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.

关键词： 微小RNA   椎间盘退变   基因诊断   基因治疗  

摘要： 目的总结微小RNA(micro RNA)在椎间盘退变中的研究进展,分析其在椎间盘退变中的研究方向和临床应用潜能。方法广泛查阅国内外有关micro RNA在椎间盘退变中作用的相关文献并进行综述。结果micro RNA是通过对基因表达进行调控,从而影响椎间盘细胞的增殖与凋亡、增加退变椎间盘组织的炎性介质和蛋白酶等方式在椎间盘退变中发挥重要作用。结论 micro RNA是椎间盘退变领域的研究新热点,对micro RNA的研究将有助于明确椎间盘退变的发病机制,同时为椎间盘退变疾病的诊断与治疗提供了新思路。

关键词： periodontal ligament   extracellular matrix   growth factors   cell differentiation   cell proliferation   BMP-2  

摘要： PLAP-1 is an extracellular matrix protein that is predominantly expressed in the periodontal ligament within periodontal tissue. It was previously revealed that PLAP-1 negatively regulates bone morphogenetic protein 2 and transforming growth factor activity through direct interactions. However, the interaction between PLAP-1 and other growth factors has not been defined. Here, we revealed that PLAP-1 positively regulates the activity of fibroblast growth factor 2 (FGF-2), a critical growth factor in tissue homeostasis and repair. In this study, we isolated mouse embryonic fibroblasts (MEFs) from Plap-1(-/-) mice generated in our laboratory. Interestingly, Plap-1(-/-) MEFs exhibited enhanced responses to bone morphogenetic protein 2 but defective responses to FGF-2, and Plap-1 transfection into Plap-1(-/-) MEFs rescued these defective responses. In addition, binding assays revealed that PLAP-1 promotes FGF-2-FGF receptor 1 (FGFR1) complex formation by direct binding to FGF-2. Immunocytochemistry analyses revealed colocalization of PLAP-1 and FGF-2 in wild-type MEFs and reduced colocalization of FGF-2 and FGFR1 in Plap-1(-/-) MEFs compared with wild-type MEFs. Taken together, PLAP-1 positively regulates FGF-2 activity through a direct interaction. Extracellular matrix-growth factor interactions have considerable effects;thus, this approach may be useful in several regenerative medicine applications.

关键词： Research Article  

摘要： Background Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-beta 1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications. Methods and Findings Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1 beta, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-beta 1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold;p = 0.01) and metastatic properties (3-fold;p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold;p = 0.001). Assessment of asporin expression and patient outcome (n = 60;10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87;95% CI 0.78-0.96;p = 0.0001). Survival analysis, based on gene expression (n = 375;25-y follow-up), confirmed that low asporin levels are associated with a