生物学因子p-p38、MMP-3、MMP-13对大鼠关节软骨的破坏作用

目的 检测大鼠骨性荚节炎(OA)中对软骨起破坏作用的细胞因子.方法 大鼠20只,以任一后腿膝关节作为实验组(OA),另一侧为手术对照组.OA组离断大鼠前交叉韧带及切除内外侧半月板前半部分建立大鼠骨性关节炎模型;对照组只切开关节囊,打开关节腔,缝合切口即可.饲养8周摄X线片后取关节软骨观察形态学的改变,免疫组化检测p-p38以及基质金属蛋白酶3(MMP-3)、基质金属蛋白酶13(MMP-13)的表达.结果 与对照组相比,OA组膝关节p-p38、MMP-3及MMP-13阳性细胞强表达,软骨被破坏,关节表面粗糙;对照组p-p38、MMP-3、MMP-13表达明显减少,软骨完整,关节表面光滑.结论 大鼠骨性关节炎软骨细胞中因生物学因子p-p38、MMP-3、MMP-13的过度表达与骨性关节炎软骨破坏有关.     

骨关节炎关节软骨自然退变过程中Dvl2及β-catenin表达变化

目的 探讨Dishevelled (Dvl)和 β-catenin在骨性关节炎(OA)关节软骨细胞自然退变中的表达情况。方法 将膝关节置换的OA患者关节软骨细胞进行体外分离培养,采用自然传代的方法建立关节软骨退变模型。倒置显微镜观察P2、P6代关节软骨细胞的形态,流式细胞仪检测软骨细胞的凋亡情况。利用Q-PCR及WB检测Dvl2和β-catenin基因和蛋白的表达情况。结果 成功分离并进行体外培养OA患者关节软骨细胞。P2代关节软骨细胞呈三角形或多角形;P6代时形态则变为梭形,并出现明显的纤维化。P6代时凋亡率显著高于P2代的凋亡率(16.34% VS 8.63%)。P2代Dvl2的mRNA相对表达量为P6代的3.629倍(p=0.004),蛋白表达量为P6代的2.548倍(p=0.03;β-catenin的mRNA相对表达量为P6代的5.087倍(p=0.001), 蛋白表达量为P6代的4.4倍(p=0.003)。     

CP-25通过抑制GRK2活性改善小鼠骨关节炎的机制

目的 研究芍药苷-6-氧-苯磺酸酯(CP-25)通过抑制GRK2活性对骨关节炎(osteoarthritis, OA)小鼠膝关节软骨的保护作用。方法 内侧半月板失稳(destabilization of the medial meniscus, DMM)手术诱导构建小鼠骨关节炎模型,实验分为假手术组、模型组、CP-25给药组和帕罗西汀给药组。术后开始灌胃给药。给药12周处死动物,Micro-CT成像观察膝关节软骨退变、骨重塑异常等情况,番红固绿染色观察小鼠关节组织病理,免疫组化、免疫荧光检测软骨组织相关分子表达水平的影响。Western blot检测CP-25用药后软骨细胞的膜蛋白及总蛋白表达水平。结果 模型小鼠关节软骨严重退变。CP-25可显著降低关节软骨骨赘数量及软骨下板厚度,促进软骨基质再生,减少软骨基质降解蛋白表达,对膝关节软骨有明显的保护作用。免疫组化和免疫荧光结果显示,CP-25治疗可显著降低膝关节组织中GRK2、ADAMTS5、MMP13的表达,并且升高膝关节组织中ColⅡ、Aggrecan表达。体外实验结果表明,CP-25给药可以显著降低GRK2的膜蛋白及总蛋白表达水平...     

