目的 建立骨痹体外软骨细胞炎症模型,为研究骨关节炎(osteoarthritis,OA)的中医药治疗机制提供细胞学基础.方法 采用胶原酶法进行人软骨细胞的分离培养,观察其形态学,Ⅱ型胶原(collagenⅡ,COLⅡ)免疫荧光染色法鉴定软骨细胞,细胞计数试剂(cell counting kit-8,CCK-8)检测各浓度梯度的脂多糖(lipopolysaccharide,LPS)对软骨细胞生长活性的影响,选择浓度分别为500、100、20、10、2 ng·mL-1的LPS经0、4、6、12、24 h诱导软骨细胞后,Elisa法检测各组细胞上清液中白细胞介素6(interleukin-6,IL-6)和基质金属蛋白酶-3(matrix metalloproteinase-3,MMP-3)的含量,筛选最佳干预浓度及时间.结果 分离培养的细胞光镜下观察符合软骨细胞形态学,COLⅡ免疫荧光染色呈阳性,具备软骨细胞特征.与对照组比较,加入差异浓度LPS的各实验组软骨细胞存活率(survival rate,SR)随LPS浓度升高呈逐渐下降趋势,而抑制率(inhibition rate,IR)则逐渐上升,呈浓度依赖性.其中高浓度的LPS对软骨细胞生长有一定的抑制作用,与对照组比较差异有统计学意义(P<0.05),其他各组与对照组比较差异均无统计学意义(P>0.05).软骨细胞上清液中IL-6和MMP-3在LPS浓度为10 ng·mL-1,干预时间为24 h时达到最大浓度(P<0.05).结论 本研究成功分离培养软骨细胞,并提供了一种建立骨痹体外软骨细胞炎症反应模型的优选方式,有利于中医药治疗策略在骨痹中的进一步研究.

目的:探讨舒筋活络方(SHC)对Toll样受体(TLRs)介导的滑膜炎症信号转导通路及其下游炎症因子的调节作用及其相关性.方法:将高、中、低浓度的SHC分别加入脂多糖(LPS)诱导的滑膜成纤维细胞(FLS),采用流式细胞术(FCM)对固有免疫代表性元件TLR2、TLR4的表达进行测定,一氧化氮(NO)一步法、酶联免疫吸附测定(ELISA)法对下游炎症因子NO和白介素6(IL-6)浓度进行测定,采用SPSS 20.0软件进行相关性分析.结果:与空白组比较,模型组TLR2和TLR4表达显著增强(P＜0.05),NO、IL-6释放量显著升高(P＜0.05);与模型组比较,SHC各浓度组均不同程度抑制TLR2、TLR4表达及NO、IL-6释放(P＜0.05),TLR2、TLR4阳性比例及NO、IL-6释放量随SHC浓度升高而降低;TLR2、TLR4和NO、IL-6之间均呈显著的正相关性(P＜0.05).结论:SHC通过调控固有免疫反应关联网络中互相紧密联系的各元件来调节骨关节炎(OA)进程,为中药复方影响OA病理机制的阐明提供了新的数据.

目的:测定舒筋活络方(SHC)对骨关节炎(OA)患者滑膜成纤维细胞(FLS)增殖、迁移、侵袭的影响,明确其作用机制.方法:对胶原酶法分离培养的FLS进行形态学观察,通过MTT比色法、划痕实验、Transwell实验、ELISA法等技术测定SHC对FLS增殖、迁移、侵袭等的影响.结果:体外分离的FLS生长状态良好,具备成纤维细胞形态特性;所选药物浓度范围对FLS增殖影响较小,与空白组比较,较高浓度和低浓度的SHC对FLS增殖的影响差异均有统计学意义(P<0.05),FLS的增殖与SHC呈浓度依赖性;镜下观察到OA-FLS的迁移现象,其中,模型组48 h、72 h的迁移率与0 h时比较,差异有统计学意义(P<0.05),SHC组72 h的迁移率与0 h时比较差异有统计学意义(P<0.05),与模型组比较,48 h、72 h时,SHC组FLS迁移能力比较,差异有统计学意义(P<0.05);SHC组FLS侵袭穿过小室膜的细胞个数较空白组明显减少(P<0.05);SHC各浓度组均对MMP3的释放有不同程度的抑制作用(P<0.05).结论:SHC调节FLS增殖、抑制FLS的迁移和侵袭,维护和修复OA患者关节内组织完整性,为OA的关节保护提供新的预防和治疗策略.

目的:研究差异浓度的舒筋活络方(SHC)对骨关节炎(OA)滑膜成纤维细胞(FLS)的活性、凋亡及细胞周期的影响,进一步明确SHC治疗OA的作用机制.方法:以差异浓度的SHC干预胶原酶法消化分离培养的FLS,通过细胞增殖-毒性检测试剂盒(CCK-8)和流式细胞术(FCM)分别检测FLS的活性、凋亡和细胞周期.结果:与对照组比较,高、低浓度的SCH均不同程度的抑制FLS活性(P＜0.05),呈浓度依赖性,中浓度的SCH则对FLS活性有部分促进作用;SCH的各浓度组均不同程度的诱导FLS早期凋亡与晚期凋亡(P＜0.05),提高总凋亡率(P＜0.05);并且延长FLS的G0/G1期(P＜0.05),同时缩短G2/M期和S期(P＜0.05),呈明显的浓度依赖性,其中,高浓度组对FLS凋亡的诱导及周期的阻滞作用最明显.结论:差异浓度的SHC通过对FLS活性、凋亡和周期的调控抑制FLS的浸润性生长,减缓OA滑膜炎症反应.

