关键词： 椎间盘退变   慢病毒   Fas基因   Sox9基因   基因治疗  

摘要： 目的:通过慢病毒介导Fas-si RNA和Sox9体内转染兔退变髓核细胞,观察单基因转染与双基因联合转染对退变椎间盘的生物学效应。研究方法:将健康成年新西兰大白兔随机分为5组:***组,B.空病毒组,***-si RNA组,***9组,E.双基因组;针刺椎间盘建立新西兰大白兔退变椎间盘模型,使用微量注射器分别向5组实验兔椎间盘内注入20μL的PBS磷酸盐缓冲液、慢病毒、r LV-h Fas-sh RNA、r LV-h Sox9、r LV-h Fas-sh RNA+r LV-h Sox9;转染后第8周、12周、24周分别对实验动物进行MRI扫描观察其椎间盘信号变化。然后处死实验动物,取出退变椎间盘,应用Western-Blot技术检测椎间盘内II型胶原、蛋白多糖水平,应用RT-PCR技术检测Fasm RNA和Sox9 m RNA表达水平。结果:转染后第8周、12周、24周对实验动物行MRI扫描,观察椎间盘MRI T2加权像信号强度,结果显示:A、B组实验动物退变椎间盘的MRI T2加权像信号较C、D、E组显著降低,差异有统计学意义(P<0.05),A、B两组退变椎间盘MRI T2加权像信号强度相差不大,差异无统计学意义(P>0.05),E组退变椎间盘的MRI T2加权像信号强度高于C、D组,差异有统计学意义(P<0.05);处死实验动物,取出退变椎间盘后,应用Western-Blot技术检测各组椎间盘II型胶原水平,结果显示:C、D、E组椎间盘II型胶原水平与A、B组相比均显著提高,差异有统计学意义(P<0.05),C、D组椎间盘II型胶原水平相差不大,差异无统计学意义(P>0.05),E组椎间盘II型胶原水平均高于C、D组,差异有统计学意义(P<0.05);应用Western-Blot技术检测各组椎间盘蛋白多糖水平,结果显示:C、E组分别与D、A、B组两两比较,椎间盘内蛋白多糖水平均明显升高,差异均有统计学意义(P<0.05);A、B、D组椎间盘内蛋白多糖水平相差不大,差异无统计学意义(P>0.05);E组椎间盘内蛋白多糖水平较C组明显升高,差异有统计学意义(P<0.05);应用RT-PCR技术检测各组椎间盘Fas m RNA和Sox9 m RNA的表达情况,结果显示:C、E组与A、B、D组比较椎间盘Fas m RNA表达水平明显降低,差异有统计学意义(P<0.05),A、B、D组椎间盘Fas m RNA表达水平相差不多,差异无统计学意义(P>0.05),E组椎间盘Fas m RNA的表达水平低于C组,差异有统计学意义(P<0.05);C、D、E组椎间盘Sox9 m RNA的表达水平明显高于A、B组,差异有统计学意义(P<0.05),A、B组椎间盘Sox9 m RNA表达水平相差不多,差异无统计学意义(P>0.05),D组椎间盘Sox9 m RNA的表达水平高于C组,差异有统计学意义(P<0.05),E组椎间盘Sox9 m RNA的表达水平高于D组,差异有统计学意义(P<0.05)。结论:慢病毒介导Fas-si RNA和Sox9体内转染兔退变椎间盘可以抑制细胞外基质中II型胶原、蛋白多糖的降解,减少髓核细胞凋亡,增加细胞外基质中II型胶原、蛋白多糖含量,从而延缓椎间盘的退变。双基因联合转染较单基因转染延缓椎间盘退变的效果更好,双基因联合转染对延缓椎间盘退变具有协同作用。

