关键词： Asporin   PDAC   侵袭   迁移   EMT  

摘要： 背景:胰腺癌是恶性程度最高的人类肿瘤之一,被称之为“癌中之王”。由于胰腺癌早期诊断困难,现有的治疗手段有限,就目前而言,胰腺癌患者的预后依旧很差。临床上近90%的胰腺癌病理类型为胰腺导管腺癌,因此,寻找具有临床意义的肿瘤生物学标记物来预测胰腺导管腺癌的发生、发展以及设计、研发针对肿瘤标记物的靶向药物,对于改善胰腺导管腺癌患者的预后就显得非常迫切和必要。目的:1.探讨Asporin、CD44在胰腺导管腺癌组织中的表达、定位及其临床意义2.探讨Asporin/CD44生物轴对胰腺癌侵袭和迁移生物学行为的影响及机制方法:第一部分:Asporin、CD44在胰腺导管腺癌组织中的表达、定位及其临床意义收集132例胰腺导管腺癌和50例癌旁石蜡组织标本、临床、病理和预后资料,采用IHC和免疫荧光分别评估Asporin和CD44在胰腺导管腺癌组织和癌旁组织中的表达和定位,SPSS16.0统计软件对结果进行分析。第二部分:体外实验Asporin/CD44生物轴对胰腺导管腺癌侵袭和迁移生物学行为的影响及机制***在人活化和去活化PSCs中的表达和定位:通过outgrowth法、油红染色法和α-SMA免疫荧光染色法,分离、鉴定并获得人源性PSCs后,使用ARTA诱导PSCs去活化,通过基因芯片、RNA测序、RT-PCR、Western Blot、细胞免疫荧光染色检测Asporin在活化和去活化PSCs中的表达和定位。2.旁分泌Asporin对胰腺癌细胞系（Mia PaCa-2和PANC-1）侵袭、迁移能力的影响:siRNA敲低PSCs中的Asporin,观察Asporin敲低前后,PSCs上清对胰腺癌细胞系侵袭和迁移能力的影响。*** 和 CD44 在胰腺癌细胞系（AsPc-1,BxPC-3,Mia PaCa-2 和 PANC-1）中的表达及两者之间相互作用的验证:RT-PCR、Western Blot、细胞免疫荧光染色检测Asporin和CD44在胰腺癌细胞系中的表达,并利用CO-IP验证两者之间的相互作用。4.旁分泌Asporin影响胰腺癌侵袭和迁移生物学行为的机制:1)使用rhAsporin、CD44中和抗体或NF-KB转录因子抑制剂处理Mia PaCa-2和PANC-1后,检测其侵袭、迁移能力、NF-KB转录活性、EMT相关蛋白及下游信号通路AKT和ERK的变化;2)使用rhAsporin、CD44中和抗体、AKT抑制剂或ERK抑制剂处理Mia PaCa-2和PANC-1后,检测NF-KB转录活性的变化。5.自分泌Asporin对胰腺癌侵袭、迁移能力的影响极其机制:使用siRNA敲低Mia PaCa-2和PANC-1细胞中的Asporin后,观察Asporin敲低前后,两株细胞系侵袭、迁移能力、EMT相关蛋白、TIMP/MMP家族蛋白、AKT、ERK信号通路及NF-K B转录因子的变化。第三部分:体内实验Asporin对胰腺癌侵袭和迁移生物学行为的影响使用慢病毒转染获得的Asporin低表达胰腺癌细胞株和对照细胞株,构建胰腺癌裸鼠原位肿瘤模型,观察两种细胞株在裸鼠胰腺内的侵袭和迁移情况。结果:第一部分:Asporin、CD44在胰腺导管腺癌组织中的表达、定位及其临床意义Asporin主要在胰腺癌肿瘤上皮邻近的间质细胞胞浆中表达,阳性率为81.06%（107/132）,也可在部分胰腺癌肿瘤上皮的胞浆中表达,阳性表达率为24.2%（32/132）,在正常胰腺组织导管上皮和间质细胞中不表达或弱阳性表达。CD44主要在胰腺肿瘤上皮细胞的胞膜上高表达,阳性率为31.06%（41/132）,也可在一小部分胰腺癌的间质细胞中高表达,在正常胰腺的间质和导管上皮中,呈弱表达或不表达。单因素和多因素分析提示:Asporin在肿瘤间质细胞中阳性表达预示患者预后不良（P=0.012）,而肿瘤细胞中阳性表达虽和分化之间存在相关性（P＜0.000）,但与患者的预后无明显相关性（P=0.772）;CD44阳性表达与患者的预后相关（P=0.004）。第二部分:体外实验Asporin/CD44生物轴对胰腺癌侵袭和迁移生物学行为的影响及机制***在活化PSCs中的表达明显高于去活化PSCs;2.活化PSCs上清对胰腺癌细胞侵袭和迁移的诱导作用,在Asporin敲低后明显减弱;***和CD44之间存在物理上的相互作用;***/CD44生物轴可通过激活AKT和ERK信号通路来促进Mia PaCa-2和PANC-1细胞内NF-κB的核蓄积,NF-κB的核蓄积进一步通过诱导EMT的发生来促进Mia PaCa-2和PANC-1细胞的侵袭和迁移;***敲低后,Mia PaCa-2和PANC-1细胞的侵袭和迁移能力明显

