Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma

Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA(cf DNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction(PCR) and direct sequencing. A locked nucleic acid(LNA) probe specificto the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfD NA, while not detected in postoperative plasma cfD NA. This technique will be useful for monitoring translocation-derived diseases such as SS.

Characterization of smooth muscle, enteric nerve, interstitial cells of Cajal, and fibroblast-like cells in the gastric musculature of patients with diabetes mellitus

AIM To investigate histologic abnormalities in the gastric smooth muscle of patients with diabetes mellitus(DM).METHODS Full-thickness gastric specimens were obtained from patients undergoing surgery for gastric cancer. H&E stain and Masson's Trichrome stain were performed to assess the degree of fibrosis. Immunohistochemical staining using various antibodies was also performed [antibodies against protein gene product 9.5(PGP9.5), neuronal nitric oxide synthase(n NOS), vasoactive intestinal peptide(VIP), neurokinin-1(NK1) receptor, c-Kit, and platelet-derived growth factor receptor-alpha,(PDGFRα)]. Immunofluorescent staining and evaluation with confocal microscopy were also conducted.RESULTS Twenty-six controls and 35 diabetic patients(21 shortduration patients and 14 long-duration patients) were included. There were no significant differences in basic demographics between the two groups except in mean body mass index(BMI)(higher in the DM group). Proportions of moderate-to-severe intercellular fibrosis in the muscle layer were significantly higher in the DM group than in the control group(P < 0.01). On immunohistochemical staining, c-Kit- and PDGFRα-positive immunoreactivity were significantly decreased in the DM group compared with the control group(P < 0.05). There were no statistically significant differences in PGP9.5, n NOS, VIP, and neurokinin 1 expression. On immunofluorescent staining, cellularity of interstitial cells of Cajal(ICC) was observed to decrease with increasing duration of DM.CONCLUSION Our study suggests that increased intercellular fibrosis, loss of ICC, and loss of fibroblast-like cells are found in the smooth muscle of DM patients. These abnormalities may contribute to changes in gastric motor activity in patients with DM.

Xeno-free culture of human spermatogonial stem cells supported by human embryonic stem cell-derived fibroblast-like cells

Spermatogonial stem cells （SSCs） divide continuously to support spermatogenesis throughout postnatal life and transmit genetic information to the next generation. Here, we report the successful establishment of the method for the isolation and identification of human SSCs from testicular tissue, and to determine the culture conditions required to expand SSCs on human embryonic stem cell-derived fibroblast-like cells （hdFs）. Large-scale cultures of SSCs were maintained on hdF feeder layers and expanded in the presence of a combination of cytokines and glial cell line-derived neurotrophic factor for at least 2 months. Cell surface marker analysis showed that SSCs retained high levels of alkaline phosphatase activity and stained strongly for anti-stage-specific embryonic antigen （SSEA）-1, OCT4 and CD49f. They also expressed the genes OCT4, SOX3 and STRA8 as detected by reverse transcription polymerase chain reaction （RT-PCR） analysis. These data clearly illustrate a novel approach for the growth of human SSCs using hdFs as feeder cells, potentially eliminating xenogeneic contaminants. This system provides a new opportunity for the study of the regulatory mechanism of the ‘niche＇ that governs SSC self-renewal, and will be a valuable source of SSCs for potential clinical applications.

Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami （Cypriniformes： Cyprinidae）

Though Yurman province contains some 562 known species of fish, no cell lines from any of these have been made available to date. To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami, a fish endemic to Fuxian Lake, Yunnan, China, we established and characterized the major features of a continuous cell line （AGF II） from the caudal fin tissue of A. grahami. This AGF II cell line consists of fibroblast-like cells and has been subeultured more than 60 times over the course of a year. The cell line was maintained in DMEM/F12 supplemented with 10% FBS, with a cellular doubling time of 51.1 h. We continued with more experiments to optimize the culture and storage conditions, and found a variety of interesting results： cells could grow at temperature between 24 ~C and 28 ~C, with the optimal temperature of 28 ~C. Likewise, the growth rate ofA. grahami fin cells increased when the FBS proportion increased from 5% to 20%, with the optimal growth at the concentrations of 20% FBS, cells were able to grow in L-15 and DMEM/FI2 with optimal growth at L-15; DMSO is a better eryoprotectant than Glycerol, EG and MeOH for AGFII cells with optimal concentration of 5% DMSO. Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52, with a modal peak at 48 chromosomes, accounting for 39.8% of all cells. Using the same primer pairs specific to mtDNA, the AGF II cell sequences obtained by PCR were identical to those from muscle tissues ofA. grahami. Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A. grahami, also indicating that that current long-term artificial propagation ofA. grahami has been successful. Finally, we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid, bright fluorescent signals were observed, suggesting that this cell line may be suitable for use in transfection and future gene expression studies.

Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami(Cypriniformes:Cyprinidae)

Though Yunnan province contains some 562 known species of fish,no cell lines from any of these have been made available to date.To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami,a fish endemic to Fuxian Lake,Yunnan,China,we established and characterized the major features of a continuous cell line(AGF II)from the caudal fin tissue of A.grahami.This AGF II cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year.The cell line was maintained in DMEM/F12 supplemented with 10%FBS,with a cellular doubling time of 51.1 h.We continued with more experiments to optimize the culture and storage conditions,and found a variety of interesting results:cells could grow at temperature between 24℃and 28℃,with the optimal temperature of 28℃.Likewise,the growth rate of A.grahami fin cells increased when the FBS proportion increased from 5%to 20%,with the optimal growth at the concentrations of 20%FBS;cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15;DMSO is a better cryoprotectant than Glycerol,EG and MeOH for AGFII cells with optimal concentration of 5%DMSO.Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52,with a modal peak at 48 chromosomes,accounting for 39.8%of all cells.Using the same primer pairs specific to mtDNA,the AGF II cell sequences obtained by PCR were identical to those from muscle tissues of A.grahami.Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A.grahami,also indicating that that current long-term artificial propagation of A.grahami has been successful.Finally,we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid,bright fluorescent signals were observed,suggesting that this cell line may be suitable for use in transfection and future gene expression studies。

Synovial White Cell Count in the Diagnosis of Septic Arthritis: Are Current Diagnostic Practices Appropriate?

Introduction & aims: Septic arthritis is an emergency, potentially causing irreversible joint destruction and disability. Synovial WCC and polymorphonuclear cell percentage are the best predictors of septic arthritis likelihood. Yet, synovial white cell and differential count are not routinely assessed. We aim to investigate the incidence of failure to perform these tests, and to develop correct synovial fluid analysis practices. Method: This is a retrospective analysis of native joints having undergone arthrocentesis for suspicion of septic arthritis at Box Hill Hospital (BHH) during September 2011 and September 2013 inclusive. Recruitment was from the Eastern Health Decision Support Service (DSS), a database compiled from all systems within Eastern Health, of which BHH is a member. The study was limited to large joints, including hip, knee and shoulder. All prosthetic joints were excluded from the patient population. All patient histories were examined for suspicion of septic arthritis and subsequent arthrocentesis. Pathology records were accessed to determine incidence of cell count and differential. Results: One hundred and thirty-six cases of joint aspirations were identified within the time frame, of which sixty-seven fitted our criteria for evaluation. All but two cases were delivered using the DSS, which was limited to data compiled only until June 2013. The two remaining cases were identified with a manual search of the radiology and pathology databases from June to September 2013. 22 of the 67 joint aspirates studied did not have a cell count carried out. Four of these 22 cases had a diagnosis of septic arthritis. In five aspirates, there was a failure to confirm a definite diagnosis and they were thus conservatively treated as a septic joint. The remaining acute joints in which no cell count was done were gout (7 cases), pseudogout (5 cases) and rheumatoid arthritis (1 case). Cell counts were not routinely detected for a variety of reasons. Eleven aspirates were deemed too viscous, and in eight cases the sample had clotted prior to pathologist assessment. Two cases had insufficient volume, and one sample was too bloodstained to calculate a cell count and differential;likely due to traumatic aspiration. Conclusions: 33% of acute monoarthritis’ evaluated over the study period failed to have a synovial fluid WCC and differential. This may be due to inadequate samples, or lack of appropriate collection tube. Better education is required for appropriate collection and test requesting wherein a diagnosis of septic arthritis is in question.