关键词： 胰腺癌导管腺癌   细胞间质蛋白   肿瘤增殖   PD-L1受体   PD-1受体   组织表达   临床预后  

摘要： 本文从以下几个方面进行论述：　　第一部分 细胞间质蛋白Asporin对胰腺导管腺癌增殖和凋亡的影响　　目的：　　胰腺癌是消化系统恶性程度最高的肿瘤之一，而且由于其在体内位置隐蔽，不易觉察，大多数病人诊断时已经处于中晚期;目前，传统的治疗手段虽然不断改进，但是对胰腺癌的治疗效果并未取得明显的效果。因此深入研究胰腺癌的发生发展机制，寻找新的治疗方法成为目前非常迫切和必要的问题。本文通过研究胰腺癌微环境中细胞间质蛋白Asporin对肿瘤细胞增殖和凋亡生物行为的影响，从而寻找治疗胰腺癌的治疗途径。　　方法：　　1.细胞间质蛋白Asporin在胰腺导管腺癌组织、细胞中的表达情况　　选择胰腺导管腺癌组织、胰腺导管腺癌细胞系（PANC-1、MIA PaCa-2和AsPC-1，BxPC-3）分别进行免疫组化和免疫荧光探讨细胞间质蛋白Asporin在胰腺导管腺癌组织和细胞中定位，再用Westblot方法探讨细胞间质蛋白Asporin在细胞系PANC-2、MIA PaCa-2、AsPC-1和BxPC-3中的表达量。　　2.胰腺星形细胞来源的细胞间质蛋白Asporin与胰腺导管腺癌细胞中表达的细胞间质蛋白Asporin之间的相互作用　　收集活化星形细胞的上清处理胰腺癌细胞，利用Westblot检测胰腺癌细胞中细胞间质蛋白Asporin的表达情况;收集PANC-1和MIA PaCa-2细胞系上清处理胰腺星形细胞，利用Westblot检测星形细胞中细胞间质蛋白Asporin的表达情况。　　3.通过体外实验和体内实验探讨细胞间质蛋白Asporin对胰腺导管腺癌增殖和凋亡的影响。　　通过CCK-8实验和平板克隆实验探讨细胞间质蛋白Asporin对胰腺导管腺癌增殖的影响;通过流式细胞技术、TUNEL实验和Hoechst实验探讨细胞间质蛋白Asporin对胰腺导管腺癌凋亡的影响;通过稳转技术得到细胞间质蛋白敲低的稳转细胞系，然后通过原位种植在体内验证细胞间质蛋白 Asporin对胰腺癌细胞增殖和凋亡的影响。　　4.探讨细胞间质蛋白Asporin对胰腺癌细胞对增殖和凋亡的影响的机制　　利用Westblot检测NFκ B/REGγ信号通路相关蛋白在敲低细胞、过表达细胞和对照细胞的表达情况。　　结果：　　1.通过免疫组化发现细胞间质蛋白既可以表达于胰腺导管腺癌的癌细胞中，又可以表达于胰腺导管腺癌的细胞间质中;通过Westblot发现在胰腺导管腺癌细胞系PANC-1和MIA PaCa-2中高表达细胞间质蛋白Asporin，在AsPC-1和BxPC-3中低表达，通过免疫荧光发现无论在胰腺癌细胞中还是星形细胞中，细胞间质蛋白都表达于细胞质中。　　2.星形细胞来源的Asporin能够促进胰腺导管细胞分泌细胞间质蛋白Asporin;而胰腺导管腺癌来源的细胞间质蛋白Asporin能够促进胰腺星形细胞分泌细胞间质蛋白Asporin。　　3.通过CCK8和平板克隆实验证明细胞间质蛋白Asporin促进胰腺导管腺癌的增殖;通过流式细胞实验、TUNEL实验和Hoechst实验证明细胞间质蛋白抑制胰腺导管细胞腺癌凋亡，通过动物实验证明细胞间质蛋白Asporin抑制胰腺导管腺癌细胞凋亡，促进增殖。　　4.细胞间质蛋白Asporin可能通过NF-κ B/REGγ信号通路调节胰腺导管腺癌的增殖和凋亡生物学行为。　　结论：　　细胞间质蛋白Asporin能够促进胰腺导管腺癌的增殖，抑制凋亡等生物行为，因此它有可能成为胰腺导管腺癌治疗的一个分子靶点。　　第二部分 PD-1/PD-L1在胰腺导管腺癌表达状况及对预后的影响　　目的：　　在许多实性肿瘤中，PD-L1(Programmed death ligand1)被确认为一个有效的治疗靶点。在胰腺导管腺癌中，PD-L1对预后的影响已经被大量研究证实。本研究目的是通过应用体外检测方法检测PD-L1在中国人中的表达情况，从而进一步研究PD-1(programmed cell death protein)作为一个治疗靶点的潜在应用价值。　　方法：　　本研究回顾性收集了373例胰腺导管腺癌的癌组织及癌旁组织的石蜡标本。通过组织芯片仪做成芯片，然后通过免疫组化评估PD-L1和PD-1的表达情况，并进一步评估PD-L1和PD-1表达于患者临床特征的关系。　　结果：　　在373例胰腺导管腺癌中，PD-L1阳性共12例，阳性率为3.2％，PD-1阳性共27例，阳性率为7.5％。同时我们还发现PD-L1主要表达与肿瘤细胞的细胞膜上，而在正常组织未观察到阳性病例。通过统计学分析发现，PD-L1表达与淋巴结转移相关。通过单因素分析发现PD-L1阳性表达与无病生存期和总生存期相关，可以作为患者的预后指标，而在多因素分析中，仅无病生存期与患者预后相关。PD-1表达于淋巴细胞，但