关键词： aggrecan   asporin   collagen II alpha 1   human intervertebral annulus cells   TGF-beta 1  

摘要： Background/Objective: Asporin is associated with osteoarthritis and lumbar disk degeneration. Previous studies in chondrocytes showed that asporin can bind to transforming growth factor-beta 1 (TGF-beta 1) and downregulate matrix biosynthesis. However, this has not been studied in intervertebral disk (IVD) cells. This study aimed to inspect the expression of asporin under TGF-beta 1 stimulation and its effect on TGF-beta 1-induced matrix biosynthesis in human intervertebral annulus cells. Methods: Human intervertebral annulus cells were obtained from the pathological region of IVD in eight patients. After primary culture and redifferentiation in alginate beads, cells were reseeded and treated with different concentrations (5 ng/mL, 10 ng/mL, and 15 ng/mL) of TGF-beta 1 for up to 24 hours. Total RNA extracted from the cells and those with asporin knockdown were subjected to real-time polymerase chain reaction analysis to examine the expression of asporin and extracellular matrix genes. Results: TGF-beta 1 stimulation induces asporin transcription significantly in a dose-and time-dependent manner. Knockdown of endogenous asporin leads to the upregulated expression of collagen II alpha 1 and aggrecan. Conclusion: Our results have verified a functional feedback loop between TGF-beta 1 and asporin in human intervertebral annulus cells indicating that TGF-beta 1-induced annulus matrix biosynthesis can be significantly upregulated by knockdown of asporin. Therefore, asporin could be a potential new therapeutic target and inhibition of asporin could be adopted to enhance the anabolic effect of TGF-beta 1 in human intervertebral annulus cells in degenerative IVD diseases. (C) 2016 The Authors. Published by Elsevier (Singapore) Pte Ltd on behalf of Chinese Speaking Orthopaedic Society.

关键词： Asporin   Dystrophin   Laminin   Lumican   Myosin light chain kinase   Periostin  

摘要： Cardiomyopathy is a serious complication in Duchenne muscular dystrophy, an X-linked neuromuscular disease of childhood that is triggered by primary abnormalities in the dystrophin gene. In order to directly correlate the deficiency in the membrane cytoskeletal protein dystrophin to secondary abnormalities in the dystrophic heart, this study has used label-free mass spectrometry to compare protein expression patterns in the aged mdx-4cv heart model of dystrophinopathy versus wild type heart. This report is the first successful identification of members of the cardiac dystrophin glycoprotein complex by comparative whole tissue proteomics. The mass spectrometric analysis confirmed the loss of dystrophin and concomitant reduction of syntrophin and sarcoglycans in the dystrophin-deficient heart. Proteomic profiling of secondary changes identified distinct alterations in the basal lamina component laminin, the Ca2+-binding protein sarcalumenin, the matricellular protein periostin, the proteoglycans asporin and lumican, the cardiac-specific myosin light chain kinase, heat shock proteins and a large number of mitochondria] and glycolytic enzymes. The proteomic findings indicate that the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy is highly complex and involves impairments, modulations and/or adaptations of mitochondria] metabolism, glycolysis, protein chaperoning and ion homeostasis, as well as the maintenance of the contractile apparatus, the intracellular cytoskeleton and the extracellular matrisome. Significance: The X-linked inherited disorder Duchenne muscular dystrophy is the most frequently inherited neuromuscular disease of childhood. Primary abnormalities in the dystrophin gene trigger progressive skeletal muscle wasting and impaired cardiorespiratory functions. In order to improve our general understanding of the molecular pathogenesis of muscular dystrophy-associated cardiomyopathy and to identify new marker candidates of cardiac chang

