关键词： 椎间盘退变   circRNA   基因治疗   水凝胶微球   细胞外基质代谢  

摘要： 背景:腰痛严重影响患者的生活质量和工作能力,并给社会带来了沉重的经济负担。研究表明,椎间盘退变引起的相关疾病是导致腰痛的最常见原因。目前对于椎间盘退变性疾病治疗方案包括:休息、理疗、药物治疗、椎间盘射频消融等介入治疗以及各种手术治疗。尽管这些治疗方法均取得了不错的短期疗效,但长期仍受高概率复发性疼痛的影响。近年来,基于生物分子、细胞和组织工程的生物疗法,因其在恢复椎间盘结构方面的巨大潜力而成为研究的焦点。但研究人员开发新的疗法时,普遍忽视了椎间盘营养供应不足的影响。这些促进细胞增殖和细胞外基质合成代谢活动的椎间盘修复策略,进一步加剧了退变椎间盘对营养的需求,从而降低了椎间盘细胞的生存能力。因此,有必要开发一种聚焦于椎间盘营养受限的修复策略,从而为治疗椎间盘退变提供新思路。在生物治疗领域中,相较于蛋白质、多肽和生长因子等传统疗法,基因治疗有望通过一次注射的方法,从根源上长期修复椎间盘退变。环状RNA(circular RNA,circ RNA)是一类新兴的RNA家族成员,通过影响转录或转录后水平的基因表达在调控细胞功能(例如增殖,分化,侵袭,迁移和代谢)方面起着关键作用。circ RNA具有种类丰富、结构稳定、序列保守以及细胞或组织特异性等特点,使其在开发椎间盘退变修复策略方面比其他RNA更具优势。然而,单纯的基因药物易被核酸酶降解和转染效率低而难以进入细胞发挥其功效,必须借助适宜的基因载体实现其有效递送。阳离子脂质体能够包装DNA或RNA并吸附在细胞膜上而被广泛用作基因载体。但是,直接注射到靶器官会导致基因载体迅速分散和丢失而难以靶向局部病灶。并且,基于载体的核酸生物学表达是瞬时性的,并不总是适用于需要进行长期持续再生修复的研究。此外,基因药物的不可控性表达,可能产生过量的细胞外基质导致病理性纤维化。因此,急需开发一种高效、持续和可控的基因递送系统来局部靶向修复椎间盘退变。先进生物材料引导的基因载体传递允许以时空精准的方式控制和传递基因药物,是一种新兴的、极具吸引力的靶向修复治疗方案。基于微流控技术制备的单分散、粒径均一、携载生物活性物的水凝胶微球,可以微创注射的方式减少组织损伤并实现精准治疗。另外,水凝胶微球可以保护和缓释封装在其内部的生物活性物,并以较低的局部药物浓度和施用频率,就可以提供安全和有效的治疗。因此,本研究拟从营养受限诱导椎间盘退变的病理机制出发,根据微阵列芯片筛选出的可靠circ RNA靶点,结合先进的可注射水凝胶微球技术,期望通过化学接枝的方式构建一种理想的局部靶向递送系统,从而实现退变椎间盘的长期修复。目的:1.筛选和鉴定营养受限诱导椎间盘退变过程中的关键circ RNA;2.评估circ RNA沉默基因工程化的可注射水凝胶微球的相关性能;3.评估circ RNA沉默基因工程化的可注射水凝胶微球在体内外修复椎间盘退变的能力。方法:第一部分:葡萄糖剥夺培养髓核细胞模拟椎间盘营养受限微环境。葡萄糖剥夺培养髓核细胞0 h、6 h、12 h和24 h后,收集样本,利用circ RNA芯片检测每个时间点髓核细胞中环状RNA表达量。STEM软件对时间序列进行聚类分析,筛选随着培养时间连续性变化的差异表达circ RNAs。功能富集分析用于预测连续性上调和下调circ RNAs的潜在功能。选择连续性上调最显著的5个circ RNAs和下调最显著的5个circ RNAs构建circ RNA-mi RNA调控网络,筛选关键的circ RNA。q RT-PCR检测关键circ RNA在退变髓核组织和葡萄糖剥夺培养的髓核细胞中的表达情况。Sanger测序和RNase R耐受实验用于验证关键circ RNA的结构。Western Blot实验检测沉默关键circ RNA后,葡萄糖剥夺培养的髓核细胞外基质成分的表达情况。第二部分:薄膜分散法制备1,2-二油酰-3-三甲基氨基丙烷/胆固醇/1,2-二油酰-sn-甘油-3-磷酸乙醇胺(1,2-dioleoyl-3-trimethylammonium-propane/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,DOTAP/Chol/DOPE)阳离子脂质体。根据DOTAP的氮原子比上p DNA的磷酸盐配制不同N/P比的基因-脂质体复合物(lipoplexes)。动态光散射(Dynamic light scattering,DLS)和透射电子显微镜(Transmission electron microscopy,TEM)观察阳离子脂质体和不同N/P比的lipoplexes的尺寸、Zeta电位以及形貌。凝胶电泳阻滞实验检测阳离子脂质体聚合p DNA的能力。流式细胞仪检测GFP阳性细胞百分率,CCK-8实验和q RT-PC