Synovial mesenchymal stem cells in vivo:Potential key players for joint regeneration

Unlike bone marrow(BM)mesenchymal stem cells(MSCs),whose in vivo identity has been actively explored in recent years,the biology of MSCs in the synovium remains poorly understood.Synovial MSCs may be of great importance to rheumatology and orthopedics because of the direct proximity and accessibility of the synovium to cartilage,ligament,and meniscus.Their excellent chondrogenic capabilities and suggested transit through the synovial fluid,giving unhindered access to the joint surface,further support a pivotal role for synovial MSCs in homeostatic joint repair.This review highlights several unresolved issues pertaining to synovial MSC isolation,topography,and their relationship with pericytes,synovial fibroblasts,and synovial fluid MSCs.Critically reviewing published data on synovial MSCs,we also draw from our experience of exploring the in vivo biology of MSCs in the BM to highlight key differences.Extending our knowledge of synovial MSCs in vivo could lead to novel therapeutic strategies for arthritic diseases.

Cell Proliferation Ability of Mouse Fibroblast-Like Cells and Osteoblast-Like Cells on a Ti-6Al-4V Alloy Film Produced by Selective Laser Melting

Successful regeneration of tissues and organs relies on the application of suitable substrates or scaffolds in scaffold-based regenerative medicine. In this study, Ti-6Al-4V alloy films (Ti alloy film) were produced using a three-dimensional printing technique called Selective Laser Melting (SLM), which is one of the metal additive manufacturing techniques. The thickness of produced Ti alloy film was approximately 250 μm. The laser-irradiated surface of Ti alloy film had a relatively smooth yet porous surface. The non-irradiated surface was also porous but also retained a lot of partially melted Ti-6Al-4V powder. Cell proliferation ability of mouse fibroblast-like cells (L929 cells) and mouse osteoblast-like cells (MC3T3-E1 cells) on both the surfaces of Ti alloy film was examined using WST assay. Both L929 and MC3T3-E1 cells underwent cell proliferation during the culture period. These results indicate that selective laser melting is suitable for producing a cell-compatible Ti-6Al-4V alloy film for biomaterials applications.

Solitary primary pulmonary synovial sarcoma:A case report

BACKGROUND Synovial sarcoma(SS)is an uncommon and highly malignant soft tissue sarcoma in the clinic,with primary pulmonary SS(PPSS)being extremely rare.Here,we describe the clinical characteristics,diagnosis,and treatment of a solitary PPSS case confirmed via surgical resection and fluorescence in situ hybridization(FISH).CASE SUMMARY A 33-year-old man was admitted because of intermittent coughing and hemoptysis for one month,with lung shadows observed for two years.Wholebody positron emission tomography-computed tomography(PET-CT)revealed a solitary mass in the upper lobe of the right lung,with uneven radioactivity uptake and a maximum standardized uptake value of 5.6.The greyish-yellow specimen obtained following thoracoscopic resection was covered with small multinodulated structures and consisted of soft tissue.Hematoxylin and eosin staining revealed spindle-shaped malignant tumor cells.Immunohistochemistry indicated these tumor cells were CD99 and BCL-2-positive.Furthermore,the FISH test revealed synovial sarcoma translocation genetic reassortment,which confirmed the diagnosis of SS.CONCLUSION PPSS is extremely rare and tends to be misdiagnosed as many primary pulmonary diseases.PET-CT,histologic analysis,and FISH tests can be used to differentiate PPSS from other diseases.Surgical resection is regularly recommended for the treatment of solitary PPSS and is helpful for improving the prognosis.

Study of synovialization of non-synovial tendon and its significance

The non-synovial part of the tendon of the profound digital flexor of rabbits was put into the knee joint cavity aseptically and the non-synovial tendon of the digital flexor of a human fetus was cultured with synovial cells in vitro.It was found the non-synovial tendon of rabbits was covered with a sheet of membrane-like tissue exhibiting the morphological features of the synovial membrane and the implanted tendon was kept free in the joint cavity without adhesion to the surrounding tissues.The surface of the human non-synovial tendon was covered with a layer of synovial cells.It is concluded that synovialization of a nonsynovial tendon can be achieved either in an environment full of synovial fluid in vivo or through tissue culture in vitro.