关键词： 椎间盘退变   基因治疗   载体  

摘要： 椎间盘退变是成人下腰痛的主要原因之一,保守治疗和手术治疗常常不能取得满意的疗效。椎间盘退变分子病理机制涉及多种分子水平上的改变,相关的生物治疗研究亦越来越受到重视。目前生物治疗的材料包括细胞因子、基因以及细胞,分别对应细胞因子治疗、基因治疗以及组织工程。其中基因治疗又分为直接基因治疗和间接基因治疗。本综术主要围绕基因治疗靶点和各种基因载体来探讨基因治疗在椎间盘退变相关研究中的应用进展。

关键词： lentivirus   intervertebral disc degeneration   aggrecan   type. collagen  

摘要： The present study examined the effects of transforming growth factor (TGF) -beta 3, connective tissue growth factor (CTGF) and tissue inhibitor of metalloproteinase 1 (TIMP1) gene transduction, using a lentiviral vector, on rabbit intervertebral disc degeneration in vivo, with the intention of investigating their potential use in gene therapy. A model of lumbar intervertebral disc degeneration was created by needle puncture into the annulus fibrosus of 15 New Zealand white rabbits. Empty lentivirus or recombinant lentiviral plasmid lenti-TGF beta 3-P2A-CTGF-T2A-TIMP1 was injected into degenerative lumbar intervertebral discs (representing the control and experimental groups, respectively), whilst untreated degenerative lumbar intervertebral discs served as the puncture group. After 16 and 20 weeks, magnetic resonance imaging (MRI) was conducted and the changes in intensity on micrographs of degenerative intervertebral discs were measured. The mRNA levels of aggrecan and type II collagen in nucleus pulposus tissue were determined by reverse transcription-polymerase chain reaction, and protein expression levels of type II collagen and aggrecan were determined by western blot analysis. MRI results indicated that intervertebral disc degeneration was ameliorated in the experimental group when compared with the control and the puncture group. Furthermore, the expression levels of type II collagen and aggrecan in the puncture and control groups were significantly lower than in the experimental group (P<0.05). In conclusion, lenti-TGF beta 3-P2A-CTGF-T2A-TIMP1 co-transduction can promote synthesis of aggrecan and type II collagen in degenerative intervertebral discs, thereby delaying intervertebral disc degeneration. These results indicate the potential of gene therapy in treatment of intervertebral disc degeneration.

关键词： Mandibular asymmetry   Condylar cartilage   Subchondral bone   Asporin   TGF-   beta 1  

摘要： Objective: To examine the effects of imbalance of masticatory muscle activity of the rat mandible on the condylar cartilage and subchondral bone during the growth period. Design: Forty 5-week-old male Wistar rats were randomly divided into experimental (n = 20) and control (n= 20) groups. In the experimental group, the left masseter muscles were resected. The rats were sacrificed at 7 or 9 weeks of age in both groups. Microcomputed tomography was used to determine the three-dimensional morphology and cancellous bone structure. For histological and histochemical examination, 5-mu m-thick serial frontal sections of the condyle were stained with toluidine blue and immunostained with asporin and TGF-beta 1 to evaluate the promotion and inhibition of chondrogenesis. Results: In the experimental group, microcomputed tomography analysis showed asymmetric growth;the resected side condyles showed degenerative changes. Histological analysis showed that the total cartilage in the central region of the resected side was significantly thinner than in the non-resected side in the experimental group, as well as in the control group. Compared with the control group, the expression of asporin was significantly higher in the resected side, and significantly lower in the non-resected side. In contrast, the expression of TGF-beta 1-immunopositive cells in the non-resected side was significantly higher than in the resected side and the control group. Conclusions: These findings imply that lateral imbalance of masseter muscle activity lead to inhibition of chondrogenesis and induce asymmetric formation of the condyle during the growth period. (C) 2015 The Authors. Published by Elsevier Ltd.

