Aucubin prevents interleukin-1 beta induced inflammation and cartilage matrix degradation via inhibition of NF-κB signaling pathway in rat articular chondrocytes

Proinflammatory cytokine interleukin-1β (IL-1β) plays a crucial role in the pathogenesis of Osteoarthritis (OA) by stimulating several mediators contributed to cartilage degradation. Aucubin, a natural compound derived from plants which has been shown to possess diverse biological activities including anti-inflammatory property, may benefit the IL-1β stimulated chondrocytes. The present study was aimed to investigate the effects of Aucubin on IL-1β stimulated rat chondrocytes. Rat chondrocytes were cultured and pretreated with Aucubin (1, 10, 20, 50 μM), and then stimulated with or without IL-1β (10 ng/ml). Gene and protein expression of MMP-3, MMP-9, MMP-13, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) was determined by real-time PCR and Western blotting respectively. Nitric oxide (NO) production was quantified by Griess reagent. Phosphorylation and nuclear translocation of p65 were detected by western blotting and immunofluorescence, respectively. We found that Aucubin significantly reversed the elevated gene and protein expression of MMP-3, MMP-9, MMP-13, iNOS, COX-2 and the production of NO induced by IL-1β challenge in rat chondrocytes. Furthermore, Aucubin was able to suppress the IL-1β-mediated phosphorylation and nuclear translocation of p65, indicating Aucubin may possibly act via the NF-κB signaling pathway. The present study proposes that Aucubin may be a potential therapeutic choice in the treatment of OA due to its anti-inflammatory and chondroprotective features.     

Differently polarized macrophages affect the viability and growth of NSPCs by regulating the expression of PACAP

ObjectiveTo explore the influence of differently polarized macrophages, M1 or M2, to viability and growth of NSPCs and its possible mechanism, especially the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in it.MethodSpinal cord-derived NSPCs were co-cultured with M1 or M2 through a transwell system. Growth of NSPCs in both groups was observed through an inverted microscope within 3 days. NSPCs viability of each group, represented as intracellular ATP levels, was measured using the Cellular ATP Kit HTS following co-culture for 24 h. PACAP levels in the co-cultured NSPCs was alleviated with immunofluorescence and Western blot analysis.ResultsMorphologically NSPCs demonstrated a long spindle shape with short sprout on 3rd day when cultured together with M2. NSPCs cultured with M1 showed a small circle or oval shape with no obvious sprout. Expression of PACAP was observed in NSPCs co-cultured with M2 through immunofluorescence. In contrast, NSPCs did not demonstrate PACAP staining in the presence of M1 or cultured alone. PACAP in the NSPCs co-cultured with M2 was upregulated significantly compared with that co-cultured with M1 according to Western blot method. The relative ATP level of NSPCs co-cultured with M1 was markedly decreased while that with M2 was elevated significantly. That trend could be relieved by exogenous PACAP or PACAP 6–38. Viability change of NSPCs by M1/M2 correlated with apoptosis.ConclusionDifferently polarized macrophages could affect the growth and viability of NSPCs by regulating the expression of PACAP.     

异丙酚对大鼠脊髓背角神经元TTX敏感型钠通道失活和去失活效应的影响

目的：观察异丙酚对急性分离的大鼠脊髓背角神经元TTX敏感型钠通道电流的影响及相关机制。方法：取出生后7d的雄性SD大鼠,击昏后断头取脊髓腰膨大部位,分离背角神经元,应用全细胞膜片钳记录模式记录钠电流。距离神经细胞100μm施加0.3-30μmol/L不同浓度的异丙酚,应用-10mV（持续30 ms）刺激电压诱发钠电流,求出异丙酚对钠电流的半效抑制浓度（IC50）。观察异丙酚对钠通道失活效应的影响时,应用从-80mV至-20mV的预刺激电压（阶差为10mV,持续30ms）,再应用0mV刺激电压（持续30ms）诱发钠电流产生,向神经细胞施加IC50水平的异丙酚。观察异丙酚对钠通道去失活效应的影响时,应用-10mV的预刺激电压（持续30ms）,再给予-10mV、持续30 ms的刺激电压诱发钠电流产生,两刺激电压的间隔时间为2-24ms。结果：异丙酚可浓度依赖性地抑制TTX敏感型钠电流,其IC50为（5.35±0.25）μmol/L。钠通道失活实验中,预刺激电压在-60mV到-40mV范围内,IC50水平的异丙酚可显著抑制由刺激电压（0mV）诱发的钠电流（P〈0.05）。钠通道去失活实验中,两刺激的间隔时间在2-6ms之间时,IC50水平的异丙酚可显著性抑制由刺激电压（-10mV）诱发的钠电流（P〈0.01,P〈0.05）。结论：异丙酚可抑制大鼠脊髓背角电压依赖性钠通道电流,这一作用与促进钠通道的失活和抑制钠通道的去失活有关。     