Transcriptome sequencing-based study on the mechanism of action of Jintiange capsules(金天格胶囊)in regulating synovial mesenchymal stem cells exosomal miRNA and articular chondrocytes mRNA for the treatment of osteoarthritis

OBJECTIVE: To corroborate the efficacy of Jintiange capsules(JTGs)( 金天格胶囊) in the treatment of osteoarthritis(OA) by exploring the potential mechanism of action of synovial mesenchymal stem cell exosomes(SMSC-Exos) and articular chondrocytes(ACs) through transcriptome sequencing(RNA-seq). METHODS: Type Ⅱ collagenase was used to induce OA in rats. The efficacy of JTGs was confirmed by macroscopic observation of articular cartilage, micro-CT observation, and safranin fast green staining. After SMSC-Exos and ACs were qualified, RNA-seq was used to screen differentially expressed mi RNAs and m RNAs. The target genes of differentially expressed mi RNAs in Synovial mesenchymal stem cells(SMSCs) were predicted based on the multi Mi R R package. The codifferentially expressed genes of SMSC-Exos and ACs were obtained by venny 2.1.0. The mi RNA-m RNA regulatory network was constructed by Cytoscape software. Based on the Omic Share platform, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed on the m RNA regulated by key mi RNAs. Expression trend analysis was performed for co-differentially expressed genes. Correlation analysis was performed on micro-CT efficacy indicators, co-differentially expressed genes mRNA and miRNA. RESULTS: The efficacy of each administration group of JTGs was significant compared with the model group. SMSC-Exos and ACs were identified by their characteristics. The expression of rno-mi R-23a-3p, rnomi R-342-3p, rno-miR-146b-5p, rno-miR-501-3p, rnomiR-214-3p was down-regulated in OA pathological state, and the expression of rno-mi R-222-3p, rno-mi R-30e-3p, rno-mi R-676, and rno-miR-192-5p expression was upregulated, and the expression of all these mi RNAs was reversed after the intervention with JTGs containing serum. The co-differentially expressed genes were enriched in the interleukin 17 signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor-β signaling pathway, etc. The expression trends of Ccl7, Akap12, Grem2, Egln3, Arhgdib, Ccl20, Mmp12, Pla2g2a, and Nr4a1 were significant. There was a correlation between micro-CT pharmacodynamic index, m RNA, and mi RNA. CONCLUSION: JTGs can improve the degeneration of joint cartilage and achieve the purpose of cartilage protection, which can be used for the treatment of OA. SMSCs-related mi RNA expression profiles were significantly altered after the intervention with JTGs containing serum. The 9 co-differentially expressed genes may be the key targets for the efficacy of JTGs in the treatment of OA rats, which can be used for subsequent validation.

靶向成纤维细胞生长因子受体1信号改善类风湿关节炎的骨破坏

背景:尽管科研人员已注意到成纤维细胞生长因子受体1在类风湿关节炎骨破坏中展现出巨大潜力,但尚未有学者对成纤维细胞生长因子受体1在类风湿关节炎骨破坏中的研究进展作全面综述。目的:通过查阅国内外相关文献,综合分析成纤维细胞生长因子受体1在类风湿关节炎骨破坏中的机制。方法:以“成纤维细胞生长因子受体1,类风湿关节炎,骨破坏,骨细胞,成骨细胞,破骨细胞,软骨细胞,巨噬细胞,滑膜成纤维细胞,T细胞,血管内皮细胞”为检索词检索中国知网数据库,以“fibroblast growth factor receptor 1,rheumatoid arthritis,bone destruction,osteocytes,osteoblasts,osteoclasts,chondrocytes,macrophages,synovial fibroblasts,T cells,endothelial cells”为检索词检索PubMed数据库,检索时间范围重点为1992年4月至2024年1月。通过阅读文献题目、摘要及全文,根据纳入与排除标准进行筛选,最后纳入82篇文献进行综述。结果与结论:成纤维细胞生长因子受体1广泛表达于骨组织相关细胞,包括骨细胞、成骨细胞、破骨细胞等,可以通过调控这些细胞的功能来影响骨重塑过程和维持骨稳态,促进类风湿关节炎骨破坏的发生和发展。成纤维细胞生长因子受体1还可以在滑膜成纤维细胞和巨噬细胞中参与炎症反应,在内皮细胞中调控滑膜血管生成,从多个方面促进骨破坏。成纤维细胞生长因子受体1可能是类风湿关节炎骨破坏的一个重要参与因素,为进一步研究类风湿关节炎治疗靶点提供依据。