关键词： Degenerative hip diseases   Gene expression analysis   Asporin   Adiponectin   HIF 1 alpha   Oxidative stress  

摘要： Objective: Osteonecrosis of the femoral head (ONFH) is a devastating disease of the hip joint. Its early diagnosis is crucial to increase the chances of joint preserving, yet difficult due to similarities with osteoarthritis (OA) of the hip in its clinical appearance. The purpose of this study was to enhance the understanding of ONFH and its pathologic processes in contrast to OA and to identify serum biomarkers helping to improve the diagnosis of the disease. Design: Bone and bone marrow samples were collected from 24 patients diagnosed with OA and 25 patients with ONFH during total hip replacement surgery. RNA was isolated, histological examination, determination of free reactive oxygen species as well as gene expression and biomarker analysis were performed. Results: Histological analysis revealed differences in the structural and cellular pattern between the groups. Gene expression analysis revealed a significant upregulation for the genes ASPN, COL1A1, COL2A1 and IL6 and a significant downregulation for HIF1A in ONFH compared to OA group. Analysis of serum biomarkers showed significant differences between the groups for asporin and adiponectin. A final logistical regression model including the parameters adiponectin, asporin and HIF 1 alpha was overall significant, explained 34.5 % of variance and classified 74.5 % of the cases correctly. Conclusion: The combination of adiponectin, asporin and HIF 1 alpha as serum biomarkers revealed a classification accuracy of 74.5 %. The information provided in this study may help to enhance the understanding of pathologic processes in ONFH and to elaborate further aspects of prediction and treatment. (C) 2021 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

关键词： asporin   biochemical analysis   internal derangement   temporomandibular joint disorders   Wilkes classification  

摘要： Background Understanding the pathogenesis of temporomandibular joint disorder (TMD) is important for diagnosis and treatment planning. Thus, biochemical analysis is usually used for the detection of tissue damage. Objective In this study, we aimed to investigate the serum asporin levels in patients with TMD. Methods Our study was planned to be performed on 43 healthy individuals (control group) without any joint problems and 43 patients with temporomandibular joint internal derangement (TMJ-ID;patients group) according to the Wilkes classification (stages 3, 4 and 5). Serum asporin levels were determined by the enzyme-linked immunosorbent assay (ELISA) method and compared between groups. Asporin levels were analysed according to the demographic and clinical characteristics of the patients, and the differences between them were demonstrated. Results Asporin levels were found to be significantly increased in the patients group compared to control group (p = .0303). The age and gender distributions of the samples in the control and patients groups were homogeneous, and there was no statistically significant difference between the groups. In addition, while there was no significant change in asporin levels in females in the patients group compared with the control group, the asporin levels were significantly increased in males in the patients group (p = .0403). Conclusions Consequently, asporin seems to be an important biomarker in the pathobiology of TMJ-ID as it is significantly upregulated in these patients.

