies to α5 and αVβ5 integrins and CD47. These studies suggested that αVβ5 integrin plays a role in the regulation of chondrocyte responses to biomechanical stimulation [48]. In vitro stud-ies showed that the primary monolayer cultures of human chondrocytes have an electrophysiological response after intermittent pressure-induced strain characterized by a membrane hyperpolarization of approximately 40 %. The cultured chondrocyte’s hyperpolarization was found to be inhibited by RGD peptides and antibodies to the α5 and β1 integrin subunits [49], and the hyperpolarization response was associated with opening of small conductance (SK) calcium-dependent K+ channels via α5β1 integrin stretch activated ion channels and a number of integrin-associated signaling molecules including the involvement of the actin cytoskeleton and tyrosine phosphorylation [50]. Thus, α5β1 is an important chondrocyte mechanoreceptor and a potential regulator of chondrocyte function [49]. Integrin α1β1 is a key participant in chondrocyte transduction of a hypo-osmotic stress. Furthermore, integrin α1β1 influences osmotransduction is independent of matrix binding, but likely dependent on the chondrocyte osmosensor transient receptor potential vanilloid-4 [51].Treatment of chondrocytes with interleukin-1 (IL-1) resulted in diminished synthesis and enhanced catabolism of matrix proteoglycans [52]. Within chondrocytes, expo-sure of interleukin-1β (IL-1β) induces the release of nitric oxide (NO) and prostaglandin E2 (PGE2) via activation of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) enzymes, independently of integrins [53, 54]. This effect can be reversed by integrin with the applica-tion of dynamic compression three-dimension agarose con-structs. Mechanical loading and IL-1β influence the release of NO and PGE2 from articular chondrocytes. The integ-rin-binding peptide, GRGDSP, abolishes or reverses the compression-induced alterations in the presence or absence of IL-1β. Thus, integrins act abrogating the NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2 in the presence and absence of IL-1β in three-dimension agarose constructs [55, 56].In the mechanical stress stimuli situation, integrins regu-late responses of human articular chondrocytes to mechani-cal stimulation via several pathway or downstream com-ponents. For example, mechanical signals control SOX-9, VEGF, and c-Myc expression and cell proliferation dur-ing inflammation via integrin-linked kinase, B-Raf, and ERK1/2-dependent signaling in articular chondrocytes [43]. Integrin-associated protein (CD47/IAP) is necessary for chondrocyte mechanotransduction. Through interac-tions with α5β1 integrin and thrombospondin, CD47/IAP may modulate chondrocyte responses to mechanical signals [57]. Furthermore, ankle joint chondrocytes appeared to show significant differences in levels of the integrin-asso-ciated proteins CD98, CD147, and galectin 3, PKC gamma, and differences in responses to glutamate were seen. This might be related to modified integrin-dependent mecha-notransduction as a result of changes in the expression of integrin regulatory molecules such as CD98 or differen-tial expression and function of downstream components of the mechanotransduction pathway such as PKC or NMDA receptors [58]. RACK1-mediated translocation of activated PKCα to the cell membrane and modulation of integrin-associated signaling are likely to be important in regula-tion of downstream signaling cascades controlling chon-drocyte responses to mechanical stimuli [59]. Recently, Whitney et al. [60] found that ultrasound (US) has emerged as a technique to deliver mechanical stress, and their find-ings suggested US signals through integrin receptors to the MAPK/Erk pathway via a mechanotransduction pathway involving FAK, Src, p130Cas, and CrkII.Integrins regulate cells proliferation and differentiationCell–cell interactions play an important role in the develop-ment of cartilage. Heterologous and homologous cell–cell interactions are critical for chondrogenic differentiation during development. Chondrocyte survival and in situ dif-ferentiation are integrin-mediated [61]. Integrin β1, β5, and α5 might be involved in signal transmission for the chon-drocyte survival and dedifferentiation [62, 63]. The lack of β1 integrins on chondrocytes leads to severe chondrodys-plasia associated with high mortality rate around birth [64]. Deletion of β1 integrins in the limb bud results in multi-ple abnormalities of the knee joints; however, it neither accelerate articular cartilage destruction, perturb cartilage metabolism, nor influence intracellular mitogen-activated proteins kinase (MAPK) signaling pathways [64]. When β1 integrin gene is inactivated in the mutant mice chondro-cytes, chondrodysplasia of various severity is developed in mice. β1-deficient chondrocytes have an abnormal shape, and they are failed to arrange into columns in the growth plate [65]. This is caused by the lack of motility, which is in turn caused by a loss of adhesion to collagen type II, reduced binding to and impaired spreading on fibronectin, and an abnormal F-actin organization. In addition, mutant chondrocytes show decreased proliferation caused by a defect in G1/S transition and cytokinesis. Altogether, these findings establish that β1-integrin-dependent motility and proliferation of chondrocytes are mandatory events for endochondral bone formation to occur [65].Cell–cell interactions between articular chondrocytes and synovial fibroblasts have enhanced binding between these two cell types compared to background binding of the labeled cells to the tissue culture plastic surface andRheumatol Intchondrocytes, specifically bound to synovial fibroblasts through RGD-dependent receptors. Therefore, β1 integrins are involved in this adhesion process, and these heterolo-gous cell interactions appear to have a negative influence on chondrogenic differentiation [66]. Articular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, α5β1 integrin was found to be involved in the induction of type I and type III pro-collagen expression. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regu-late the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin (a potent disintegrin) inhibits dedifferentiation of monolayer-cultured chondrocytes [67]. In chondrocytes, during expan-sion for tissue engineering, a candidate for signal transmis-sion during dedifferentiation is integrin α5β1 in conjunction with its ligand fibronectin [68]. Other receptors, like vitron-ectin and OPN (αVβ3) or laminin (α6β1) or their ligands, do not seem to be involved in signal transmission for dedi-fferentiation. In addition, the GPIIb/IIIa receptor seems to assist the process of dedifferentiation. Intracellularly, ILK, ICAP1, and CD47 might assist the transduction of the inte-grin-dependent signals [68]. In tissue engineering research, it was confirmed that the mesenchymal stem cells (MSCs) with high chondrogenic differentiation potential are highly α10 positive and propose α10 as a potential marker to pre-dict the differentiation state of MSCs [69].The signaling cascades involved in these processes of integrin regulating cells proliferation and differentiation mainly were MAPK, and GTPases as Ras and Raf, and subsequent apoptosis in human articular chondrocytes. Ras activation stimulates the extracellular signal-regulated kinase (ERK) MAPK cascade [70]. Loss of chondrogenic potential is accompanied by reduced expression in key signaling proteins of the MAPK pathway and apoptosis [71]. Activation of the chondrogenic transcription factor Sox-9 seems to be mediated by the MAPK pathway [72]. Ras-activated Raf–MEK–ERK signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines [73]. In articular chondrocytes, the affinity of αVβ5 integrin for ligands was regulated by the small GTPase R-Ras. R-Ras was gradually activated in monolayer-cultured chondro-cytes after plating, which caused a gradual decline in the cartilage matrix gene expression through enhanced Vβ5 integrin activation and the subsequent increase in ERK signaling [74].Integrins in cartilage homeostasisOsteoarthritis-affected cartilage exhibits enhanced expres-sion of FN and OPN mRNA. Ligation of α5β1 using acti-vating mAb JBS5 (which acts as agonist similar to FN N-terminal fragment) up-regulates the inflammatory medi-ators such as NO and PGE2, as well as the cytokines, IL-6, and IL-8. In contrast, αVβ3 complex-specific function-blocking mAb (LM609), which acts as an agonist similar to OPN, attenuates the production of IL-1β, NO, and PGE2 in a dominant negative fashion by osteoarthritis-affected carti-lage and activated bovine chondrocytes. These demonstrate a cross talk in signaling mechanisms among integrins and show that integrin-mediated “outside–in” and “inside–out” signaling very likely influences cartilage homeostasis, and its deregulation may play a role in the pathogenesis of oste-oarthritis [75]. In the α1-KO mice, more severe cartilage degradation, glycosaminoglycan depletion, and synovial hyperplasia were found as compared with the wild-type (WT) mice [76]. MMP-2 and MMP-3 expressions were increased in the OA-affected areas. In cartilage from α1-KO mice, the cellularity was reduced and the frequency of apoptotic cells was increased. Therefore, deficiency in the α1 integrin subunit is associated with an earlier deregula-tion of cartilage homeostasis and an accelerated, aging-dependent development of OA [76].Integrin α1β1 plays a vital role in mediating chondrocyte responses to two contrasting factors that are critical play-ers in the onset and progression of OA—inflammatory IL-1 and anabolic TGF-β [77]. In a rat OA experimental model, an increased expression of α5 and α2 integrins was found at OA late stages, which was correlated with the changes in the ECM content, as a consequence of the increased MMPs activity. In addition, in the rat OA experimental model, the presence of α4 integrin since OA early stages was corre-lated with the loss of proteoglycans and clusters formation [78]. However, at late OA stages, the increased expression of α4 integrin in the middle and deep zones of the cartilage was also correlated with the abnormal endochondral ossi-fication of the cartilage through its interaction with OPN. Finally, these findings concluded that ECM–chondrocytes interaction through specific cell receptors is essential to maintain the cartilage homeostasis. However, as the integ-rins cell signaling is ligand-dependent, changes in the ECM contents may induce the activation of either anabolic or catabolic processes, which limits the reparative capacity of chondrocytes, favoring OA severity [78].Fibroblast growth factor (FGF) and insulin-like growth factor (IGF) have been implicated as contributing factors in cartilage homeostasis [79, 80]. FGF-18 most likely exerts anabolic effects in human articular chondrocytes by induc-ing ECM formation, chondrogenic cell differentiation, and inhibiting cell proliferation [79, 81]. The role of FGF-8 has been identified as a catabolic mediator in rat and rab-bit articular cartilage [82]. IGF-1 is a major growth factor involved in cartilage matrix synthesis and repair. IGF-1 promotes synthesis of collagen type II, proteoglycans, and other matrix components [83]. Chondrocytes from。
基底膜的分子组成与功能
细胞外基质蛋白复合体。
6
基底层（basal lamina） 细胞外基质蛋白复合体
A model of the molecular structure of a basal lamina. The basal lamina is formed by specific interactions between the proteins type IV collagen（IV型胶原）, laminin（层 粘连蛋白）, and nidogen(巢蛋白）, and the proteoglycan perlecan（基底膜蛋白聚 糖）. Transmembrane laminin receptors (integrins ) in the plasma membrane are thought 7 to organize the assembly of the basal lamina.