淫羊藿苷含药血清促进3种细胞共培养体系中软骨细胞增殖和干细胞成软骨分化

背景:关节软骨损伤修复能力十分有限,组织工程技术为修复受损软骨提供了新的治疗方案,其中软骨细胞、骨髓间充质干细胞和滑膜间充质干细胞之间的相互影响和诱导作用是自体软骨损伤愈合的基础。目的:构建软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞共培养体系用以模拟软骨细胞体内微环境,并探究其最佳细胞接种比例,同时观察淫羊藿苷含药血清对该体系中软骨细胞增殖和干细胞成软骨分化的影响。方法:提取、培养、鉴定大鼠膝关节软骨细胞、骨髓间充质干细胞和滑膜间充质干细胞,按不同细胞接种比例构建软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞非接触共培养体系,共培养72 h后观察软骨细胞增殖活性和表型能力,选择综合效应最佳的共培养体系;用淫羊藿苷溶液(0.25 mg/m L)灌胃新西兰大白兔制备淫羊藿苷含药血清,对照组共培养体系用含体积分数为10%胎牛血清、1%双抗的高糖DMEM培养液培养,实验组共培养体系在此基础之上加入体积分数为10%淫羊藿苷含药血清进行干预,24,48 h后检测两组软骨细胞增殖活性和Ⅱ型胶原表达,14 d后采用免疫荧光染色检测骨髓间充质干细胞、滑膜间充质干细胞成软骨分化情况。结果与结论:(1)3种细胞以不同比例共培养时均可正常贴壁生长,当软骨细胞、骨髓间充质干细胞、滑膜间充质干细胞接种比例为2∶1∶1时,共培养体系中软骨细胞表现出最佳增殖活性和表型能力;(2)与对照组相比,实验组培养24 h后软骨细胞增殖活性和Ⅱ型胶原表达显著升高(P<0.01),48 h后两组仍有差异(P<0.05),培养14 d后两组骨髓间充质干细胞和滑膜间充质干细胞出现明显的软骨分化,部分细胞呈现圆形或椭圆形,胞浆Ⅱ型胶原免疫荧光染色为阳性,实验组荧光强度明显高于对照组(P<0.01);(3)结果表明,以非接触共培养方法可以成功建立软骨细胞-骨髓间充质干细胞-滑膜间充质干细胞共培养体系且细胞比例为2∶1∶1时软骨细胞增殖活性和表型能力最佳;同时淫羊藿苷含药血清具有促进该体系中软骨细胞增殖及骨髓间充质干细胞、滑膜间充质干细胞成软骨分化的作用。

miR-27a通过TLR4/NF-κB信号通路对类风湿关节炎滑膜细胞生物学行为的影响

目的:探讨miR-27a通过Toll样受体(TLR)4/NF-κB信号通路对类风湿关节炎(RA)滑膜细胞生物学行为的影响。方法:选择行膝关节置换术的30例RA患者(RA组)和同期因创伤急诊截肢的18例患者(对照组)的滑膜组织,采用qRT-PCR法检测miR-27a的表达。将RA成纤维样滑膜细胞MH7A分为4组:空白对照组,不进行任何处理;TNF-α组,加入终浓度为20μg/L的TNF-α处理24 h;TNF-α+miR-NC组,加入终浓度为20μg/L的TNF-α处理后转染miR-NC;TNF-α+miR-27a mimic组,加入终浓度为20μg/L的TNF-α处理后转染miR-27a mimic,采用qRT-PCR法检测组织或细胞中miR-27a的表达量,CCK-8法检测细胞增殖情况,克隆形成实验检测细胞克隆形成能力,Transwell法检测细胞侵袭和迁移能力,Annexin V/PI双染法检测细胞凋亡情况,双荧光素酶报告实验验证TLR4 mRNA与miR-27a的靶向关系,Western blot法检测细胞中TLR4、NF-κB、磷酸化TLR4(p-TLR4)和磷酸化NF-κB(p-NF-κB)蛋白的表达情况。结果:对照组和RA组滑膜组织中miR-27a的表达量分别为(1.00±0.08)和(0.36±0.05),RA组低于对照组(P<0.001)。与空白对照组比较,TNF-α组和TNF-α+miR-NC组细胞中miR-27a表达量下降,TNF-α+miR-27a mimic组miR-27a表达量升高;与TNF-α组和TNF-α+miR-NC组比较,TNF-α+miR-27a mimic组细胞中miR-27a表达量升高(P<0.05)。与空白对照组比较,TNF-α组和TNF-α+miR-NC组细胞增殖、克隆形成、侵袭和迁移能力增强,细胞凋亡率降低;与TNF-α组和TNF-α+miR-NC组比较,TNF-α+miR-27a mimic组细胞增殖、克隆形成、侵袭和迁移能力减弱,细胞凋亡率升高(P<0.05)。双荧光素酶报告实验证实TLR4是miR-27a的靶基因。与空白对照组比较,TNF-α组和TNF-α+miR-NC组p-TLR4/TLR4、p-NF-κB/NF-κB升高;与TNF-α组和TNF-α+miR-NC组比较,TNF-α+miR-27a mimics组p-TLR4/TLR4、p-NF-κB/NF-κB降低(P<0.05)。结论:miR-27a可能通过抑制TLR4/NF-κB信号通路,降低RA成纤维样滑膜细胞的增殖、侵袭和迁移能力,促进其凋亡。