目的:观察推拿治疗结合等速肌力训练对膝关节骨性关节炎的治疗效果.方法:从膝关节骨性关节炎(KOA)患者中随机选取60例作为观察对象,60例患者随机分为推拿结合等速治疗组(试验组)和单纯推拿治疗组(对照组).分别将两组治疗前后的膝关节西安大略和麦克马斯特大学骨关节炎指数(WOMAC)、等速屈伸肌力测试对比分析.结果:两组治疗后膝关节WOMAC评分均较治疗前有显著改善(P＜0.01),其中推拿结合等速组膝关节WOMAC评分由(62.93±16.17)降为(26.20±12.68),改善较单纯推拿组更为显著(P＜0.01).两组治疗干预前等速肌力测试各指标比较没有显著性差异.经过8周治疗后推拿加等速训练组膝关节伸肌峰力矩、峰力矩/体重、达峰时间、平均功率,膝关节屈肌峰力矩、达峰时间与本组治疗前比较明显改善,差异有显著性(P＜0.01);膝关节伸肌平均功率与本组治疗前比较有改善,差异有显著性(P＜0.05);推拿加等速训练组膝关节伸肌峰力矩、峰力矩/体重、达峰时间、膝关节屈肌达峰时间、屈肌/伸肌比单纯推拿组治疗后改善明显,差异有显著性(P＜0.05);推拿加等速训练组膝关节屈肌峰力矩与单纯推拿组治疗后比较提高明显,差异有显著性(P＜0.01).结论:推拿结合等速肌力训练可以提高KOA患者膝关节屈伸肌肉力量,对于KOA的治疗具有良好的临床疗效,且明显优于单纯推拿治疗.

目的:通过通络止痛方中君药的活性成分桂皮醛(CA)和白芍总苷(TGP)的对滑膜成纤维细胞(FLS)的干预,研究固有免疫系统中TLR2和TLR4及其介导的信号通路的下游产物白介素1β (IL-1β)和基质金属蛋白酶(MMP-13)在骨关节炎滑膜炎性反应中的表达及其相互关系.方法:将高、低浓度的CA和TGP分别对经脂多糖(LPS)活化的FLS进行干预,收集细胞上清采用ELISA法测定IL-1 β和MMP-13的浓度;收集细胞采用流式细胞术测定FLS表面TLR2和TLR4的表达,并分析它们之间的相关性.结果:LPS对滑膜炎性反应有显著的诱导活化作用,高、低CA和TGP均可不同程度抑制IL-1β、MMP-13的释放和TLR2、TLR4的表达,其中CA高浓度组抑制作用最强(P＜0.05).TLR2、TLR4、IL-1β、MMP-13之间均存在显著的正相关性,具体为:TLR2与TLR4(r=0.862,P＜0.05),IL-1β与MMP-13(r=-0.994,P＜0.05),TLR2与IL-1β(r=0.754,P＜0.05),TLR2与MMP-13(r=-0.717,P＜0.05),TLR4与IL-1β(r=0.929,P＜0.05),TLR4与MMP-13(r=0.929,P＜0.05).结论:CA和TGP在通过调控固有免疫应答进程进而抑制滑膜炎性反应的作用,明确了固有免疫效应网中不同环节的元件的相关性,为中药有效成分作用靶点的探寻及未来防治OA有效方案的制定提供数据支持及新的思路.

随着年龄的增加,有些人常常感觉颈项酸痛、手麻无力、头痛头晕、耳鸣耳聋、视物不清、记忆力减退、失眠、反复落枕等,有人会认为这是上了年纪的缘故,没有办法,只能对症吃点药.其实不然,应该到医院去检查—下自己的颈椎,看看是否得了颈椎病.

Objective:To investigate the influence of miRNA-146a gene disturbances and cinnamic aldehyde (CA) on TLR4,NO,and MMP-13,which was released by synovioblast,and to explore their roles in the molecular mechanism of synovial inflammation in osteoarthritis (OA).Methods:Plasmids of miR-146a gene mimics and inhibitors were loaded into LPS-induced FLS using lipofection transfection.Then the interferential FLS were treated with CA,and the release amount of TRL4,NO and MMP-13 were detected.Results:After the miRNA-146a related plasmid transfection,the variation tendency of the concentrations of TLR4,NO and MMP-13 were almost the same.Compared with control group,the inhibitor group was elevated,and mimics group was declined (P ＜ 0.05).Compared with the control group,when the interferential FLS was treated with CA alone,TLR4,its release of TLR4,NO and MMP-13 were significantly decreased (P ＜ 0.05),whereas the mimics and CA coordinated intervention,the release was significantly decreased (P ＜ 0.05).Conclusion:Both of the miR-146a and CA have effects on synovial inflammation in OA,especially when mimics and CA were combined,the inhibition of inflammation was best.It could provide an experimental basis to clarify molecular mechanism,identify and develop novel drug targets of OA.

[Objective] To discuss the regulatory mechanism of total glucosides of paeonia (TGP) on proliferation, apoptosis and cell cycle of fibroblast-like synovial cells (FLS) in the process of osteoarthritis (OA). [Methods] With FLS being the object of action, Methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to detect the proliferation of FLS, and flow cytometry (FCM) was used to detect the apoptosis and cell cycle. [Results] The concentration dependence of FLS was existed in the concentration of TGP. In addition, the inhibitory effect of TGP on FLS mainly through inducing late apoptosis, as well as prolonging G2 phase (P<0.05) and blocking S phase (P<0.05) in the cell cycle of FLS. [Conclusion] TGP could inhibit the abnormal growth of FLS by regulating its proliferation, apoptosis and cell cycle. The findings above laid the groundwork for confirming the regulatory mechanism and curative effect of active components of Chinese medicine.