基底膜的分子组成与功能
浙江大学医学院
2013
1
肾小球基底膜
肾小球毛细血管壁三层结构
内皮细胞层 基底膜
上皮细胞层
2
Basal laminae (yellow) surround certain cells as in the kidney glomerulus. Note that, in the kidney glomerulus（肾小球）, both cell sheets have gaps in them, so that the basal lamina serves as the permeability barrier determining which molecules will pass into the urine from the blood.
细胞生物学50名词解释-北医本科
1.Microsome 微粒体2.N-end rule N端原则3.Signal peptide 信号肽4.SRP 信号识别颗粒5.SRP受体6.translocon转位子7.Stop-transfer anchor sequence 止步锚定序列8.Signal-anchor sequence 信号锚定序列9.Glycosylation 糖基化10.Protein disulfide isomerase 蛋白二硫异构酶11.Chaperone 分子伴侣12.KDEL/HDEL信号序列13.Vesicular transport 囊泡运输14.MTOC微管组织中心15.Cell differentiation 细胞分化16.Integrins 整合素17.Translational translocation 翻译共转位18.26S proteasomes 蛋白酶体19.Basal lamina 基膜20.Nuclear skeleton 核骨架21.Apoptotic body 凋亡小体22.Apoptosome 凋亡体23.Lipofuscin 脂褐素24.CDK 周期蛋白依赖性蛋白激酶25.细胞表面26.奢侈基因27.Motor protein马达蛋白28.亲核蛋白29.Cell determination细胞决定30.Collagen 胶原31.紧密连接（TJ）32.缝隙连接33.细胞外基质（ECM）34.线粒体的半自主性35.核孔复合体36.Receptor mediated endocytosis 受体介导的内吞作用37.Biosynthetic-secretory pathway 生物合成—分泌通路38.Check point细胞周期检验点39.Dolichol 多萜醇40.Targeting sequence 导肽41.踏车运动42.Synchronization of cells 细胞同步化43.Centre cycle 中心体周期44.Sequential induction 依次诱导45.Anchorage dependent growth 锚定性依赖生长46.Anokis 失巢凋亡现象47.Cell recognition 细胞识别48.Cell communication 细胞通讯49.Cell adhesion 细胞粘附50.Adhesion plaque 黏着斑----------下面是答案，祝大家明天考试顺利1.Microsome 微粒体破碎细胞膜形成的近球形囊泡状结构。
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Cell culture
Mouse pancreatic carcinoma cells(Pan02, also known as Panc02) murine endothelial cells (bEnd.3) mouse embryonic fibroblasts (MEFs)
Tumor growth in vivo
Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and can represent the cause for the reduction in angiogenesis and tumor growth. These led us to the discovery that, in vitro, Fbln5 regulates ROS production in both fibroblasts and endothelial cells through a mechanism that is dependent on β1 integrin binding to fibronectin.
Pan02 cells were injected subcutaneously or orthotopically into WT and Fbln5–/– mice. Only single-cell suspensions with greater than 90% viability (as determined by trypan blue exclusion) were used for injections. A successful subcapsular intrapancreatic injection of tumor cells was identified by the appearance of a fluid bleb without intraperitoneal leakage.