关键词： 胶质母细胞瘤   牙周韧带相关蛋白-1   替莫唑胺   患者预后  

摘要： 目的观察胶质母细胞瘤(GBM)组织中牙周韧带相关蛋白-1(Asporin)的表达变化,并分析其与患者替莫唑胺(TMZ)耐药、预后的关系。方法选取GBM组织40例份,正常脑组织10例份,采用Western blotting法和免疫组化法检测各脑组织Asporin,根据Asporin的表达情况将GBM标本分为高表达者和低表达者,分析Asporin表达与患者对TMZ耐药性、预后的关系。结果 GBM组织、正常脑组织Asporin相对表达量分别为1.90±1.20、0.34±0.20,免疫组化染色得分分别为(8.01±4.38)、(2.00±1.15)分,两者比较,P均<0.05。接受MGMT启动子甲基化检测的17例GBM患者中,12例MGMT启动子未甲基化(对TMZ耐药),5例MGMT启动子甲基化(对TMZ敏感),12例对TMZ耐药者、5例对TMZ敏感者瘤组织Asporin相对表达量分别为3.07±0.61、1.04±0.18,两者比较,P<0.05;接受MGMT启动子甲基化检测的17例GBM患者根据瘤组织免疫组化染色评分结果分为Asporin高表达者(13例)、Asporin低表达者(4例),Asporin高、低表达者中对TMZ耐药者分别为10、2例,两者比较,P<0.05。30例GBM患者根据瘤组织Asporin染色评分结果分为高表达者(27例)、低表达者(13例),Asporin高、低表达者中位生存期分别为(12.0±0.7)、(23.0±3.9)个月,两者比较,P<0.05。结论 GBM组织Asporin的表达升高,Asporin表达水平越高,对TMZ耐药者越多,患者预后越差。

摘要： Asporin (ASPN) presents in the tumor microenvironment and exhibits a cancer-promoting effect as a stroma protein. Even though ASPN has already been observed inside cancer cells, the functions of intracellular ASPN and its underlying mechanisms remain unknown. Here we reported that ASPN was upregulated in different stages of gastric cancer (GC), and associated with a poor prognosis. Moreover, we found that ASPN markedly inhibited GC cell apoptosis and promoted cell growth in vitro and in vivo. Further mechanism investigations revealed that ASPN directly binding to lymphoid enhancer-binding factor 1 (LEF1) and promoted LEF1-mediated gene transcription independent of beta-catenin, the classic co-factor in the Wnt/LEF1 pathway. We also demonstrated that ASPN selectively facilitated LEF1 binding to and activating the promoters of PTGS2, IL6, and WISP1 to promote their transcription. The suppression of cell apoptosis by ASPN overexpression could be attenuated by LEF1 knockdown or 100 mu M aspirin (PTGS2 inhibitor), and siASPN mediated apoptosis could be rescued by LEF1 ectopic expression or adding recombinant IL6. Therefore, we concluded that ASPN repressed GC cell apoptosis via activating LEF1-mediated gene transcription independent of beta-catenin, which could serve as a potential prognostic biomarker in GC patients.

