摘要： Disc degeneration (DD) is a multifaceted chronic process that alters the structure and function of intervertebral discs. The pathophysiology of degeneration is not completely understood, but the consensus is that changes in genes encoding extracellular matrix (ECM) proteins in the disc are the leading factors contributing to DD. Asporin is an ECM protein that has been shown to be increased in degenerated intervertebral discs, but little is known about how asporin is regulated during DD. In exploring the intricate mechanism, we confirmed that asporin was abundantly increased in patients' degenerated nucleus pulposus. Consistently, the increased asporin expression with degeneration was also proved by rabbit intervertebral disc degeneration (IDD) model. Mechanistically, IL-1 beta upregulated asporin expression by activating the p65 pathway in human nucleus pulposus cells. Furthermore, p65 mediated asporin expression by binding to -41/-31 bp on asporin promoter. Functionally, asporin was the intermediator of IL-1 beta-inhibited aggrecan and collagen. expression and played a negative role in TGF-beta- induced aggrecan and collagen. formation in human nucleus pulposus cells. Therefore, identifying asporin as a negative regulator of aggrecan and collagen. and elucidating its induction mechanisms in human nucleus pulposus cells provides new insight for asporin induction during IDD.

关键词： prostate cancer   tumour stroma   stromal markers   asporin  

摘要： Background: Prostate cancer shows considerable heterogeneity in disease progression and we propose that markers expressed in tumour stroma may be reliable predictors of aggressive tumour subtypes. Methods: We have used Kaplan-Meier, univariate and multivariate analysis to correlate the expression of Asporin (ASPN) mRNA and protein with prostate cancer progression in independent cohorts. We used immunohistochemistry and H scoring to document stromal localisation of ASPN in a tissue microarray and mouse prostate cancer model, and correlated expression with reactive stroma, defined using Masson Trichrome staining. We used cell cultures of primary prostate cancer fibroblasts treated with serum-free conditioned media from prostate cancer cell lines to examine regulation of ASPN mRNA in tumour stromal cells. Results: We observed increased expression of ASPN mRNA in a data set derived from benign vs tumour microdissected tissue, and a correlation with biochemical recurrence using Kaplan-Meier and Cox proportional hazard analysis. ASPN protein localised to tumour stroma and elevated expression of ASPN was correlated with decreased time to biochemical recurrence, in a cohort of 326 patients with a median follow up of 9.6 years. Univariate and multivariate analysis demonstrated that ASPN was correlated with progression, as were Gleason score, and clinical stage. Additionally, ASPN expression correlated with the presence of reactive stroma, suggesting that it may be a stromal marker expressed in response to the presence of tumour cells and particularly with aggressive tumour subtypes. We observed expression of ASPN in the stroma of tumours induced by p(53) inhibition in a mouse model of prostate cancer, and correlation with neuroendocrine marker expression. Finally, we demonstrated that ASPN transcript expression in normal and cancer fibroblasts was regulated by conditioned media derived from the PC3, but not LNCaP, prostate cancer cell lines. Conclusions: Our results suggest

关键词： 椎间盘退变   微小RNA   非病毒载体   基因治疗  

摘要： 目的总结椎间盘退变(intervertebral disc degeneration,IDD)中微小RNA(micro RNA,mi RNA)及其非病毒载体的研究进展,探讨非病毒载体递送mi RNA的临床应用潜能,为IDD治疗提供新思路。方法广泛查阅国内外有关mi RNA在IDD中作用及其非病毒载体相关的文献并进行总结分析。结果退变椎间盘组织中明显差异性表达的mi RNA通过对基因表达的调控参与多种病理生理过程,许多类型非病毒载体递送mi RNA的基因治疗方式在一些疾病治疗中已取得了理想效果。结论 mi RNA在IDD细胞分子机制中发挥重要作用,非病毒载体作为一种安全有效的基因治疗策略,为通过mi RNA治疗IDD提供了新的可能。