滑膜巨噬细胞在类风湿关节炎中的研究进展

类风湿关节炎(RA)是以侵蚀性、对称性多关节炎为主要临床表现的自身免疫性疾病,基本病理改变为滑膜炎和血管翳形成。在RA患者的滑膜组织中有多种免疫细胞浸润,包括巨噬细胞、T淋巴细胞和B淋巴细胞等。滑膜巨噬细胞(SMs)在RA的发生和发展中起至关重要的作用。目前SMs主要有两种来源,一种来源于外周血单核细胞浸润到滑膜组织的巨噬细胞,另一种是滑膜组织驻留巨噬细胞。由于SMs的异质性,其在RA的发生和发展中起不同作用,SMs可以加速RA的疾病进展,但具有不同细胞表面标志物的SMs亚群也可以形成保护屏障,延缓RA的发生和发展。因此,未来应深入探讨SMs的来源及不同细胞表面标志物的SMs在RA疾病进展中的作用机制,以为RA的治疗提供依据。

类风湿关节炎患者关节滑液及滑膜组织中CCP/AST表达水平及临床意义

目的探究类风湿关节炎(rheumatoid arthritis,RA)患者关节滑液及滑膜组织中环瓜氨酸肽抗原特异性T细胞(cyclic citrullinated peptide antigen-specific T cells,CCP/AST)表达水平及临床意义。方法选取石家庄市中医院2021年1月~12月128例RA患者为RA组,并取同期在该院因关节疼痛需做关节镜检查者50例为对照组。其中RA患者轻度组46例,中度组52例和重度组30例;通过蛋白印迹分析各组受试者滑膜组织类风湿因子(rheumatoid factors,RF)和抗瓜氨酸蛋白抗体(anticitrullinated protein antibodies,ACPA)蛋白表达水平;通过流式细胞仪分析受试者关节滑液中CCP/AST频率;双重免疫荧光染色/激光共聚焦扫描观察滑膜组织中CCP/AST染色强度;Pearson相关性分析评估关节滑液、滑膜组织中CCP/AST表达与RF和ACPA的相关性;采用Logistic回归分析RA发生的危险因素。结果对照组、轻度组、中度组和重度组患者滑膜组织中RF(1.01±0.01,1.53±0.03,2.01±0.08,2.66±0.12kDa)和ACPA蛋白(1.03±0.01,1.61±0.03,2.04±0.10,2.59±0.13 kDa)依次升高,差异具有统计学意义(F=14.207,12.446,均P<0.05)。对照组、轻度组、中度组和重度组患者关节滑液中CCP/AST表达依次升高(8.26%±1.68%,22.46%±3.28%,33.58%±4.37%,46.15%±5.44%),差异具有统计学意义(F=25.306,P<0.05)。对照组、轻度组、中度组和重度组患者滑膜组织CCP/AST染色强度依次升高(1.05±0.26,1.35±0.89,2.04±0.56,2.78±0.15分),差异具有统计学意义(F=70.67,P<0.05)。RA患者关节滑液、滑膜组织中CCP/AST表达与RF呈明显正相关(r=0.861,0.934,均P<0.05),与ACPA呈明显正相关(r=0.854,0.913,均P<0.05)。Logistic回归分析结果显示,高血压(OR=3.241,95%CI:1.491~6.752)、糖尿病(OR=2.565,95%CI:1.126~5.813)以及关节滑液(OR=4.450,95%CI:1.652~11.622)、滑膜组织(OR=5.629,95%CI:2.474~12.390)中CCP/AST表达均为RA发生的独立危险因素(P<0.05)。结论RA患者关节滑液及滑膜组织中CCP/AST呈现高表达,且与疾病活动度及关节破坏有关,可用于评估此类患者临床关节活动度及骨破坏程度。

