关键词： 椎间盘退变   腰背痛   再生治疗   生长因子   细胞治疗   基因治疗   病毒载体   非病毒载体   组织工程  

摘要： 背景:多因素引起的椎间盘退行性病变所导致的严重的腰背痛是致残率最高的脊柱疾病,随着医学分子生物学的发展及椎间盘退变机制研究的深入,椎间盘退变的再生疗法为恢复这种难治性疾病提够了可能。目的:文章对近年来国内外各研究者关于椎间盘退变的相关分子和各种再生疗法在椎间盘退变中应用的研究进行归纳总结。方法:使用计算机检索PubMed、Embase、Web of science 数据库和中国知网、万方、维普数据库在1995年1 月至2019 年3 月发表的关于椎间盘退变再生治疗的相关文献,其中中文的检索关键词包括“椎间盘退变、腰背痛、再生治疗、生长因子、细胞治疗、基因治疗、病毒载体、非病毒载体”,英文的关键词为“Intervertebraldisc degeneration,low back pain,regenerative therapy,growth factor,cell therapy;gene therapy,viralvector,non-viral vector”,筛选出相关文献后再进一步归纳总结。结果与结论:研究显示,近20 多种基因影响着髓核细胞的变性,其中就包括各种非编码RNA(如microRNA、lincRNA 和lncRNA)。目前,生长因子疗法、细胞疗法和基因疗法等再生研究已经取得了预期效果。非病毒载体转导的基因治疗能避免感染的风险和靶细胞突变的可能,将成为恢复或缓解椎间盘退变的强大工具,具用广阔的应用前景。然而,这些再生疗法也有一定的局限性,在其应用于临床之前,需进行大量的基础研究和临床实验,以便评估其疗效和对逆转退变的影响。

关键词： 椎间盘退变   生长因子治疗   细胞治疗   基因治疗   组织工程治疗  

摘要： 下腰痛是骨科常见的疾病之一,而椎间盘髓核退变是引起下腰痛的主要原因。目前对于椎间盘退变的治疗方式主要是保守治疗和手术治疗,然而这些治疗方法的目的是缓解症状,并非是针对椎间盘退变发生机制的病因治疗。对椎间盘髓核退变发生机制的详细了解有助于开展生物学再生修复。目前,针对髓核退变的分子机制和背景,发展出不同的髓核退变生物学再生修复的方法。文章从生长因子治疗、基因治疗、细胞治疗、组织工程化髓核等方面对髓核退变生物学再生修复进行综述。

关键词： asporin   collagen fiber   osteoclastogenesis   periodontitis   PLAP-1  

摘要： Background and ObjectivePeriodontalligament-associated protein-1 (PLAP-1) is an important regulator of osteogenic differentiation ofperiodontalligament cells and plays important role in the homeostasis of periodontal tissues. But the role of PLAP-1 in periodontitis is poorly understood. Expressions of PLAP-1 in experimental periodontitis are observed to elucidate whether PLAP-1 gets involved in the pathogenesis of periodontitis. Material and MethodsWistar rats were randomly allocated to two groups (n=6/group): Ligation group and Control group. PLAP-1 expression in experimental periodontitis was assessed by immunohistochemistry and collagen fibers in periodontal ligament were observed using picrosirius red staining. Expressions of PLAP-1 and CD68 in periodontitis were colocalized by double-labelled immunofluorescence. To further examine the relationship between PLAP-1 and osteoclastogenesis in experimental periodontitis, acute periodontal inflammatory infiltration and alveolar bone destruction were induced by administering ligated rats with 10ng/mL tumor necrosis factor alpha (TNF-;ligation+TNF- group, n=6). Alveolar bone loss was observed by micro-computed tomography (Micro-CT), and osteoclasts were identified by tartrate-resistant acid phosphatase staining (TRAP). Expressions of PLAP-1 in TNF- stimulated human periodontal ligament cells were also detected at 24 and 48hours by western blotting. ResultsPLAP-1 expression levels in periodontal ligament cells and collagen fibers were lower in the ligation group,compared with the control group. Similarly, TNF- decreased PLAP-1 expression in human periodontal ligament cells in vitro. Degradation or destruction of collagen fibers accompanied the reduced PLAP-1 expression in the periodontal ligament in the ligation group. Colocalization of PLAP-1 and CD68 revealedthe positive relationship between PLAP-1 and CD68+ infiltrating cells in periodontitis. More PLAP-1-positive inflammatory cells were found in the ligation+TNF- gro