关键词： 椎间盘退变   基因治疗   BMP-2   细胞分化抑制因子1  

摘要： 目的研究兔腰椎间盘髓核内注射慢病毒携带的BMP-2和细胞分化抑制因子1(inhibitor of differentiation 1,Id1)基因能否有效改善椎间盘退变进程。方法取4月龄新西兰兔32只,体质量2.0~2.5 kg;以L3、4、L4、5、L5、6为目标椎间盘,经腹细针穿刺法制造椎间盘退变模型。将30只造模成功的实验动物随机分为5组,每组6只;造模后4周于各组目标椎间盘中分别注射Lv-BMP-2病毒(A组)、Lv-BMP-2+Lv-Id1病毒(B组)、LvId1病毒(C组)、Lv-绿色荧光蛋白空载体病毒(D组)及PBS(E组)。于注射后2、4、8周各组分别随机取2只动物行T2-mapping MRI检查获得确切的T2弛豫时间(T2值);之后处死动物取椎间盘组织,应用实时荧光定量PCR、ELISA法检测Ⅱ型胶原及蛋白聚糖的m RNA相对表达量及含量。结果 T2-mapping MRI检查示,注射后0、2周各组间T2值比较差异均无统计学意义(P>0.05);注射后4周,A、B组T2值显著高于C、D、E组(P<0.05),A、B组间比较差异无统计学意义(P>0.05);注射后8周,B组T2值显著高于其余各组(P<0.05)。实时荧光定量PCR及ELISA检测示,注射后2、4、8周B组Ⅱ型胶原及蛋白聚糖的m RNA相对表达量及含量均显著高于其余各组(P<0.05)。结论联合应用BMP-2和Id1基因在体内实验中能有效延缓兔腰椎间盘退变的进程。

关键词： Osteoarthritis   Knee   Asporin   D13 and D14 alleles   Polymorphism  

摘要： Objective: Studies have assessed the association between aspartic acid (D)-repeat polymorphism in the gene encoding Asporin (ASPN) and knee osteoarthritis (KOA) risk, but the results were inconclusive and contradictory. Therefore, we performed a meta-analysis to investigate the association between ASPN gene D-repeat polymorphism and KOA risk. Methods: Eligible studies were identified by searching several electronic databases for relevant reports published before September 2016. The pooled odds ratios (ORs) for the association between ASPN polymorphism and KOA and their corresponding 95% confidence intervals (Cls) were estimated using the random- or fixed-effect model. Results: A total of eleven case-control studies in ten publications with 4610 KOA cases and 3621 controls were included for the ASPN D-repeat polymorphism. Overall, no significant association was detected for D14 allele carrier (D14 vs. D13: OR = 1.10, 95% CI = 0.90-1.36, p = 0.32). Meta-analysis of D14 vs. other alleles and D13 vs. other alleles showed the same pattern of KOA association as the D14 vs. D13 (OR = 1.30, 95% CI = 1.00-1.70, p = 0.06;OR = 0.93, 95% CI = 0.82-1.06, p = 0.33, respectively). Also, in the stratified analysis by ethnicity, no significant association of this polymorphism with risk of KOA was found in the European and Asians populations (OR = 1.05, 95% CI = 0.91-1.21, p = 0.49;OR = 0.98, 95% CI = 0.78-1.23, p = 0.88, respectively). Conclusions: The present meta-analysis suggests that the ASPN D-repeat polymorphism is not associated with an increased KOA risk. However, future large studies with gene gene and gene environment interactions are needed to validate these findings. Level of evidence: Level III diagnostic study. (C) 2017 Turkish Association of Orthopaedics and Traumatology. Publishing services by Elsevier B.V.

关键词： 椎间盘退变   NF-κB   Asporin   IL-1β  

摘要： 椎间盘退变(Intervertebral disc degeneration,IVDD)是一系列脊柱退行性疾病的前提和基础病理过程,其病因众多,机制复杂,目前无有效的治疗措施。我们前期利用生物信息学技术分析比较了退变椎间盘与正常椎间盘的基因表达谱芯片的结果,发现椎间盘退变主要涉及炎症因子(IL-1β、TNF-α等)相关的NF-κB信号通路,且富集分析显示Asporin等基因在退变椎间盘中表达异常升高。我们使用JASPAR数据库发现Asporin启动子序列中存在2个NF-κB的结合位点,因此我们推测炎症因子(IL-1β、TNF-α等)通过NF-κB信号通路刺激Asporin表达,并促进椎间盘的退变发生。为此,本课题主要研究内容和方法如下:***基因在椎间盘髓核细胞中的表达情况用IHC、rt-PCR、Western-blot方法检测病人、动物模型中椎间盘髓核组织Asporin基因的表达变化。***基因在人髓核细胞中的调控机制(1)用IF检测IL-1β和NF-κB/P65特异阻断剂Bay11-7082刺激人髓核细胞后,NF-κB/P65信号通路的变化。(2)在NF-κB/P65信号通路被病毒沉默、质粒高表达、化学阻断的情况下,用IF、rt-PCR、Western-blot、Elisa方法检测人髓核细胞在IL-1β刺激下Asporin基因表达变化。(3)用DLR探究NF-κB信号通路对Asporin启动子活性调节机制***基因在人髓核细胞退变中的作用在Asporin基因被病毒沉默、质粒高表达的情况下,用Elisa、rt-PCR、Western-blot检测IL-1β、TGF-β1对人髓核细胞的Aggrecan、CollagenⅡ的表达影响。主要研究结果:***基因在退变椎间盘髓核细胞中表达上调***-1β通过NF-κB信号通路刺激人髓核细胞Asporin基因表达上调***通过拮抗TGF-β1的作用引起人髓核细胞退变结论:IL-1β通过NF-κB信号通路促进人髓核细胞Asporin基因表达上调,进而促进人椎间盘的退变。