芒果苷抑制类风湿关节炎成纤维样滑膜细胞增殖、迁移及炎性因子的表达

背景:芒果苷是一种从芒果叶、树皮、根中提取的双苯吡酮类化合物,前期研究表明芒果苷可以通过NF-κB、JAK/STAT等转录因子的活化发挥抗全身性炎症作用。目的:探讨芒果苷对类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis fibroblast-like synovial cells,RA-FLS)增殖、迁移和炎症因子表达的影响及机制。方法:RA-FLS分为空白组、R848(TLR7/8激动剂)刺激组、芒果苷低、中、高浓度组(2,4,8μg/mL)、阳性对照组(Cu-CPT8,TLR8通路抑制剂)。CCK-8法检测不同质量浓度芒果苷对RA-FLS毒性的影响并筛选最终细胞用药质量浓度,细胞克隆形成实验检测RA-FLS的增殖能力,划痕实验及Transwell迁移实验检测RA-FLS的迁移能力,qRT-PCR检测RA-FLS中白细胞介素1β、白细胞介素6、肿瘤坏死因子αmRNA的表达。结果与结论:①与空白组比较,2-10μg/mL的芒果苷干预组对RA-FLS活力有抑制趋势,但差异不显著(P>0.05),说明对RA-FLS的毒性影响很小;②与R848刺激组比较,芒果苷低、中、高浓度组RA-FLS克隆数、划痕愈合率及迁移细胞数均降低(P<0.01);芒果苷中浓度和高浓度组白细胞介素1β、白细胞介素6及肿瘤坏死因子αmRNA的表达降低(P<0.01);③与R848刺激组比较,阳性对照组细胞克隆数、划痕愈合率及迁移细胞数以及白细胞介素6、肿瘤坏死因子αmRNA表达水平显著降低(P<0.05,P<0.01);白细胞介素1βmRNA表达水平无明显差异;④结果表明,芒果苷可能通过TLR7/8信号通路抑制RA-FLS增殖、迁移以及炎症因子表达发挥抗类风湿关节炎的作用。

滑膜细胞及软骨细胞释放HMGB1流体力学阈值的研究

目的探究流体流动剪切力(fluid flow shear stress,FFSS)下,滑膜细胞及软骨细胞释放高迁移率族蛋白1(high⁃mobility group box 1,HMGB1)时流体力学的阈值;阐明异常机械力刺激下,滑膜细胞及软骨细胞破坏的先后顺序,为了解颞下颌关节骨关节炎的发病机制及病理研究提供实验基础。方法获得医院动物实验伦理委员会的审批,于SD大鼠膝关节获取滑膜组织及软骨组织块,消化培养获得滑膜细胞及软骨细胞,取3~4代滑膜细胞及软骨细胞,通过流体剪切力学装置对软骨及滑膜细胞施加FFSS,根据不同大小的FFSS值分组,分别采用1、3、5、10 dyn/cm^(2)的FFSS刺激滑膜细胞1 h,采用4、8、12、16 dyn/cm^(2)的FFSS刺激软骨细胞1 h,静息培养(0 dyn/cm^(2))作为对照组,观察细胞的形态学变化、免疫组化检测HMGB1及白细胞介素⁃1β(interleu⁃kin⁃1β,IL⁃1β)的细胞表达分布,ELISA检测上清液中HMGB1与IL⁃1β的水平;Western blot检测细胞HMGB1与IL⁃1β蛋白表达水平。结果随着FFSS负载的增大,滑膜细胞及软骨细胞逐渐肿胀,破裂,且细胞数量减少。随着FFSS负载的增大,HMGB1与IL⁃1β的表达由胞核逐渐转移至胞浆。在滑膜细胞中,与对照组相比,1、3、5、10 dyn/cm^(2)干预组中的HMGB1和IL⁃1β在上清液及细胞内的表达水平明显升高(P<0.01)。在软骨细胞中,与对照组相比,4、12、16 dyn/cm^(2) FFSS干预组中HMGB1在上清液中的表达水平增加(P<0.05),细胞HMGB1蛋白表达水平明显增加(P<0.01);而在8 dyn/cm^(2) FFSS干预组中HMGB1在上清液中的表达水平明显增加(P<0.01),但细胞HMGB1蛋白表达减少(P<0.01);与对照组相比,4、8、12、16 dyn/cm^(2) FFSS干预组中IL⁃1β在上清液中表达水平逐渐增加(P<0.01);除4 dyn/cm^(2)组外,随着FFSS负载增大,细胞中IL⁃1β的蛋白表达水平逐渐升高。结论滑膜细胞及软骨细胞随FFSS负载增大出现溶胀破裂,释放HMGB1流体力学阈值分别接近于1 dyn/cm^(2)和8 dyn/cm^(2);即在受到机械力刺激时,滑膜细胞先于软骨细胞损伤。