关键词： Osteoarthritis   Asporin   Cartilage intermediate layer protein   Genetic association   Functional variant  

摘要： Background: Osteoarthritis (OA) is the most common degenerative joint disease afflicting people in the Western world and has a strong genetic influence. The aim of this study was to examine the association of two known functional polymorphisms in the TGF-beta inhibiting genes, asporin (ASPN) and cartilage intermediate layer protein (CILP), with hand OA and potential gene-occupational hand loading interaction. Results: Statistically significant interaction of the CILP rs2073711 T and ASPN D15 alleles with hand OA was observed (OR = 2.48, 95% CI 1.27-4.85, p = 0.008) in a Finnish hand. OA cohort of 543 women (aged. 45-63). When stratified by variation in working tasks, low variation of working tasks increased the risk further (OR = 3.00, 95% CI 1.35-6.66, p = 0. 007). Based on the analysis of ASPN and CILP protein-coding regions, functional studies were performed with one observed variant, rs41278695 in the ASPN 'gene. Analyses showed that bone morphogenetic protein 2 (BMP2) mediated expression of aggrecan (Agc1) and type II collagen (Col2a1) was significantly suppressed (p = 0.011 and p = 0.023, respectively) in a murine chondrocytic cell line (ATDC5) with cells stably expressing ASPN rs41278695. Conclusions: The carriage of either ASPN D15 or CILP rs2073711 TT is associated, with increased risk of symmetrical hand. OA, particularly in individuals with low variation in work tasks. ASPN rs41278695 SNP had an effect on Agc1 and. Col2a1 gene expression when induced with BMP-2 suggesting an effect on the cartilage extracellular matrix composition.

关键词： Asporin   Endochondral growth   Growth plate   Mandibular condylar cartilage   TGF-β  

摘要： Mandibular condylar cartilage (MCC) exhibits dual roles both articular cartilage and growth center. Of many growth factors, TGF-β has been implicated in the growth of articular cartilage including MCC. Recently, Asporin, decoy to TGF-β, was discovered and it blocks TGF-β signaling. Asporin is expressed in a variety of tissues including osteoarthritic articular cartilage, though there was no report of Asporin expression in MCC. In the present study, we investigated the temporal and spatial expression of Asporin in MCC. Gene expression profile of MCC and epiphyseal cartilage in tibia of 5 weeks old ICR mice were firstly compared with microarray analysis using the laser capture microdissected samples. Variance of gene expression was further confirmed by real-time RT-PCR and immunohistochemical staining at 1,3,10, and 20 weeks old. TGF-β and its signaling molecule, phosphorylated Smad-2/3 (p-Smad2/3), were also examined by immunohistochemical staining. Microarray analysis revealed that Asporin was highly expressed in MCC. Real-time RT-PCR analysis confirmed that the fibrous layer of MCC exhibited stable higher Asporin expression at any time points as compared to epiphyseal cartilage. This was also observed in immunohistochemical staining. Deeper layer in MCC augmented Asporin expression with age. Whereas, TGF-β was stably highly observed in the layer. The fibrous layer of MCC exhibited weak staining of p-Smad2/3, though the proliferating layer of MCC was strongly stained as compared to epiphyseal cartilage of tibia at early time point. Consistent with the increase of Asporin expression in the deeper layer of MCC, the intensity of p-Smad-2/3 staining was decreased with age. In conclusion, we discovered that Asporin was stably expressed at the fibrous layer of MCC, which makes it possible to manage both articular cartilage and growth center at the same time. © 2017 The Authors

关键词： asporin gene   association study   Mexican Mestizo population   osteoarthritis of the knee  

摘要： BackgroundAsporin is a novel extracellular matrix protein (ECM) with an important role in the development of osteoarthritis (OA), because it has been reported that functional polymorphisms in the aspartic acid repeat (D) of the asporin gene (ASPN) are associated with susceptibility to OA. AimThis study was planned to investigate the association of the ASPN polymorphism with primary OA of the knee in a Mexican population, including several countryside regions. MethodsWe conducted a case-control study in which 93 cases with primary OA of the knee and 118 controls were included. Cases included patients > 40 years of age, with a body mass index (BMI) 27 and a radiologic score for OA of the knee of 2. Controls were subjects > 40 years of age with a radiologic score of < 2. The D repeat polymorphism was genotyped and logistic regression was developed to evaluate risk magnitude. ResultsThe D14 allele was more common in our cases and was associated with an increased risk for developing OA, while the frequencies of the remaining alleles did not exhibit differences. ConclusionOur data suggest that the D14 allele of the ASPN polymorphism could exert an influence on primary OA of the knee etiology in a Mexican Mestizo population.

