TFDG对IL-1β体外诱导大鼠软骨细胞炎性损伤的保护作用研究

体外分离培养SD雄性大鼠膝关节软骨细胞,经甲苯胺蓝及Ⅱ型胶原(Col Ⅱ)免疫荧光染色鉴定后,加入白介素-1β(IL-1β)诱导建立骨关节炎(OA)细胞模型,探讨茶黄素双没食子酸酯(TFDG)对OA软骨细胞的保护作用。细胞形态观察发现TFDG能明显改善OA软骨细胞形态。实时荧光定量PCR(Real-time PCR)结果显示,TFDG不仅可以上调软骨细胞分子标志物Col Ⅱ mRNA的表达,还可以下调炎症因子IL-1β、IL-6mRNA的表达。酶联免疫吸附实验(ELISA)检测结果进一步表明TFDG可明显降低炎症因子的分泌。免疫印迹(Western blot)检测结果证明,TFDG预干扰可降低炎症诱导酶环氧化酶COX-2蛋白表达量。这些结果说明TFDG通过减弱炎症反应,从而对IL-1β体外诱导大鼠软骨细胞炎性损伤起到保护作用。     

兔关节软骨细胞的获取及培养

试验采用组织块法和酶消化法获取兔关节软骨细胞,并对这两种方法取得的效果进行对比分析。在酶消化方法上,运用Ham-F12培养液和无Ca2 、Mg2 的PBS液配制2g/L的Ⅱ型胶原酶进行消化,同时结合酶阶段性消化法收获软骨细胞,对细胞成活率进行测定。对最终收获的细胞进行培养观察,测定软骨细胞的分泌活性。结果表明:在获取细胞的数量上,酶消化法明显优于组织块法;在成活率比较试验中,Ham-F12配制组明显高于常规的PBS液(无Ca2 、Mg2 )配制组,两组之间存在显著差异(P<0.05);对体外培养的软骨细胞进行鉴定,结果显示软骨细胞仍具有较强的分泌基质能力。在此试验消化分离体系下,可得到大量的、高活性的软骨细胞,且在体外能维持良好的生长特性。     

玻璃化冻存对羊软骨细胞的影响

对玻璃化冻存后羊Ovis aries软骨细胞的存活率和琥珀酸脱氢酶（SDH）活性进行了初步研究。结果表明：细胞存活率随着冻存时间的延长而逐渐下降,但下降速度不明显;SDH活性在玻璃化冻存1 ~ 20 d变化幅度较大,20 ~ 30 d下降趋势减弱,保持相对稳定。从各项指标来看,认为玻璃化溶液VSb组的冻存效果最好,在经过5,10, 15,20和30 d的冻存后,其存活率分别为85.47% ± 1.78%,80.73% ± 1.81%,78.62% ± 2.06%, 76.35% ± 2.58%和73.83% ± 1.49%,细胞SDH活性吸光度值分别为0.49 ± 0.064,0.444 ± 0.073,0.394 ± 0.039,0.354 ± 0.082和0.339 ± 0.053。该玻璃化冻存方法对长期冻存羊软骨细胞具有一定的实际应用意义。图4表2参17     

Effect of Herba Taxilli extracts combined with miR-375 on viability and apoptosis of osteoarthritic chondrocytes

AIM To study the effects of extracts of Herba Taxilli (Sangjisheng, SJS) on the viability and apoptosis of osteoarthritic chondrocytes and the underlying mechanism. METHODS Human primary osteoarticular chondrocytes (RPOC) were divided into control group, interleukin-1β (IL-1β) group, IL-1β+low-dose extracts of SJS (SJS-L) group, IL-1β+medium-dose extracts of SJS (SJS-M) group, IL-1β+high-dose extracts of SJS (SJS-H) group, IL-1β+anti-miR-NC group, IL-1β+anti-miR-375 group, IL-1β+SJS-H+miR-NC group, IL-1β+SJS-H+miR-375 group. The cell viability was measured by MTT assay, apoptosis was analyzed by flow cytometry, miR-375 expression was detected by qPCR, and the protein levels of cyclin D1, P21, Bcl-2, Bax and caspase-3 were determined by Western blot. RESULTS Compared with control group, the viability of RPOC at 24 h, 48 h and 72 h and the protein expression levels of cyclin D1 and Bcl-2 were significantly decreased (P<0.05), the protein levels of P21, Bax and caspase-3, the apoptotic rate and the expression level of miR-375 were remarkably increased in IL-1β group(P<0.05). Compared with IL-1β group, the cell viability at 24 h, 48 h and 72 h and the protein expression of cyclin D1 in the RPOC were greatly increased (P<0.05), while the expression of P21 was significantly decreased in IL-1β+SJS-M group and IL-1β+SJS-H group(P<0.05).The apoptotic rate, Bax, caspase-3 protein and miR-375 expression were obviously decreased (P<0.05), and Bcl-2 protein level was significantly increased in IL-1β+SJS-H group compared with IL-1β group(P<0.05). Compared with IL-1β+anti-miR-NC group, the expression of miR-375, the protein levels of P21, Bax, caspase-3 and the apoptotic rate in the RPOC of IL-1β+anti-miR-375 group were markedly decreased (P<0.05), while the cell viability at 24 h, 48 h and 72 h and the protein levels of cyclin D1 and Bcl-2 were significantly increased (P<0.05). Over-expression of miR-375 reversed the effects of extracts of SJS on the viability and apoptosis of RPOC with IL-1β stimulation. CONCLUSION The extracts of Herba Taxilli promotes the viability and inhibits apoptosis of RPOC treated with IL-1β, which is related to the regulation of miR-375 expression.     