uPAR promotes tumor-like biologic behaviors of fibroblast-like synoviocytes through PI3K/Akt signaling pathway in patients with rheumatoid arthritis

Urokinase-type plasminogen activator receptor(uPAR),is a multifunctional receptor on cell surface,widely present in endothelial cells,fibroblasts,and a variety of malignant cells.Current studies have suggested that uPAR overexpressed on synovial tissues or in synovial fluid or plasma in patients with rheumatoid arthritis(RA).However,there are limited researches regarding the role of uPAR on fibroblast-like synoviocytes of rheumatoid arthritis(RA-FLSs)and its underlying mechanisms.Here,our studies show that the expression of uPAR protein was significantly higher in fibroblast-like synoviocytes(FLSs)from RA than those from osteoarthritis or traumatic injury patients.uPAR gene silencing significantly inhibited RA-FLSs cell proliferation,restrained cell transformation from the G0/G1 phase to S phase,aggravated cell apoptosis,interfered with RA-FLSs cell migration and invasion,and reduced activation of the PI3K/Akt signaling pathway,which may be associated withβ1-integrin.Cell supernatants from uPAR gene-silenced RA-FLSs markedly inhibited the migration and tubule formation ability of the HUVECs(a human endothelial cell line).Therefore,we demonstrate that uPAR changes the biological characteristics of RA-FLSs,and affects neoangiogenesis of synovial tissues in patients with RA.All of these may be associated with theβ1-integrin/PI3K/Akt signaling pathway.These results imply that targeting uPAR and its downstream signal pathway may provide therapeutic effects in RA.

新风胶囊通过结合Wnt5a经Wnt/β-catenin信号通路抑制类风湿性关节炎

目的:本研究将探讨Wnt5a是否可以作为类风湿性关节炎(RA)潜在的诊断和治疗靶点,新风胶囊(XFC)如何通过Wnt5a/β-catenin信号通路改善RA。方法:在体内Adjuvant arthritis(AA)大鼠模型中采用ELISA和RT-qPCR检测炎症因子和病理基因研究XFC对AA大鼠疗效,RT-qPCR检测验证XFC调控通过网络药理学预测的核心基因和关键通路。在体外原代AA成纤维样滑膜细胞(FLS)中采用RT-qPCR、Western blot和免疫荧光等方法研究XFC对Wnt/β-catenin通路的调节机制。结果:XFC显著下调AA大鼠的关节炎评分和足爪肿胀,抑制AA大鼠关节炎症。XFC降低AA大鼠外周血中炎症因子TNF-α和IL-1水平,抑制AA大鼠关节滑膜和AA FLS中病理基因MMP3和fibronectin水平。网络药理学预测出Wnt通路与XFC治疗RA高度相关。细胞水平上,含XFC血清抑制Wnt通路相关基因β-catenin、CCND1和c-Myc的表达。分子对接结果显示XFC关键成分与Wnt5a的结合能力强,在AA FLS中Wnt5a过表达(Wnt5a-ove)干扰了XFC的作用。结论:Wnt5a在AA FLS和RA FLS中表达明显升高,XFC通过与Wn5a结合,抑制Wnt/β-catenin信号通路活化改善RA,为XFC改善RA提供新的治疗机制。