前路一期病灶清除植骨内固定治疗胸腰椎结核

目的探讨前路一期病灶清除植骨内固定治疗胸腰椎结核的临床疗效。方法 2005年1月～2010年1月采用前路一期病灶清除植骨内固定治疗34例胸腰椎结核患者,术前四联抗结核化疗4～6周,术中病灶清除取髂骨或多根肋骨植骨,钉棒或钉板内固定。术后卧床8～12周,继续正规化疗12～18月。结果本组病例获得16～30个月随访,内固定无松动、脱落,后凸畸形矫正满意,局部病灶无复发,腰背疼痛症状消失。结论对于胸腰椎结核患者行前路一期病灶清除植骨内固定可彻底地清除病灶,重建脊柱稳定性,矫正和预防脊柱后凸畸形疗效满意。     

miR-106a负调控PTEN促进骨肉瘤细胞增殖并抑制凋亡

目的:研究miR-106a在骨肉瘤组织和MG-63细胞中的表达水平及其对MG-63细胞增殖和凋亡的影响及机制。方法:用荧光实时定量PCR法检测20对骨肉瘤和相邻正常组织及MG-63和成骨细胞hFOB 1.19中miR-106a的表达。用miR-106a mimics、miR-106a antagomir 及两者相应的对照物转染MG-63细胞,然后分别用CCK-8法检测四组细胞增殖活性和FCM法检测细胞凋亡率。miR-106a mimics和mimics control与野生型或突变型PTEN 3'-UTR 重组载体共转染后,应用荧光素酶基因报告系统检测miR-106a是否与PTEN基因3'-UTR 结合。利用Western blot技术检测PTEN蛋白在上述四组转染MG-63细胞和骨肉瘤标本中的表达水平。结果:与相邻正常组织（1.19±0.15）相比,肿瘤组织miR-106a的表达水平（2.60±0.86）显著升高;同时,miR-106a在MG-63中的表达水平（2.60±0.92）明显高于hFOB 1.19（1.19±0.39）,以上差异均有统计学意义（P＜0.05）。CCK-8和FCM检测结果显示,与mimics control组相比,miR-106a mimics组的增殖率明显增加,而细胞凋亡率下降;反之,miR-106a antagomir组与antagomir control组相比,增殖率前者低于后者,而凋亡率前者高于后者,上述差异均有统计学意义（P＜0.05）。荧光素酶报告实验显示,miR-106a mimics和wt PTEN 3'-UTR共转染组的荧光强度值明显低于mimics control和wt PTEN 3'-UTR组（P＜0.05）。Western blot发现,与对照组相比,miR-106a mimics组PTEN表达下调,而miR-106a antagomir表达上调;临床标本,肿瘤组织PTEN表达明显低于正常组织,差异均有统计学意义（P＜0.05）。结论:miR-106a在骨肉瘤组织及细胞中过表达,并靶向负调控PTEN表达,促进骨肉瘤细胞增殖并抑制其凋亡,从而发挥促癌作用。因此,miR-106a可为骨肉瘤的诊治提供新的潜在分子靶点。     

骨肉瘤多药耐药表型的分化特性及矛盾相关性分析

目的：探讨肿瘤的多药耐药性和恶性程度的关系,主要研究骨肉瘤细胞在向耐药表型的转化过程中,是否出现如骨源性干细胞在成熟分化过程中相似的生物学行为,或者是更复杂的生物学表型。方法：采用不同表达程度Pgp的骨肉瘤耐药亚型细胞,并针对增殖、分化及转移关键性基因Stathmin及VEGF,应用原位杂交和RT-PCR来衡量Mdr1同Stathmin、VEGF表达的相关性。结果：Mdr1/P170表型的骨肉瘤细胞的碱性磷酸酶的活性明显高于亲本细胞,说明Mdr表型的骨肉瘤细胞出现了分化。INH和RT-PCR结果揭示Mdr1mRNA同VEGF、Stathmin mRNA表达呈负相关。结论：Mdr1/P170的表达预示着骨肉瘤细胞向成熟状态分化,为研究Mdr及其逆转开拓了新的思路,为正确认识Pgp的功能提供了新的研究方法。     