通痹灵对胶原诱导型关节炎大鼠软骨及其细胞代谢的影响

目的探讨中药通痹灵(TBL)抗软骨破坏的作用机制。方法以胶原诱导型关节炎(CIA)大鼠为动物模型,分组灌胃治疗后,做大鼠趾关节病理切片,常规HE染色,观察软骨病变;培养软骨细胞,以鼠重组白细胞介素-1β(rrIL-1β)体外刺激软骨细胞导致软骨细胞基质降解作为药理模型,加用不同浓度的通痹灵总碱,并以地塞米松作为对照组,培养48h后,收集细胞上清液,用阿利新蓝法检测葡糖胺聚糖(GAG)含量;用硝酸酶还原法检测NO的含量。结果①趾关节病理HE切片评价结果显示:通痹灵总碱组软骨破坏程度小于造模组(P<0.05〉;②不同浓度的rrIL-1β(1、10、100、500、1000ng/ml)促进了软骨细胞蛋白多糖的降解(P<0.01),且呈量效关系;③不同浓度的通痹灵总碱(200、100、50、25mg/L)及地塞米松可明显地抑制软骨细胞蛋白多糖的降解(P<0.01)及NO的产生(P<0.01)。结论通痹灵总碱可抑制CIA大鼠趾关节软骨破坏;体外实验结果显示通痹灵总碱可对抗软骨细胞蛋白多糖的降解,其作用途径可能是通过抑制NO的生成来实现的。     

p38在大鼠骨性关节炎模型中的表达

目的 检测大鼠骨性关节炎﹙OA﹚软骨细胞中丝裂素活化蛋白激酶(MAPK)p38的表达.方法 取大鼠20只,雌雄各半,以任一后腿膝关节作为实验组(OA)组,另一侧为手术对照(C)组.OA组离断大鼠前交叉韧带及切除内外侧半月板前半部分建立大鼠骨性关节炎模型;C组仅打开关节腔,缝合切口.饲养8周后摄X线片后取关节软骨观察,通过HE、甲苯胺蓝(PG)观察软骨形态学的改变,免疫组织化学染色及免疫印迹(Western blot)检测磷酸化p38(P-p38).结果 与C组相比,OA组膝关节软骨表面粗糙,软骨被破坏;C组关节表面光滑.免疫组织化学染色及Western blot检测OA组膝关节P-p38阳性细胞表达明显增多.结论 大鼠骨性关节炎模型软骨细胞中p38 MAPK被激活.p38 MAPK被激活可能是造成骨性关节炎的又一环节.     

Wnt通路相关蛋白在胃癌前病变组织中的表达和临床意义

目的 探讨Wnt通路相关蛋白在胃癌前病变发生、发展中的作用以及在胃癌早期诊断中的意义.方法 选取2012年6月-2015年6月胃癌前病变胃镜活检标本256例(20例轻度异型增生、44例中度异型增生、52例重度异型增生、140例肠上皮化生),同时选取45例正常胃黏膜组织标本作为对照组.采用免疫组织化学方法对各组组织标本中Wnt通路相关蛋白Wnt1、β-连环蛋白(β-catenin)、结肠腺瘤性息肉病蛋白(APC)和细胞周期蛋白D1(cyclinD1)蛋白表达水平进行检测.分析各组Wnt通路相关蛋白的表达水平及阳性表达率.结果 各观察组Wnt1、β-catenin和cyclin D1蛋白表达水平高于对照组,且APC蛋白水平显著低于对照组(P＜0.05).中度和重度异型增生组Wnt1、β-catenin和cyclin D1蛋白表达水平高于轻度异型增生组和肠上皮化生组,而APC蛋白水平低于轻度异型增生组和肠上皮化生组(P＜0.05).重度异型增生组Wnt1、β-catenin和cyclin D1蛋白表达水平高于中度异型增生组,而APC蛋白水平低于中度异型增生(P＜0.05).与对照组比较,Wnt1、β-catenin和cyclin D1在各观察组中的阳性表达率增加(P＜0.05),APC阳性表达率下降(P＜0.05);中度和重度异型增生的各蛋白阳性表达率与轻度异型增生组差异比较具有统计学意义(P＜0.05).结论 Wnt通路相关蛋白在胃癌前病变组织中呈异常表达,从而激活了Wnt通路,参与了胃癌的发生和发展,对其进行联合检测有助于胃癌的早期诊断.     