关键词： Aspartic acid   Osteoarthritis   Knee   Gender   Ethnicity   Meta-analysis   Polymorphism  

摘要： Background: Knee osteoarthritis (KOA) is the most prevalent form of knee joint disease and characterized by the progressive degeneration of articular cartilage. Although pathology of KOA remains unknown, genetic factors are considered to be the major cause. Asporin is a group of biologically active components of extracellular matrix (ECM) in articular cartilage, and asporin gene (ASPN) D-repeat polymorphism was reported to be associated with KOA. Thus, our meta-analysis is aimed at investigation of the association between asporin D-repeat polymorphism and susceptibility of KOA. Methods: We gathered data from MEDLINE, Embase, OVID, and ScienceDirect to search relevant published epidemiological studies through April 2017. Compared with previous studies, our meta-analysis is the first study to investigate the association of ASPN D15, D16, and D17 alleles and KOA susceptibility by ethnic- and sex-stratified subgroup analysis. Results: We found no significant association between D15 allele and susceptibility to KOA (OR = 1.05, 95% CI 0.95-1.17) in overall population. The same results were observed in the analysis of D16 (OR = 1.01, 95% CI 0.80-1.28) and D17 alleles (OR = 1.28, 95% CI 0.91-1.80). The ethnic- and sex-subgroup analyses did not alter the ORs. However, significant association was detected in the sensitivity analysis of D17 in overall population (OR = 1.05, 95% CI 0.95-1.17) and Asian population (OR = 1.78, 95% CI 1.02-3.11, P < 0.05). Conclusion: Our results indicated that D-repeat polymorphism of ASPN may not play a major role in susceptibility of KOA in ethnic- and sex-specific analysis. Because of the limitations of the present meta-analysis, firm conclusions could not be drawn based on the current evidence, and further studies are required to detect genuine role of ASPN.

关键词： Asporin   Nlrp3 inflammasome   CAECs   ROS  

摘要： OBJECTIVE Recent studies have demonstrated that the Nlrp3 inflammasome serve as a central role in the pathogenesis of cardiovascular diseases and endothelial dysfunction occurs in association with several cardiovascular risk *** the demonstrated anti-inflammatory effects of aspirin,the present study was designed to test whether aspirin diminish NLRP3 inflammasome activation and prevent endothelium injury and associated coronary artery damage during *** Mouse carotid arterial endothelial cells(CAECs)were cultured and treated with 0.1-3 mmol·L^(-1) of aspirin in response to LPS(2μg·mL^(-1))*** 24 h,the Nlrp3 inflammasome complexes consist of varied proteins were analyzed by *** and T-AOC in the supernatant was detected by *** reactive oxygen species(ROS)generation for 24 h was observed by DCF *** mice were treated with aspirin(12.5 mg·kg^(-1) per day,62.5 mg·kg^(-1) per day,125 mg·kg^(-1) per day)and dexamethasone(0.0182 mg·kg^(-1)per day)for 7 *** level of IL^(-1)β,IL^(-1)8 protein was detected by *** Immunofluorescence results showed the colocalization of Nlrp3 with ASC or caspase 1decrease in a concentration-dependent ***,the expression of Nlrp3 and caspase 1protein was decreased with the concentration of aspirin,but no changes the expression of ASC ***3 protein levels in CAECs were 0.33-0.8-fold and cle-caspase 1 protein levels in CAECs were 0.48-1-fold compared to those in LPS stimulation when treated with 0.1-3 mmol·L^(-1) aspirin for 24 h(P<0.01).Aspirin significantly antagonized the effect of LPS on NO(1.22-1.91-fold that of LPS stimulation,P<0.01)and T-AOC expression(1.02-1.90-fold that of LPS stimulation,P<0.01).As the different concentration of aspirin treated,the generation of ROS was 0.51-1.10-fold that of LPS stimulation(P<0.01).In vivo data shown the level of IL^(-1)β,IL^(-1)8 protein from serum are in concordance with the level of Nlrp3 inflammasome activation.