摘要： Previous studies revealed that Asporin (ASPN) is a potential mediator in the development of various types of cancer as a secreted stroma protein, but the function of ASPN inside the cancer cells remains largely unknown. Here, we demonstrated a higher expression level of ASPN in colorectal cancer (CRC) than matched normal tissues, and 25% (2/8) CRC showed copy number variation (CNV) gain/amplification in ASPN gene. Both higher ASPN expression levels and ASPN CNV gain/amplification indicated a worse prognosis in CRC patients. ASPN can promote proliferation, migration, and invasion of CRC cells, and inhibit apoptosis by activating Akt/Erk and TGF-beta/Smad2/3 signalings. Further investigations revealed that ASPN interacts with Smad2/3, facilitates its translocation into nucleus, and up-regulates the expression of Epithelial-mesenchymal transition (EMT) related genes. Rescue assays confirmed that TGF-beta signaling is essential for the effects of ASPN on promoting CRC cell migration and invasion. In conclusion, ASPN promotes the migration and invasion of CRC cells via TGF-beta/Smad2/3 pathway and could serve as a potential prognostic biomarker in CRC patients.

关键词： 糖尿病心肌病   D-松醇   氧化应激   糖自噬   PI3K/Akt/mTOR信号通路   D-松醇   血管平滑肌细胞   Asporin   细胞外基质  