共同培养骨髓基质干细胞对髓核细胞增殖分化的影响

目的:针对椎间盘退行性变的组织修复目前集中于细胞移植治疗,实验在体外联合培养条件下观察骨髓基质干细胞对髓核细胞增殖分化的影响。方法:实验于2005-12/2006-09在华中科技大学同济医学院附属同济医院骨科实验室和同济医院卫生部重点实验室进行。①实验方法:4月龄健康新西兰大耳白兔3只,麻醉后于髂嵴处作骨髓腔穿刺,抽吸6mL骨髓,分离骨髓基质干细胞;相同实验兔抽取骨髓后立即处死,取腰骶部脊柱之椎间盘,切开纤维环,取出髓核组织,分离椎间盘髓核细胞,同时设立单独髓核细胞培养组。②实验评估:在相同的培养条件下,定期观察两组髓核细胞在光镜下形态学变化及电镜下细胞超微结构的改变;于诱导培养7,14d通过RT-PCR法、Western blot法和免疫组化法法检测两组髓核细胞Ⅱ型胶原蛋白和聚集蛋白聚糖的表达。结果:①髓核细胞大体形态学及超微结构变化:髓核细胞与骨髓基质干细胞共同培养后,髓核细胞很快就呈现为圆形或多角形,且细胞形体变大、折光性好,贴壁时间早,数量增殖快,超微结构细胞表面可见许多短而粗的微绒毛突起,胞质内有大量扩张的粗面内质网、丰富的线粒体和高尔基体,细胞合成代谢旺盛。单独培养的髓核细胞胞体小,贴壁、增殖慢,细胞器不丰富。②髓核细胞Ⅱ型胶原蛋白与聚集蛋白聚糖检测结果:RT-PCR和Western blot检测显示诱导培养7,14d骨髓基质干细胞与髓核细胞共同培养组的Ⅱ型胶原蛋白、聚集蛋白聚糖条带均明显亮于单独髓核细胞培养组,吸光度值比较差异有显著性意义(P<0.01)。免疫组化检测显示骨髓基质干细胞与髓核细胞共同培养组中Ⅱ型胶原蛋白和聚集蛋白聚糖在细胞胞浆中的表达随时间延长而逐渐增加,2周达到高峰,单独髓核细胞培养组中此两种蛋白表达很低,且培养第7,14天无明显变化。结论:骨髓基质干细胞在体外与髓核细胞共同培养时,能激活髓核细胞并促进其增殖分化,增加髓核细胞外基质的合成。提示骨髓基质干细胞与髓核细胞联合培养有利于退变椎间盘的髓核细胞修复、再生。     

膝关节骨性关节炎注射玻璃酸钠穿刺点选择的临床分析

目的研究膝关节注射玻璃酸钠时穿刺点的选择对中老年骨性关节炎疗效的影响。方法共60例中老年骨性关节炎患者纳入研究。受试者被随机按穿刺点分为髌上囊组、膝眼组及髌旁组,各20例,分析各组注射时视觉模拟评分法(VAS)评分、关节积液抽吸量及当周lysholm评分。结果各组随着治疗时间、lysholm评分呈正相关增长,组间评分比较差异无显著性。髌上囊组及膝眼组在注射4周后,关节积液抽吸人均量分别为(14.75±3.1)ml和(15.0±1.7)ml,与髌旁组的(1.75±0.30)ml相比差异有统计学意义(P<0.05)。注射时VAS疼痛评分结果示:髌上囊组,膝眼组,髌旁组各组失败率分别为15.63%,10.23%及0。前两组与髌旁组评分相比差异有统计学意义(P<0.05)。结论膝关节穿刺点的选择不会影响关节注射的效果。推荐常规髌上囊及膝眼穿刺注射,注射失败首选髌旁注射补救。     

新型国产髋关节假体用于人工髋关节置换的随机对照研究

目的 通过随机对照临床试验,评价一种新型国产髋关节假体用于人工全髋关节置换术(total hip arthroplasty,THA)的临床疗效和安全性.方法 本研究采用多中心、随机、单盲、阳性平行对照设计,在全国5家医院共招募72例受试者,分别纳入试验组和对照组,各36例.试验组使用新型国产髋关节假体,对照组使用成熟的...