关键词： Amino acids   Anatomy   Animal models   Basic amino acids   Biochemistry   Biology and life sciences   Carbohydrates   Cell biology   Cellular structures and organelles   Chemical compounds   Chemistry   Collagens   Dermis   Disaccharides   Experimental organism systems   Extracellular matrix   Extracellular matrix proteins   Glycobiology   Integumentary system   Lysine   Medicine and health sciences   Model organisms   Mouse models   Organic chemistry   Organic compounds   Physical sciences   Proteins   Proteoglycans   Research and analysis methods   Research Article   Skin  

摘要： The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs' effects on collagen and extracellular matrix in vivo have been elucidated;one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn(-/-) mouse model and the investigation of the Aspn(-/-) skin phenotype. Functionally, Aspn(-/-) mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn(-/-) mice (not statistically significant). Several matrix genes were upregulated, including collagens (Col1a1, Col1a2, Col3a1), matrix metalloproteinases (Mmp2, Mmp3) and lysyl oxidases (Lox, Loxl2), while lysyl hydroxylase (Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn(-/-) skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn(-/-) skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties.

关键词： Asporin   Pancreatic cancer   Invasion   Migration   EMT  

摘要： Pancreatic cancer is histopathologically characterized by excessive desmoplasia induced by pancreatic stellate cells (PSCs). Asporin, an extracellular matrix (ECM) protein, is highly expressed in cancer associated fibroblasts (CAFs). Asporin expression in PSCs and its roles in PSC-pancreatic cancer cell (PCC) interaction remain unclear. The present study firstly showed that Asporin is highly expressed in activated PSCs and is involved in PSC-mediated invasion and migration of PCCs. Exogenous Asporin interacted with the transmembrane receptor CD44 on PCCs to activate NF-kappa B/p65 and promoted the epithelial mesenchymal transition (EMT) in PCCs. Furthermore, AKT and ERK pathways participated in Asporin/CD44-induced NF-kappa B/p65 activation in pancreatic cancer. Asporin had similar effects on PCCs via an autocrine mechanism. Consistent with our in vitro experiments, we showed that Asporin in peritumoral stroma of pancreatic cancer tissues was associated with poor clinical outcome. In conclusion, this is the first study to show that Asporin promotes EMT, invasion, and migration of PCCs by activating CD44-AIT/ERK-NF-kappa B pathway in paracrine and autocrine manners. Moreover, our results indicate that Asporin may be a prognostic marker and suggest that targeting the tumor microenvironment represents a promising therapeutic strategy in pancreatic cancer. (C) 2017 Elsevier B.V. All rights reserved.

关键词： 椎间盘   转化生长因子β1   细胞外基质   信号传导  

摘要： 目的探讨asporin在椎间盘基质合成和分泌中的分子调控机理，明确其在椎间盘退行性病变中的作用。方法取因严重椎间盘突出症（有典型临床症状、体征及Pfirrmann’s Ⅲ级以上）入院行椎间融合术患者的椎间盘组织标本共8例，其中男6例，女2例，年龄11-28岁，平均（20．25±3．37）岁。利用酶消化法提取椎间盘组织中的原代细胞，在藻酸盐珠中悬浮培养2周后播种于培养皿中。细胞予以不同浓度TGF-β1刺激6、12、18和24h，提取细胞RNA后行实时荧光定量PCR，检测asporin的mRNA表达；细胞经siRNA沉默内源性asporin表达后，以TGF—β1刺激细胞24h，提取细胞RNA行实时荧光定量PCR，检测asporin、Ⅱ型胶原蛋白和蛋白聚糖的mRNA表达；细胞经特异性p38抑制剂或ERK抑制剂处理12h后，以TGF-β1刺激细胞24h，提取细胞蛋白质后行蛋白印迹实验，检测asporin蛋白水平。结果在原代椎间盘细胞实验中，TGF-β1诱导下asporin表达水平显著增高，TGF—β1呈现剂量与时间正依赖关系，刺激24h后不同浓度（5，10，15ng／m1）之间有显著差异，分别为2．754±0．24、3．651±0．319和4．583±0．38（F=24．782，P=0．001）。经敲除内源性asporin表达后，TGF-β1诱导合成的基质蛋白，即蛋白聚糖（t=7．387，P=0.002）和Ⅱ型胶原蛋白（t=4．443，P=0．011）显著增加。利用特异性p38抑制剂阻断p38磷酸化后，TGF-β1对asporin的诱导作用明显地被抑制。结论在椎间盘细胞中asporin与TGF-β1呈现功能反馈环：TGF—β1上调asporin表达，而asporin通过负反馈抑制TGF-β1信号传导通路，进而抑制TGF-β介导的细胞外基质的合成；在椎间盘细胞中TGF-β1通过p38途径上调asporin表达。