摘要： 第一部分D-松醇对STZ诱导的SAMP-8小鼠糖尿病心肌病的保护作用及机制研究背景糖尿病心肌病(DCM)是糖尿病重要并发症之一,是指不能用高血压、冠状动脉粥样硬化及其他心脏病变来解释的发生于糖尿病患者的心肌疾病。DCM被认为是引起糖尿病患者心力衰竭及死亡率升高的驱动因素。目前的研究认为DCM的病因主要包括心肌细胞代谢紊乱、心肌细胞钙转运缺陷、冠状动脉微循环障碍、心肌间质纤维化及心脏的自主神经病变等。主要表现是在代谢紊乱及微血管病变的基础上引发心肌广泛灶性坏死,出现亚临床的心功能异常,最终进展为心力衰竭、心律失常及心源性休克,重症患者甚至猝死。目前对DCM的发病机制尚未完全阐明,亦无特异性治疗药物。血糖控制不佳导致的氧化应激反应增加、自噬调节的紊乱,目前被普遍认为是DCM的发病机制之一。D-松醇是D-手性肌醇的衍生物,近年的研究发现,D-松醇能发挥胰岛素类似物的作用,调控血糖,但是它在DCM中的具体作用及机制尚未明确。因此本实验通过研究D-松醇对STZ诱导的糖尿病SAMP-8小鼠心肌氧化应激、心肌细胞凋亡、糖自噬及相关信号通路的作用,探讨D-松醇对糖尿病心肌病的作用及机制。研究目的1.研究D-松醇对SAMP-8小鼠糖尿病心肌病的作用;2.研究D-松醇对SAMP-8小鼠糖尿病心肌损伤的保护作用机制。研究方法1.糖尿病模型的建立:应用快速衰老型小鼠(SAMP-8),采用低剂量的链脲佐菌素(STZ,50mg/kg/day)连续腹腔注射5天,分别于1、2周后通过剪尾法采血测量小鼠血糖,确定建模是否成功。2.分组及灌胃治疗:20只衰老抵抗小鼠(SAMR-1)为正常对照组(SAMR-1组)。SAMP-8小鼠60只分为3组:分别为衰老对照组(SAMP-8组)(建模时腹腔注射等量STZ溶解剂,n=20);糖尿病组(DM组)(建模成功后,每日等体积双蒸水灌胃,n=20);糖尿病治疗组(DMT组)(建模成功后,D-松醇100mg/kg/day灌胃,共10周,n=20)。3.记录血糖、体重的情况:分别记录建模0周,2周,4周,8周,12周各组小鼠血糖及体重情况。4.心脏超声检查:建模后第13周,对各组小鼠麻醉后行经胸心脏超声检测,测量左室射血分数等相关指标。5.心肌氧化应激及自噬等相关指标的检测:Masson染色检测心肌胶原纤维合成情况;ELISA检测心肌组织SOD、GSH、GPx及MDA含量;TUNEL检测各组心肌细胞凋亡情况;透射电镜检测心肌超微结构及自噬体情况;PCR检测各组心肌组织BNP、ANP、α-MHC及β-MHC表达;免疫印迹法检测心肌Nrf2、bcl-2、caspase 3、caspase 9、STBD1、LC3B、Beclin-1、p62、Atg8、PI3K、Akt 及 mTOR 等蛋白表达。研究结果1.建模后第4周、8周及12周,DMT组血糖显著低于DM组;建模后第8周及12周,DMT组体重较DM组升高;2.实验结果发现,与SAMP-8组相比,DM组小鼠左室射血分数减低,心肌肥厚增加,心肌纤维排列紊乱,胶原纤维合成增多,心肌细胞凋亡增加,抗氧化因子合成减少,氧化因子合成增加,自噬体形成增多;3.与DM组相比,DMT组小鼠心脏左室射血分数增加,心肌肥厚及纤维化程度减轻,心肌细胞凋亡减少,氧化应激减轻,自噬体形成减少。PI3K/Akt/mTOR信号通路活跃度增加。结论1.D-松醇对STZ诱导的SAMP-8小鼠糖尿病心肌病具有保护作用;2.D-松醇可以通过调控血糖,改善氧化应激,抑制心肌细胞凋亡,调控糖自噬,调控PI3K/Akt/mTOR信号通路,改善糖尿病SAMP-8小鼠心肌损伤。第二部分D-松醇对晚期糖基化终末产物诱导的血管平滑肌细胞增殖和迁移的保护机制研究背景糖尿病的大血管病变是糖尿病严重的慢性并发症之一,以不断进展的动脉粥样硬化及血管重构为特征。而动脉中膜细胞外基质成分增多和平滑肌细胞增殖及其相互间的关系在糖尿病大血管病的致病过程中起着关键的作用。本实验通过研究D-松醇对晚期糖基化终末产物(AGEs)诱导的小鼠主动脉血管平滑肌细胞(VSMC)增殖和迁移的作用及内在机制,探讨D-松醇对糖尿病大血管病变的保护作用。研究目的1.研究不同浓度D-松醇对VSMC增殖及迁移的作用;2.研究D-松醇对VSMC增殖和迁移的保护作用机制。研究方法体外分离培养小鼠主动脉VSMC,应用AGEs建立血管平滑肌细胞糖基化损伤模型,观察不同浓度的D-松醇对细胞增殖和迁移的影响,应用CCK-8测定细胞增殖,细胞划痕检测细胞迁移,并测定各组细胞中TGF-β 1、p-smad2、p-smad3、Asporin蛋白的表达。研究结果***组细胞增殖、迁移、TGF-ββ1、p-smad2、p-smad3、Asporin蛋白表达水平明显高于对照组(P<0.01);