豆腐果苷对关节失稳模型骨关节炎的作用及机制北大核心CSCD

目的研究豆腐果苷对关节失稳模型骨关节炎(osteoarthritis,OA)的治疗作用,探讨豆腐果苷治疗OA的作用机制。方法通过切除大鼠膝关节内侧半月板的方法(medial meniscectomy,MMx)建立OA大鼠模型。术后给予豆腐果苷治疗后,通过HE和番红O-固绿染色法观察膝关节软骨组织的组织病理学变化;免疫组化法检测膝关节软骨和滑膜组织中瞬时受体电位香草酸亚型1(Trpv1)表达;Western blot法检测大鼠膝关节滑膜组织中Trpv1蛋白的表达情况。结果HE和番红O-固绿染色法显示,豆腐果苷治疗明显减缓膝关节软骨的退变情况。免疫组化结果显示,豆腐果苷明显降低膝关节软骨和滑膜中Trpv1蛋白的表达水平;Western blot结果证实豆腐果苷下调膝关节滑膜组织中Trpv1蛋白的表达量。结论豆腐果苷能明显抑制MMx诱导的关节软骨的破坏和软骨基质的丢失,从而发挥治疗OA作用。     

Ck2α、β-catenin、c-IAP1及 survivin 联合检测在宫颈癌诊断中的临床意义

目的：研究酪蛋白激酶2α(Ck2α)、β-连环蛋白(β-catenin)、凋亡抑制蛋白(c-IAP1)及存活素(sur-vivin)在宫颈癌组织中的表达情况及其相关性,探讨四者联合检测在宫颈癌诊断中的临床意义。方法选择重庆市大足区人民医院收治的宫颈癌74例(宫颈癌组)、宫颈上皮内瘤变(CIN)83例(CIN 组)和子宫肌瘤50例(对照组),采用免疫组化法检测3组宫颈组织中 Ck2α、β-catenin、c-IAP1及 survivin 表达情况,分析其相关性及与临床病理特征的关系。结果宫颈癌组 Ck2α、β-catenin、c-IAP1、survivin 阳性表达率高于 CIN 组和对照组,且其表达与肿瘤分化程度及 FIGO 分期有关( P <0．05),Ck2α阳性表达率与β-catenin、c-IAP1、survivin 表达水平均呈正相关( r =0.570、0．391、0.476,P <0．05),Ck2α、β-catenin、c-IAP1、survivin 阳性表达率与分化程度呈负相关(r =-0.325、-0.633、-0．397、-0.324,P <0．05),与 FIGO 分期呈正相关(r =0.349、0.634、0.621、0.391,P <0．05)。结论 Ck2α、β-catenin、c-IAP1及 survivin 在宫颈癌组织中阳性表达率较高,其与宫颈癌的发生、发展有关,四者联合检测对早期诊断宫颈癌及临床治疗有重要意义。     

钙敏感受体在人骨关节炎软骨中的表达研究

【摘要】目的 研究钙敏感受体(CaSR)在人骨性关节炎(OA)软骨中的表达强度,探讨其与OA 发病机制的关 系。方法收集5 例因膝关节OA 接受全膝关节置换术患者的股骨远端及胫骨近端标本,HE 染色后根据Collins 病 理学分级标准进行关节软骨退变分级,Collins 0 级切片8 张为软骨正常组,Collins Ⅱ级切片8 张为软骨退变组,免疫 组织化学染色后比较2 组软骨组织内CaSR 的表达强度。结果CaSR 在OA 软骨中阳性表达,CaSR 在软骨正常组 的表达强度低于退变软骨组（评分：1.63±0.95 vs 3.52±0.78,t=8.99,P＜0.05）。结论CaSR 活化与关节软骨退变 有关。     