关键词： ASPN   fibroblast   fibrosis   intercellular mechanocommunication   keloid  

摘要： Keloids are fibrotic lesions that grow unceasingly and invasively and are driven by local mechanical stimuli. Unlike other fibrotic diseases and normal wound healing, keloids exhibit little transformation of dermal fibroblasts into alpha-SMA(+) myofibroblasts. This study showed that asporin is the most strongly expressed gene in keloids and its gene-ontology terms relate strongly to ECM metabolism/organization. Experiments with human dermal cells (HDFs) showed that asporin overexpression/treatment abrogated the HDF ability to adopt a perpendicular orientation when subjected to stretching tension. It also induced calcification of the surrounding 3D collagen matrix. Asporin overexpression/treatment also prevented the HDFs from remodeling the surrounding 3D collagen matrix, leading to a disorganized network of thick, wavy collagen fibers that resembled keloid collagen architecture. This in turn impaired the ability of the HDFs to contract the collagen matrix. Asporin treatment also made the fibroblasts impervious to the fibrous collagen contraction of alpha-SMA(+) myofibroblasts, which normally activates fibroblasts. Thus, by calcifying collagen, asporin prevents fibroblasts from linearly rearranging the surrounding collagen;this reduces both their mechanosensitivity and mechanosignaling to each other through the collagen network. This blocks fibroblast activation and differentiation into the mature myofibroblasts that efficiently remodel the extracellular matrix. Consequently, the fibroblasts remain immature, highly proliferative, and continue laying down abundant extracellular matrix, causing keloid growth and invasion. Notably, dermal injection of asporin-overexpressing HDFs into murine wounds recapitulated keloid collagen histopathological characteristics. Thus, disrupted interfibroblast mechanocommunication may promote keloid progression. Asporin may be a new diagnostic biomarker and therapeutic target for keloids.

关键词： 椎间盘退变   基因治疗   病毒   基因编辑  

摘要： 椎间盘是一个低氧并且营养供应较为困难的组织,其独特的解剖和生理特性是进行基因治疗最主要的挑战之一。现阶段一些基因治疗在退变椎间盘中的应用已初见成效,而且不断有新技术的出现也让椎间盘退变的基因治疗再一次步入了全新的起点,本文依据近期发表的文献,将椎间盘退变的基因治疗进展综述,为将来的临床应用提供理论基础。

关键词： asporin   growth   differentiation factor-5   osteoarthritis   polymorphism  

摘要： Aim: Osteoarthritis (OA) is the most common chronic joint disorder, resulting from the breakdown of joint cartilage. It occurs in the knees, hands, and hips, leading to pain, stiffness, inflammation, and swelling. Methods: In this study, 100 hand and knee OA patients, meeting the American College of Rheumatology criteria were included in the case group, and 100 healthy individuals were allocated to the control group. Blood samples were collected from the participants. After DNA extraction, genotyping was carried out for GDF5 rs143383 C/T polymorphism by allele-specific polymerase chain reaction (ASPCR) and for D-repeat alleles of asporin (ASPN) by conventional PCR assay. Results: The results showed that the frequency of the D14 allele of ASPN was significantly higher than other alleles in the case group (P = .0001). Also, the frequency of the D14 allele among women was significantly higher than in men (P = .004). Moreover, the frequency of the TT allele in GDF5 rs143383 C/T polymorphism was significantly higher than the CC and CT alleles in the case group, compared with the control group (P = .001). A significant difference was found between the TT allele and other alleles in female and male patients compared with the control group (P = .02). Conclusions: The D14 allele of the ASPN gene and TT allele of the GDF5 gene (rs143383 + 104T/C) are associated with hand and knee OA in the Kurdish population, indicating that these alleles could be risk factors for OA, at least in our populations.