关键词： Asporin   小分子蛋白多糖   炎症   肿瘤  

摘要： 近年来,随着对人类疾病认识的加深,细胞外基质(extracellular matrix,ECM)蛋白引起了学者们的广泛关注。Asporin是近年来在ECM中新发现的一种富含亮氨酸的小分子蛋白多糖(small leucine rich proteoglycan,SLRP)。目前很多研究表明,A sporin在人类多种疾病(炎症性疾病、退化性疾病和肿瘤)的发生、发展中发挥着重要的作用。本文就Asporin在人类疾病中的研究现状作一综述。

关键词： Periodontal ligament   Compressive force   Orthodontic tooth movement   Asporin   PLAP-1   Sclerostin  

摘要： Objective: During orthodontic tooth movement, bone resorption and inhibition of bone formation occur on the compressed side, thereby preventing ankylosis. Periodontal ligament (PDL) cells control bone metabolism and inhibition of bone formation on the compressed side by secreting bone-formation inhibitory factors such as asporin (ASPN) or sclerostin (encoded by SOST). The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. Design: In vitro, the changes in expression of ASPN and SOST and subsequent protein release in human PDL (hPDL) cells were assessed by semi-quantitative polymerase chain reaction (PCR), real-time PCR, and immunofluorescence in hPDL cells subjected to centrifugal force using a centrifuge (45, 90, 135, and 160 x g). In vivo, we applied a compressive force using the Waldo method in rats, and examined the distribution of ASPN or sclerostin by immunohistochemistry. Results: In vitro, hPDL cells subjected to 90 x g for 24 h demonstrated upregulated ASPN and downregulated SOST expressions, which were confirmed by immunofluorescent staining. In addition, the formation of mineralized tissue by human osteoblasts was significantly inhibited by the addition of medium from hPDL cells cultured during compressive force as well as the addition of equivalent amounts of ASPN peptide. In vivo, asporin-positive immunoreactive PDL cells and osteoclasts were found on the compressed side, whereas few sclerostin-positive PDL cells were observed. Conclusions: PDL cells subjected to an optimal compressive force induce the expression and release of ASPN, which inhibits bone formation during orthodontic tooth movement on the compressed side. (C) 2015 Elsevier Ltd. All rights reserved.

关键词： 慢病毒   MMP-3   椎间盘退变   RNA干扰   基因治疗  

摘要： 目的:体外筛选设计并合成的抑制效率最佳的MMP-3shRNA慢病毒载体,并观察其对人退变髓核细胞的生物学效应。方法:采用酶序贯消化法对临床腰椎间盘突出症患者需行摘除术来源的髓核组织进行分离、单层原代细胞培养,应用细胞免疫组化法、细胞免疫荧光法等方法对髓核细胞进行鉴定;根据人MMP-3基因m RNA序列设计合成4对不同的MMP-3sh RNA序列,与酶切后LV3载体链接并转入感受态细胞,通过PCR和基因测序对扩增的阳性克隆进行鉴定,对慢病毒进行包装和滴度测定;通过荧光定量PCR、Western Blot分别检测转染人退变髓核细胞后MMP-3、Ⅱ型胶原、蛋白多糖基因和蛋白质水平的变化,从而筛选最佳抑制序列慢病毒载体及其对人退变髓核细胞的生物学效应。结果:原代髓核细胞呈三角形、多角形或短梭形等不规则形状,细胞核较大,培养约3周时,原代髓核细胞汇合可达80%以上;苏木精-伊红染色细胞核呈蓝色,胞浆呈淡红色,甲苯胺蓝染色胞核成深蓝色,胞浆呈淡蓝色,细胞免疫组化及细胞免疫荧光Ⅱ型胶原分别呈黄褐色和绿色荧光。经酶切鉴定阳性扩增片段正确,基因测序显示与设计序列完全相符,滴度检测显示大于1x108TU/ml。MMP3sh RNA慢病毒转染72和96小时后从MMP-3基因和蛋白水平综合筛选得出MMP-3sh RNA3为最佳抑制序列(P<0.05)。MMP-3sh RNA3转染人退变髓核细胞5天后检测Ⅱ型胶原和蛋白多糖m RNA及蛋白水平均增加(P<0.01)。结论:成功构建并筛选出抑制效率最佳的MMP-3sh RNA慢病毒载体;慢病毒介导的RNAi可以显著抑制人退变髓核细胞MMP-3的表达,从而增加人退变髓核细胞外基质表达量,有望成为阻止或逆转椎间盘退变的进程而治疗椎间盘退变。

关键词： periodontal ligament (PDL)   extracellular matrix (ECM)   periodontitis   LPS   ECM protein   inflammatory cytokines  

摘要： Periodontal ligament-associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2-and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-kappa B activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, I.B kinase a degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2-and TLR4induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.