骨关节炎患者骨桥蛋白、IL-1β表达水平及意义

目的检测骨原发性关节炎（OA）患者骨桥蛋白（OPN）、IL-1β表达水平的变化及意义,并探讨其与骨关节炎的相关性。方法选取2012年1月-2014年1月确诊为原发性OA患者的软骨标本20例作为OA组,以同期因意外受伤截肢的20例正常软骨标本为对照组。采用HE染色法观察组织学变化,免疫组化法检测关节软骨中OPN、IL-1β表达水平,并采用SamplePCI图象分析系统进行半定量分析。结果正常软骨细胞外基质染色均匀,分散于软骨基质之中,细胞核着色清晰均匀,胞核、胞质清晰可辨。随着OA病变程度的加重软骨细胞基质染色不均,甚至出现纤维化,细胞数目逐渐减少。经免疫染色后,OPN、IL-1β吸光值表示OPN、IL-1β表达水平均升高（P〈0.05）。相关分析得出,OPN与IL-1β呈正相关（r=O.899,P〈0.01）。软骨病变程度分级与OPN、IL-1β呈正相关（r=0.909,0.904,P均〈0.01）。结论原发性OA患者软骨组织中OPN、IL-1β表达水平升高,且与病变程度具有密切联系,为OA治疗的靶点提供了依据。     

Synthesis,Cytotoxicity and Antileishmanial Activity of Some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines

We report herein the condensation of 4,7-dichloroquinoline (1) with tryptamine (2) and D-tryptophan methyl ester (3) . Hydrolysis of the methyl ester adduct (5) yielded the free acid (6) . The compounds were evaluated in vitro for activity against four different species of Leishmania promastigote forms and for cytotoxic activity against Kb and Vero cells. Compound (5) showed good activity against the Leishmania species tested, while all three compounds displayed moderate activity in both Kb and Vero cells.     

Efficacy of gut lavage,hemodialysis, and hemoperfusion in the therapy of paraquat or diquat intoxication

Clinical and in vitro investigations were carried out to test the efficacy of gut lavage, hemodialysis, and hemoperfusion in the treatment of poisoning with paraquat or diquat. In a patient suffering from diquat intoxication 130 times more diquat was removed by gut lavage 30 h after ingestion than was removed by complete aspiration of the gastric contents.Determination of in vitro clearances for paraquat and diquat by hemodialysis showed that, at serum concentrations of 1–2 ppm, such as are frequently encountered in poisoning in man, toxicologically relevant quantities of herbicide cannot be removed from the body. At a concentration of 20 ppm, on the other hand, hemodialysis proved to be effective, the clearance being 70 ml/min at a blood flow rate of 100 ml/min. The efficacy of hemoperfusion with coated activated charcoal was on the whole better. Especially at concentrations around 1–2 ppm, the clearance values for hemoperfusion were some 5–7 times higher than those for hemodialysis.In a patient suffering from paraquat poisoning, both hemodialysis as well as hemoperfusion were carried out. The in vitro results could be confirmed: At serum concentrations of paraquat less than 1 ppm no clearance could be obtained by hemodialysis while by hemoperfusion with activated charcoal quite high clearance values were measured and the serum level dropped down to zero.

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Zusammenfassung Klinische Untersuchungen und Laboratoriumsversuche wurden durchgeführt, um die Wirksamkeit von Darmspülung, Hämodialyse und Hämoperfusion bei Paraquat- und Deiquat-Vergiftungen zu prüfen.Bei einem Patienten wurde 30 Std nach Deiquat-Aufnahme durch Darmspülung 130mal mehr Deiquat entfernt als durch vollständige Aspiration des Mageninhaltes. In vitro-Versuche ergaben, daß bei Blutserumkonzentrationen von 1–2 ppm, die bei Vergiftungen oft gemessen werden, durch Hämodialyse keine toxikologisch relevanten Paraquat- oder Deiquat-Mengen entfernt werden können. Dagegen erwies sich die Hämodialyse bei 20 ppm und einer Blutumlaufgeschwindigkeit von 100 ml/min mit einer Clearance von 70 ml/min als wirksam. Die Hämoperfusion mit beschicheter Aktivkohle war in diesen Versuchen aber eindeutig überlegen, denn insbesondere bei Konzentrationen um 1–2 ppm waren die Clearance-Werte 5–7mal höher als bei der Hämodialyse.Die in vitro-Ergebnisse wurden bei einem Patienten mit einer Paraquat-Vergiftung bestätigt: Bei Konzentrationen unter 1 ppm war die Hämodialyse wirkungslos, während durch Hämoperfusion relativ hohe Clearance-Werte erreicht wurden, so daß der Serumspiegel rasch unter die Nachweisgrenze abfiel.