TUNEL
即原位缺口末端标记技术，又称脱氧核糖核苷酸末端转移酶介 导的缺口末端法，用来检测细胞凋亡。 正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3’-OH 形成，很少能够被染色。而细胞凋亡中, 染色体DNA双链断裂或 单链断裂而产生大量的粘性3’-OH末端，可在脱氧核糖核苷酸末 端转移酶（TdT）的作用下，将脱氧核糖核苷酸和荧光素、过 氧化物酶、碱性磷酸酶或生物素形成的衍生物标记到DNA的3’末端，从而可进行凋亡细胞的检测。 可对完整的单个凋亡细胞核或凋亡小体进行原位染色，能准确 地反应细胞凋亡典型的生物化学和形态特征。可用于石蜡包埋 组织切片。冰冻组织切片、培养的细胞和从组织中分离的细胞 的细胞形态测定，并可检测出极少量的凋亡细胞
Western blot
又称蛋白质印迹 ，采用的是聚丙烯酰胺凝胶电泳，被检测物是 蛋白质，“探针”是抗体，“显色”用标记的二抗。其基本原理 是通过特异性抗体对凝胶电泳处理过的细胞或生物组织样品进行 着色。通过分析着色的位置和着色深度判断特定蛋白质的表达情 况，该技术广泛应用于检测蛋白水平的表达。
经过PAGE分离的蛋白质样品，转移到固相载体（例如硝酸纤维 素膜NC膜）上，固相载体以非共价键形式吸附蛋白质，且能保 持电泳分离的多肽类型及其生物学活性不变。以固相载体上的蛋 白质或多肽作为抗原，与对应的抗体起免疫反应，再与酶或同位 素标记的第二抗体起反应，经过底物显色或放射自显影以检测电 泳分离的特异性目的蛋白的表达。
Mice that are deficient in Fbln5 have fragmented and disorganized elastic fibers leading to loose skin, vascular anomalies and alveolar defects.
Recent studies have suggested that Fbln5 can regulate angiogenesis. The exact mechanism by which Fbln5 exerts its effect on angiogenesis is unknown. Given these results and the aberrant vessel formation in Fbln5-null animals, we sought to determine whether tumor growth is altered in Fbln5–/– mice owing to effects on angiogenesis.
Therefore, we are interested in determining the effect of the matricellular protein Fbln5 on tumor growth.
Fbln5 is a member of the fibulin family of proteins and consists of six calcium-binding epidermal growth factor-like repeats ( cbEGF ), one of which contains an integrin-binding RGD motif and a COOH-terminal fibulin-type module. Fbln5 protein is secreted into the ECM by fibroblasts, vascular smooth muscle cells and endothelial cells, and associates with cells through integrins αVβ3, α4β1 and α5β1
siRNA knockdown of Fbln5
gene knockdown即基因抑制，是一种抑制基因的方法。
Loss of fibulin-5 binding to β1 integrins inhibits tumor growth by increasing the level of ROS
Contents
Introduction Materials and Methods
Results
Discussion
We report the first results from tumor studies performed in Fbln5–/– mice,which showed the decrease of the growth of an implanted murine pancreatic cancer cell line and the reduce of blood vessel density in the tumor microenvironment. And we found an increase in the level of reactive oxygen species(ROS) in tumors grown in Fbln5–/– animals.
Immunohistological analysis(免疫组化分析)
免疫组化，是应用免疫学基本原理——抗原抗体反应，即抗原 与抗体特异性结合的原理，通过化学反应使标记抗体的显色剂 （荧光素、酶、金属离子、同位素）显色来确定组织细胞内抗原 （多肽和蛋白质），对其进行定位、定性及定量的研究。 抗体和抗原之间的结合具有高度的特异性，免疫组织化学正是 利用了这一原理。先将组织或细胞中的某种化学物质提取出来， 以此作为抗原或半抗原，通过免疫动物后获得特异性的抗体，再 以此抗体去探测组织或细胞中的同类的抗原物质。由于抗原与抗 体的复合物是无色的，因此还必须借助于组织化学的方法将抗原 抗体结合的部位显示出来，以其达到对组织或细胞中的未知抗原 进行定性，定位或定量的研究。
Introduction
The tumor microenvironment contains many components that influence tumor survival and progression. The acellular component of the tumor microenvironment, the ECM, consists of a three-dimensional network of structural proteins such as fibronectin and collagen that function as an architectural scaffold for cells, and provides the context for cell function and tissue development. The organization of, and the interaction between, these components within the tumor microenvironment are crucial determinants of tumor survival.
Materials and Methods
Study subject
Immunohistological analysis Quantification of immunohistochemistry TUNEL Western blot
Gene knockdown
Mice
Fbln5–/– mice and WT littermates aged 4-6 months old were used for tumor studies. Fbln5RGE/RGE mice (this kind of mice harbor a point mutation in the integrin-binding RGD motif of Fbln5 that it is deficient in the ability to bind to integrins . ).