关键词： 椎间盘退变   基因治疗   BMP-2   细胞分化抑制因子1  

摘要： 目的 研究兔腰椎间盘髓核内注射慢病毒携带的BMP-2和细胞分化抑制因子1(inhibitor of differentiation 1,Id1)基因能否有效改善椎间盘退变进程。方法 (1)经腹建立兔腰椎间盘细针穿刺退变模型,并在影像学和组织学上加以验证;(2)取3月龄新西兰兔32只,体质量2.0～2.5 kg;以L3/4、L4/5、L5/6为目标椎间盘,经腹细针穿刺法制造椎间盘退变模型。将30只造模成功的实验动物随机分为5组,每组6只;造模后4周于各组目标椎间盘中分别注射Lv-BMP-2病毒(A组)、Lv-BMP-2+Lv-Id1病毒(B组)、Lv-Id1病毒(C组)、Lv-绿色荧光蛋白空载体病毒(D组)及PBS(E组)。于注射后2、4、8周各组分别随机取2只动物行T2-mapping MRI检查获得确切的T2弛豫时间(T2值);之后处死动物取椎间盘组织,应用实时荧光定量PCR、ELISA法检测Ⅱ型胶原及蛋白聚糖的m RNA相对表达量及含量。结果 (1)手术操作本身对兔子的体质量无明显影响,仅一只兔子发现腹壁与肠管轻度部分粘连;术后证实出穿刺的椎间盘为目标椎间盘,其椎间盘高度指数和MRI信号强度呈进行性退变改变;穿刺后4周组织学表现为髓核区域脊索细胞显著减少而纤维细胞增生,纤维环结构紊乱。(2)T2-mapping MRI检查示,注射后0、2周各组间T2值比较差异均无统计学意义(P>0.05);注射后4周,A、B组T2值显著高于C、D、E组(P<0.05),A、B组间比较差异无统计学意义(P>0.05);注射后8周,B组T2值显著高于其余各组(P<0.05)。实时荧光定量PCR及ELISA检测示,注射后2、4、8周B组Ⅱ型胶原及蛋白聚糖的m RNA相对表达量及含量均显著高于其余各组(P<0.05)。结论 (1)经腹纤维环细针穿刺法,操作简单,目标椎间盘定位准确,手术视野更佳,椎间盘退变程度一致,并发症少;而且在影像学和组织学上符合人腰椎间盘退变情况;(2)联合应用BMP-2和Id1基因在体内实验中能有效延缓兔腰椎间盘退变的进程。

关键词： 结直肠癌   Asporin   转移   Cortactin  

摘要： 目的:检测Asporin在结直肠癌组织和癌旁组织中m RNA和蛋白的表达水平,分析其表达情况与患者临床病理资料之间的联系。进一步过表达和干扰结直肠癌细胞中的Asporin表达水平后通过一系列生物功能实验了解Asporin对结直肠癌细胞侵袭、转移能力的影响,并建立体内动物模型。最后,我们通过Western-blotting技术了解Asporin在结直肠癌中影响肿瘤转移的的作用机制。方法:应用RT-PCR、免疫组化技术技术检测200例结直肠癌患者术后标本中癌组织、癌旁组织中Asporin的表达情况,并分析标本中Asporin的表达与患者的临床指标之间的关系。应用Western-blotting技术检测Asporin在8株结直肠癌细胞HT-29、Lo Vo、Caco2、HCT116、SW1116、SW480、RKO、SW620中的表达情况,并用细胞免疫组化法验证Western-blotting的结果。构建Asporin过表达慢病毒载体,对于低表达的细胞HT-29和Lo Vo转染慢病毒后经嘌呤霉素(Puro)筛选获得稳转过表达细胞株。采用细胞划痕实验、Transwell实验、小管形成实验检测Asporin上调后HT-29和Lo Vo细胞的愈合、侵袭、迁移及小管形成能力。Western-blotting检测HT-29、Lo Vo细胞中Asporin上调后Src信号通路蛋白及VEGF蛋白的表达水平变化,同时在组织标本上检测Asporin和P-cortactin(Tyr421)的共表达情况。将HT-29/Asporin、HT-29/Vector细胞注射裸鼠皮下成瘤,取出瘤体免疫组化、Western-blotting检测VEGF的表达情况。裸鼠门静脉注射法构建肠癌肝转移动物模型,检测Asporin上调后的肠癌细胞HT-29的肝转移能力。将Asporin在RKO和SW620细胞中经sh RNA干扰后行上述系列生物功能实验和分子机制研究。结果:Asporin基因在结直肠癌组织中的m RNA和蛋白水平上的表达相比于癌旁组织明显增高(P<0.0001),Asporin的过表达与结直肠癌的TNM分期和淋巴结转移情况相关。划痕实验、Transwell实验、小管形成实验显示Asporin增加结直肠癌细胞的愈合、侵袭、迁移、小管形成能力。在裸鼠动物模型中,Asporin增加癌组织中VEGF的表达,促进结直肠癌细胞肝转移(P<0.01)。另外,我们发现Asporin可导致EGFR、Src的磷酸化,并进一步引起Cortactin Tyr421位点的磷酸化。结论:Asporin在结直肠癌组织中呈高表达,其表达水平与患者的TNM分期及淋巴转移情况相关;Asporin通过EGFR/Src/Cortactin信号通路促进结直肠癌细胞的侵袭、转移。