     

In vitro studies on sterically stabilized liposomes (SL) as enzyme carriers in organophosphorus (OP) antagonism

This study describes a new approach for organophosphorous (OP) antidotal treatment by encapsulating an OP hydrolyzing enzyme, OPA anhydrolase (OPAA), within sterically stabilized liposomes. The recombinant OPAA enzyme was derived from Alteromonas strain JD6. It has broad substrate specificity to a wide range of OP compounds: DFP and the nerve agents, soman and sarin. Liposomes encapsulating OPAA (SL)* were made by mechanical dispersion method. Hydrolysis of DFP by (SL)* was measured by following an increase of fluoride ion concentration using a fluoride ion selective electrode. OPAA entrapped in the carrier liposomes rapidly hydrolyze DFP, with the rate of DFP hydrolysis directly proportional to the amount of (SL)* added to the solution. Liposomal carriers containing no enzyme did not hydrolyze DFP. The reaction was linear and the rate of hydrolysis was first order in the substrate. This enzyme carrier system serves as a biodegradable protective environment for the recombinant OP-metabolizing enzyme, OPAA, resulting in prolongation of enzymatic concentration in the body. These studies suggest that the protection of OP intoxication can be strikingly enhanced by adding OPAA encapsulated within (SL)* to pralidoxime and atropine.     

Lung disease and PKCs

The lung offers a rich opportunity for development of therapeutic strategies focused on isozymes of protein kinase C (PKCs). PKCs are important in many cellular responses in the lung, and existing therapies for pulmonary disorders are inadequate. The lung poses unique challenges as it interfaces with air and blood, contains a pulmonary and systemic circulation, and consists of many cell types. Key structures are bronchial and pulmonary vessels, branching airways, and distal air sacs defined by alveolar walls containing capillaries and interstitial space. The cellular composition of each vessel, airway, and alveolar wall is heterogeneous. Injurious environmental stimuli signal through PKCs and cause a variety of disorders. Edema formation and pulmonary hypertension (PHTN) result from derangements in endothelial, smooth muscle (SM), and/or adventitial fibroblast cell phenotype. Asthma, chronic obstructive pulmonary disease (COPD), and lung cancer are characterized by distinctive pathological changes in airway epithelial, SM, and mucous-generating cells. Acute and chronic pneumonitis and fibrosis occur in the alveolar space and interstitium with type 2 pneumocytes and interstitial fibroblasts/myofibroblasts playing a prominent role. At each site, inflammatory, immune, and vascular progenitor cells contribute to the injury and repair process. Many strategies have been used to investigate PKCs in lung injury. Isolated organ preparations and whole animal studies are powerful approaches especially when genetically engineered mice are used. More analysis of PKC isozymes in normal and diseased human lung tissue and cells is needed to complement this work. Since opposing or counter-regulatory effects of selected PKCs in the same cell or tissue have been found, it may be desirable to target more than one PKC isozyme and potentially in different directions. Because multiple signaling pathways contribute to the key cellular responses important in lung biology, therapeutic strategies targeting PKCs may be more effective if combined with inhibitors of other pathways for additive or synergistic effect. Mechanisms that regulate PKC activity, including phosphorylation and interaction with isozyme-specific binding proteins, are also potential therapeutic targets. Key isotypes of PKC involved in lung pathophysiology are summarized and current and evolving therapeutic approaches to target them are identified.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.