关键词： Asporin   骨关节炎   细胞外基质   TGF-β1   Smad2/3  

摘要： 一、研究背景骨关节炎(Osteoarthritis，OA)是最常见的关节疾病,主要发生在膝关节,临床表现为膝关节疼痛、肿胀、僵硬和运动功能障碍。OA一直是研究的热点,但其发病机制仍不明确,目前大量研究发现构成细胞外基质蛋白重要组成部分的SLRP家族中的asporin可能与OA的发病有关,但其具体作用机制目前还不清楚。二、研究目的本研究首先明确asporin在OA关节软骨细胞中表达情况,并进一步明确软骨细胞asporin表达对OA病理改变的影响,最后明确OA病程中,关节软骨细胞asporin与TGF-β/Smad信号通路的关系,深入探讨软骨细胞asporin在骨关节炎病程中的作用及其分子机制,为防治骨关节炎提供新的潜在靶点。三、研究方法1、收集临床上OA患者关节软骨标本和因严重外伤需要截肢的正常膝关节软骨标本通过免疫组织化学方法(IHC)检测asporin在节软骨中的表达水平;同时收集骨关节炎患者与正常人血清标本,通过酶联免疫吸附试验(ELISA)检测血清中asporin表达情况;利用C57/BL6小鼠构建DMM骨关节炎模型,通过IHC检测asporin在OA发生后4周以及8周后关节软骨中的表达情况。2、构建关节软骨体外移植OA模型,通过IHC检测asporin在小鼠关节软骨中的表达情况。3、利用软骨前体细胞进行体外实验,通过特异性siRNA序列抑制软骨细胞asporin的表达,逆转录实时荧光定量PCR(RT-qPCR)以及免疫印迹(western blot)来检测软骨细胞外基质成分的表达情况;同时利用具有生物活性的asporin重组蛋白刺激软骨前体细胞,通过RT-qPCR以及western blot检测软骨细胞外基质成分的表达情况。4、对骨关节炎造模(DMM)小鼠进行关节腔内注射载有特异性asporin-siRNA序列的慢病毒,通过免疫荧光(IF)以及IHC检测软骨细胞外基质成分的表达情况,并通过HE染色和番红O-固绿染色来评估关节软骨退变情况。5、通过TGF-β1细胞因子刺激关节软骨前体细胞,并进一步观察asporin对TGF-β1下游信号分子Smad2/3磷酸化表达水平的影响以及其对软骨细胞基质主要组成成分Aggrecan以及COLL2的合成代谢的影响。四、研究结果***在OA患者及小鼠骨关节炎关节软骨中表达较正常组明显升高,同时在OA患者血清中的表达也明显升高。2.关节软骨体外移植OA模型中,与对照组相比,OA组软骨细胞中asporin表达明显升高。3.利用siRNA序列沉默asporin基因后,软骨前体细胞中Aggrecan、COLL2表达升高,而利用asporin重组蛋白刺激软骨前体细胞后Aggrecan、COLL2表达明显下降。4.关节腔内注射慢病毒抑制软骨细胞asporin的表达后,细胞外基质Aggrecan、COLL2的合成增多,关节软骨磨损程度减轻,软骨厚度增加,小鼠关节软骨骨关节炎病理改变得到明显改善,同时关节软骨中Smad2/3磷酸化水平表达升高。***重组蛋白可明显抑制TGF-β1下游信号分子Smad2/3磷酸化水平,同时抑制软骨细胞基质Aggrecan、COLL2的合成;五、研究结论随着骨关节炎病理进展,软骨细胞asporin表达上调;抑制asporin表达可促进细胞外基质成分的合成从而延缓关节软骨的退变;asporin通过抑制TGF-β/Smad2/3信号通路抑制软骨细胞外基质中Aggrecan、COLL2的合成从而参与OA病理进展。