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

摘要： 目的:通过慢病毒介导MMP-3shRNA和Fas-siRNA双基因体外转染人退变髓核细胞,探讨单基因、双基因联合转染后对人退变髓核细胞功能的影响。方法:a.体外培养人退变髓核细胞,对其进行台盼蓝染色、甲苯胺蓝染色,观察细胞活力以及细胞形态;b.分别根据人MMP-3基因与Fas基因的mRNA序列合成不同的MMP-3 shRNA与Fas-siRNA序列,与酶切后的慢载体链接并转入感受态293T细胞,对慢病毒进行包装。c.慢病毒感染退变髓核细胞,按照处理方式不同,本实验分为5组,分别为:A-空白对照组,B-AAV阴性对照组,C-AAV-MMP-3shRNA组,D-AAV-FassiRNA组,E-AAV-FassiRNA-MMP3shRNA组;d.转染72小时后CCK8测定各组之间的细胞增殖情况;72小时通过荧光定量PCR检测转染髓核细胞mRNA水平变化(MMP-3、Fas以及Ⅱ型胶原、蛋白多糖);96小时后通过Western Blot检测髓核细胞内蛋白水平变化(Ⅱ型胶原蛋白、蛋白多糖)。结果:a.细胞形态学进行观察发现初消化的成人退变髓核细胞贴壁时间约为4-5天,初贴壁时的形态表现为椭圆形、梭形、多角形,形状不规则,胞质向外伸突并随着时间延长逐渐伸长。经10-14 d以后,90%细胞呈融合状态,形状类似漩涡状或火焰状的细胞团,胞质丰富且带有一定折光性,细胞核较大轮廓清楚,呈卵圆形核,有大约1-3个核仁。经过传代后,第一代细胞之间的粘附性较原代变差,细胞形态变为长梭形,这与之前的研究相一致原代髓核细胞经甲苯胺蓝染色后呈短梭形、多角形等不规则形状,胞质均呈蓝色,细胞核位于细胞中央或偏向一侧细胞膜,颜色较周围胞质深。b.退变髓核细胞原代培养期间存活率在97%以上,第1代培养期间存活率在92-96%之间,第二代仍能维持90%左右,以后细胞存活率逐渐下降。c.72小时后用荧光显微镜检测重组慢病毒侵染人退变髓核细胞后的GFP、BFP表达情况,结果显示各组退变髓核细胞均已达到很高的转染效果。d.与空白对照组相比较,Fas干扰组、MMP3干扰组及MMP3+Fas双基因共转组Fas、MMP-3、II型胶原、蛋白多糖的mRNA表达量增加,且双基因组效果优于单基因组,与空白对照组相比较,Fas干扰组、MMP3干扰组及MMP3+Fas双基因共转组髓核细胞的蛋白多糖和Ⅱ型胶原蛋白表达量增高,且双基因组效果优于单基因组,以上结果差异均具有统计学意义(P<0.05)。结论:1.通过基因沉默技术干扰髓核细胞内Fas、MMP-3基因的表达,可以有效抑制髓核细胞的凋亡,双基因共转染具有协同作用。2.慢病毒介导的RNAi可以显著抑制人退变髓核细胞MMP-3、Fas的表达,增加人退变髓核细胞外基质表达量,且双基因具有协同效应。