药用植物淫羊藿与富血小板血浆协同对软骨细胞的增殖作用

观察淫羊藿和富血小板血浆(PRP)对SD大鼠软骨细胞增殖作用的影响,探讨两者对软骨细胞的协同作用。取3周龄SD大鼠的膝关节软骨建立软骨细胞体外培养体系,分别采用MTT法、流式细胞术对细胞增殖作用进行检测。结果表明: (1) PRP对软骨细胞增殖有显著的促进作用(P<001);(2)淫羊藿对软骨细胞有一定的增殖作用,但不显著;(3)淫羊藿联合PRP对软骨细胞的增殖作用显著下降,效果低于淫羊藿或PRP组(P<001)。淫羊藿联合PRP对软骨细胞有显著抑制作用(P<001),推断淫羊藿某些成分抑制了PRP中生长因子的活性,从而减弱了PRP对软骨细胞的作用,该推断有待进一步研究确认。     

Amentadione from the Alga Cystoseira usneoides as a Novel Osteoarthritis Protective Agent in an Ex Vivo Co-Culture OA Model

Osteoarthritis (OA) remains a prevalent chronic disease without effective prevention and treatment. Amentadione (YP), a meroditerpenoid purified from the alga Cystoseira usneoides, has demonstrated anti-inflammatory activity. Here, we investigated the YP anti-osteoarthritic potential, by using a novel OA preclinical drug development pipeline designed to evaluate the anti-inflammatory and anti-mineralizing activities of potential OA-protective compounds. The workflow was based on in vitro primary cell cultures followed by human cartilage explants assays and a new OA co-culture model, combining cartilage explants with synoviocytes under interleukin-1β (IL-1β) or hydroxyapatite (HAP) stimulation. A combination of gene expression analysis and measurement of inflammatory mediators showed that the proposed model mimicked early disease stages, while YP counteracted inflammatory responses by downregulation of COX-2 and IL-6, improved cartilage homeostasis by downregulation of MMP3 and the chondrocytes hypertrophic differentiation factors Col10 and Runx2. Importantly, YP downregulated NF-κB gene expression and decreased phosphorylated IkBα/total IkBα ratio in chondrocytes. These results indicate the co-culture as a relevant pre-clinical OA model, and strongly suggest YP as a cartilage protective factor by inhibiting inflammatory, mineralizing, catabolic and differentiation processes during OA development, through inhibition of NF-κB signaling pathways, with high therapeutic potential.     

外源褪黑素对鹿茸软骨细胞生长发育的影响

试验旨在探讨褪黑素对体外培养的鹿茸软骨细胞增殖、周期及凋亡的影响。利用甲苯胺蓝、茜素红S、阿利新蓝染色鉴定软骨细胞,采用外源添加褪黑素的方法,用不同浓度（0、400、800、1 200、1 600和2 000 pg/mL）、不同时间（24、48和72 h）处理鹿茸软骨细胞,CCK-8法检测细胞增殖。选取800 pg/mL褪黑素处理软骨细胞24 h,利用流式细胞仪检测细胞周期的分布及细胞凋亡情况,采用ELISA试剂盒检测细胞培养液中睾酮含量。结果显示,经不同浓度的褪黑素处理鹿茸软骨细胞不同时间后,800 pg/mL褪黑素处理鹿茸软骨细胞24 h细胞活力极显著增加（P<0.01）。软骨细胞经褪黑素处理后,与对照组相比,褪黑素处理组细胞G1期比例显著降低（P<0.05）;G2期比例极显著增加（P<0.01）,细胞被阻滞在G2期;褪黑素处理组细胞早期凋亡率无显著变化（P>0.05）,晚期凋亡率显著下降（P<0.05）;ELISA检测睾酮分泌水平均显著上升（P<0.05）。综上,外源添加800 pg/mL褪黑素可以促进鹿茸软骨细胞的增殖,抑制软骨细胞的晚期凋亡,促进睾酮的分泌;以上结果可为阐明褪黑素参与鹿茸生长发育机制提供参考。