关键词： 腰椎间盘退变   基因治疗   基因表达  

摘要： 腰椎间盘退变造成的腰椎疾病是影响许多人的重要问题,目前此类疾病的临床治疗主要分为保守治疗与手术治疗两种方式,但是这些方法都无法影响腰椎间盘的进一步退变。近些年来,随着临床对腰椎间盘退变的研究深入,发现基因治疗在该病的临床治疗中表现出了较好的应用效果。文章主要针对腰椎间盘退变基因治疗的研究进展展开分析。

关键词： osteoarthritis   susceptibility   asporin   polymorphism   meta-analysis  

摘要： To provide an evidence-based medical basis for the treatment of osteoarthritis, a meta-analysis was performed to assess the association between asporin (ASPN) gene polymorphism and susceptibility to osteoarthritis (OA). The current study searched the literature from January 1st, 1915 through February 1st, 2017 using the Cochrane Library, PubMed, the Excerpta Medica database (EMBASE) and three main Chinese databases (VIP, CNKI and Wan Fang). Cohort and case-control studies that explored the association between different types of ASPN alleles and OA susceptibility were evaluated. The K/L grading system, clinical and radiological diagnoses were used for OA diagnosis. A random-effects model was used in a pooled analysis to adjust for heterogeneity of the included studies, and the differences between treatment groups were reported as odds ratio (OR), 95% confidence intervals (CIs) and P-values. Begg's funnel plots and Egger's tests were used to assess publication bias in the present meta-analysis. Following document retrieval and screening, a total of 10 studies were deemed eligible, including 4,842 patients and 3,661 healthy subjects. Results of the multivariate meta-regression analysis revealed that the study sample size was a source of heterogeneity between studies. The D17 allele was a risk factor for the development of OA (OR=1.33, 95% CI: 1.02-1.73, P<0.05). The other alleles were not considered as risk factors for development of OA (P>0.05). The results of the meta-analysis verified that ASPN polymorphisms were not significantly relevant to an increased OA risk. However, the mechanisms contributing to the association between ASPN polymorphisms and OA risk still require further study.