关键词： 椎间盘退变   基因治疗   锌指蛋白A20   NF-κB通路   炎症反应   兔  

摘要： 目的探讨锌指蛋白A20对兔腰椎间盘退变的影响。方法取3月龄新西兰大白兔26只,体质量2.0~2.5 kg,经腹细针穿刺法制备L3、4、L4、5、L5、6椎间盘退变模型,其中24只术后4周MRI检查明确造模成功,随机分为4组(n=6),于目标椎间盘中分别注射锌指蛋白A20过表达腺病毒(过表达A20组)、空载体腺病毒(空载体组)、PBS液(对照组)、锌指蛋白A20干扰腺病毒(干扰A20组)。于注射前1 d及注射后1、2、3、6 d行生物反应综合评分;注射后2、4、8周,各组行MRI检查并测量T2弛豫时间(T2信号值)后,取材行阿利辛蓝染色观察椎间盘髓核细胞退变情况,免疫组织化学染色检测锌指蛋白A20以及椎间盘退变相关指标(Ⅱ型胶原、蛋白聚糖)的表达,Western blot检测锌指蛋白A20、NF-κB结合蛋白[P65、磷酸化P65(phosphate P65,P-P65)、Ⅱ型胶原、蛋白聚糖]、自噬相关蛋白[LC3(LC3Ⅱ/LC3Ⅰ)、P62]以及炎症因子(TNF-α、IL-1β)的表达。结果各组注射后各时间点生物反应综合评分均明显低于注射前1 d(P<0.05);注射后6 d干扰A20组评分明显低于其他组(P<0.05),其他组间比较差异均无统计学意义(P>0.05)。MRI检测提示,注射后2、4、8周过表达A20组T2信号值均最高(P<0.05),2、4周时干扰A20组最低(P<0.05),其余组间差异均无统计学意义(P>0.05)。阿利辛蓝染色显示,注射后4周过表达A20组蛋白聚糖含量最高(P<0.05)、干扰A20组最低(P<0.05);8周时过表达A20组蛋白聚糖含量显著高于其他组(P<0.05),其他组间比较差异无统计学意义(P>0.05)。免疫组织化学染色示,锌指蛋白A20、Ⅱ型胶原、蛋白聚糖表达过表达A20组最高(P<0.05),干扰A20组上述蛋白表达最低(P<0.05)。Western blot检测示锌指蛋白A20、蛋白聚糖、Ⅱ型胶原、LC3(LC3Ⅱ/LC3Ⅰ)蛋白相对表达量过表达A20组最高、干扰A20组最低,而P-P65、TNF-α、IL-1β、P62蛋白相对表达量过表达A20组最低、干扰A20组最高,与其他组比较差异均有统计学意义(P<0.05);各组P65蛋白相对表达量差异均无统计学意义(P>0.05)。结论锌指蛋白A20能通过抑制炎症反应,有效延缓兔腰椎间盘退变的进程。

关键词： asporin   gastric cancer   HIF1 alpha   oxidative stress   ROS  

摘要： Asporin (ASPN), a small leucine-rich proteoglycan expressed predominantly by cancer associated fibroblasts (CAFs), plays a pivotal role in tumor progression. ASPN is also expressed by some cancer cells, but its biological significance is unclear. Here, we investigated the effects of ASPN expression in gastric cancer cells. Overexpression of ASPN in 2 gastric cancer cell lines, HSC-43 and 44As3, led to increased migration and invasion capacity, accompanied by induction of CD44 expression and activation of Rac1 and MMP9. ASPN expression increased resistance of HSC-43 cells to oxidative stress by reducing the amount of mitochondrial reactive oxygen species. ASPN induced expression of the transcription factor HIF1 alpha and upregulated lactate dehydrogenase A (LDHA) and PDH-E1 alpha, suggesting that ASPN reprograms HSC-43 cells to undergo anaerobic glycolysis and suppresses ROS generation in mitochondria, which has been observed in another cell line HSC-44PE. By contrast, 44As3 cells expressed high levels of HIF1 alpha in response to oxidant stress and escaped apoptosis regardless of ASPN expression. Examination of xenografts in the gastric wall of ASPN(-/-) mice revealed that growth of HSC-43 tumors with increased micro blood vessel density was significantly accelerated by ASPN;however, ASPN increased the invasion depth of both HSC-43 and 44As3 tumors. These results suggest that ASPN has 2 distinct effects on cancer cells: HIF1 alpha-mediated resistance to oxidative stress via reprogramming of glucose metabolism, and activation of CD44-Rac1 and MMP9 to promote cell migration and invasion. Therefore, ASPN may be a new therapeutic target in tumor fibroblasts and cancer cells in some gastric carcinomas.