关键词： 椎间盘退变   慢病毒   MMP3基因   Sox9基因   基因治疗  

摘要： 目的:构建兔椎间盘退变的动物模型,利用慢病毒(lentivirus vector,LV)介导MMP3-siRNA和Sox9双基因体内转染兔退变髓核细胞,探讨单基因及双基因联合转染兔退变髓核细胞的生物学效应。方法:1.4月龄新西兰大白兔使用针刺纤维环法构建兔椎间盘退变实验动物模型,手术后4周、8周行核磁共振检查及病理学检测,观察目的椎间盘的退变情况,及目的椎间盘造模成功情况。2.30只建模成功的新西兰大白兔随机分为6组,分别为(1)空白对照组,(2)PBS对照组,(3)空病毒组,(4)MMP3组,(5)Sox9组,(6)双基因组(MMP3-siRNA+Sox9),分别于造模的椎间盘髓核用微量注射器注入20μL:PBS磷酸盐缓冲液、r LVX-mCMV-mcherry、rLVX-CMV-hMMP3-sh RNA-m CMVmcherry、rLVX-CMV-h Sox9-mCMV-mcherry、rLVX-CMV-hMMP3-sh RNA-m CMVmcherry+rLVX-CMV-hSox9-m CMV-mcherry。于注射后8、12、24周行MRI检查观察椎间盘退变情况。术后24周空气栓塞法处死实验动物,取出目的椎间盘组织,行Western-Blot检查II型胶原、蛋白多糖水平的表达,行RT-PCR检查MMP3mRNA和Sox9 mRNA表达水平。结果:1.手术成功后4周、8周对实验动物行腰椎MRI检查,获得其核磁共振的T2WI像。第8周手术组椎间盘的MRI的T2WI像信号开始减弱,病理学检测结果示椎间盘呈退变改变,提示造模成功。2.将目的基因转染到实验动物体内后8、12、24周行磁共振检查:空白对照组、PBS组、空病毒组退变椎间盘MRI的T2WI像信号强度明显低于MMP3、Sox9和双基因组。空白对照组、PBS组、空病毒组两两比较MRI的T2WI像信号强度比较无统计学意义(P>0.05);MMP3组、Sox9组MRI的T2WI像信号强度比较无统计学意义(P>0.05);MMP3组、Sox9组与双基因组MRI的T2WI像分别比较显示具有统计学意义(P<0.05)。术后第24周,Western-Blot检查各组II型胶原、蛋白多糖表达,其中MMP3组、Sox9组和双基因组的II型胶原表达量有不同程度提高,双基因组II型胶原的表达均高于其他各组(P<0.05)。MMP3组和双基因组蛋白多糖的表达高于其他组(P<0.05),Sox9组与PBS组、空病毒组比较,蛋白多糖表达未见明显升高(P>0.05),双基因组II型胶原的表达均高于其他各组(P<0.05);RT-PCR检查各组MMP3、Sox9 mRNA的表达情况,其中MMP3组、双基因组与PBS组、空病毒组、Sox9组比较MMP3mRNA表达水平明显降低(P<0.05),其中双基因组MMP3 mRNA的表达水平均低于其他各组(P<0.05)。Sox9组和双基因组与PBS组、空病毒组及MMP3组比较Sox9 mRNA的表达水平明显升高(P<0.05),其中双基因组Sox9 m RNA的表达水平均高于其他各组(P<0.05)。结论:1.针刺纤维环法构建兔退变椎间盘实验动物模型方法成功有效,术后8周实验动物椎间盘影像学及病理学检查均显示造模成功。2.慢病毒介导MMP3-siRNA和Sox9双基因体内转染对兔退变髓核细胞有明显的延缓椎间盘退变的作用,明显的促进了退变椎间盘细胞外基质中II型胶原,MMP3-si RNA基因的转染促进蛋白多糖的表达,Sox9基因过表达不会促进蛋白多糖的表达。

关键词： Cardioprotection   Extracellular matrix   Fibrosis   Mitochondria   Remodeling  

摘要： Loss of extracellular matrix architecture during ischemia greatly influences the post-ischemic remodeling process. Transforming Growth Factor (TGF)β is activated in the infarcted myocardium leading to stimulation of fibroblasts to promote fibrosis. Asporin is an extracellular matrix protein from small leucine rich proteoglycans (SLRP) family known to inhibit TGFβ signaling during osteoarthritis. However, little is known concerning the role of asporin in heart in the context of ischemia/reperfusion injury. Using microarray data analysis from publicly available GEO datasets, we recently identified asporin as one of the top up-regulated candidates in heart tissues from patients with ischemic cardiomyopathy compared to control donor hearts. In paired heart biopsies taken before and after cardiopulmonary bypass surgery involving cold cardioplegia, we detected an increase in asporin protein expression post-procedure. Additionally, we found that cardiac asporin protein expression is reduced in ischemic border and scar zone of mice administered permanent coronary artery ligation, where increased TGFβ signaling is known to stimulate fibrosis. However, asporin expression in remote (normal) zone remains unchanged, suggesting its importance in regulating TGFβ signaling in the injured region. We demonstrated that recombinant human asporin (1 μM) stimulates mitophagy in a time-dependent manner in differentiated H9c2 cells. Furthermore, Seahorse respirometry analysis showed that the mitochondrial spare respiratory capacity of H9c2 cells was increased after treatment with recombinant asporin for 24 hours. Based on our preliminary data, we developed a rodent asporin peptide against TGFβ interaction site and tested its efficacy in reducing infarct size in mice subjected to 30 min ischemia and 24hr reperfusion. We found that the peptide reduced infarct size calculated as a percent of area at risk (28.23 ± 4.63 compared to vehicle control 52.18 ± 9.814; n=4) when given 5 minutes before