神经生长因子抗体在小鼠膝关节炎疼痛模型中的作用研究

摘要 目的：探究神经生长因子（Nerve growth factor,NGF）抗体L148M在碘乙酸（Monoiodoacetate,MIA）诱导的膝关节炎（Kee osteoarthritis,KOA）小鼠模型中的作用机制。方法：随机将8周龄C57BL/6雄性小鼠分为对照组、MIA组和L148M组。采用关节腔注射20 mg/mL MIA诱导KOA小鼠模型。手术后2周,L148M组小鼠给予腹腔注射L148M（10 mg/kg）处理。通过苏木精-伊红（HE）染色、番红O染色、OARSI评分和Micro-CT分析评估小鼠膝关节软骨及软骨下骨组织形态学变化。通过qRT-PCR检测CCR2、MCP1、MMP-1、MMP-3、MMP-13、COL10、IL-1β和TNF-α的mRNA表达水平。通过Western blotting检测INOS、COX-2、Collagen II和Aggrecan的蛋白表达水平。通过免疫组织化学法分析VEGFA和Ang-1的蛋白表达水平。通过Micro-CT血管造影分析软骨下骨血管形成数量。结果：HE染色结果显示,L148M组小苏软骨细胞数和关节软骨厚度均高于MIA组。番红O染色显示,L148M组小鼠基质降解小于MIA组。L148M组OARSI评分显著低于MIA组（P<0.05）。micro-CT扫描结果表明,L148M组小鼠软骨和软骨下骨结构完整,没有明显病理损伤。qRT-PCR检测结果显示,与MIA组相比,L148M组小鼠COL10、MMP1、MMP3和MMP-13表达水平均显著降低（P<0.05）。Western blotting检测结果表明,与MIA组相比,L148M组小鼠Collagen II和Aggrecan表达水平升高,INOS和COX-2表达水平降低（P<0.05）。疼痛行为学分析显示,与MIA组相比,L148M组小鼠行进距离和机械刺激反应阈值均降低（P<0.05）。micro-CT血管造影分析显示,L148M小鼠组微血管数量和体积明显低于MIA组（P<0.05）。免疫组化检测显示,L148M组小鼠VEGFA和Ang-1蛋白表达水平均显著低于MIA组（P<0.05）。结论：L148M通过抑制软骨下骨异常血管生成,缓解关节炎症疼痛;并通过抑制炎症细胞因子表达,减轻软骨和软骨下骨病理损伤。     

钙蛋白酶抑制剂Calpeptin在脓毒症膈肌功能障碍中的作用研究

摘要 目的：探究钙蛋白酶抑制剂Calpeptin在脓毒症膈肌功能障碍中的保护作用及其分子机制。方法：通过腹腔内注射8 mg/kg LPS的方法构建大鼠脓毒症模型,将雄性SD大鼠分为3组（n=6/组）：正常对照组（Con组）、脓毒症组（Sepsis组）和钙蛋白酶抑制剂预处理组（Calpeptin组）。处死大鼠,快速分离大鼠膈肌组织,采用苏木精-伊红(HE)染色法检测膈肌组织病理学改变,通过实时荧光定量PCR法（qRT-PCR）分别检测膈肌组织中钙蛋白酶μ-Calpain、凋亡相关基因天冬氨酸特异性半胱氨酸蛋白酶-3（Caspase-3）、自噬相关蛋白Beclin-1、肿瘤坏死因子-α（TNF-α）和白介素-6（IL-6）mRNA表达水平。结果：与正常对照组相比,LPS处理24 h的脓毒症组大鼠膈肌组织HE染色未见明显膈肌萎缩改变,但膈肌收缩力下降,这与我们以往的研究结果一致。qRT-PCR法检测到脓毒症组大鼠膈肌组织中上述基因mRNA表达量明显增加（P<0.05）,而Calpeptin预处理后,上述基因mRNA表达水平显著下降（P<0.05）。结论：脓毒症时膈肌发生功能障碍,钙蛋白酶抑制剂Calpeptin可显著减轻LPS诱导的炎症、凋亡、自噬的激活。     

Sunitinib对哮喘气道重塑小鼠PDGFR-β磷酸化和MMP-9表达的影响

目的:探讨sunitinib对支气管哮喘气道重塑的干预作用及可能的作用机制.方法:BALB/c小鼠随机分为正常对照组、哮喘气道重塑组、sunitinib干预组.鸡卵清蛋白致敏和激发建立哮喘小鼠气道重塑模型;收集支气管肺泡灌洗液(BALF)做细胞计数;取右肺组织行苏木精-伊红(HE)染色和Masson三色染色;免疫沉淀(IP法)测定小鼠肺组织PDGFR-β受体磷酸化及westernblot(WB)法检测MMP-9蛋白质的表达.结果:HE和Masson三色染色提示哮喘组黏膜下层和平滑肌增厚、气道管腔狭窄、胶原纤维增生、大量炎症细胞浸润,sunitinib干预组上述改变较哮喘组为轻;哮喘组BALF中炎症细胞总数和嗜酸性粒细胞(EOS)计数在哮喘组表达较对照组为高(P＜0.05),而sunitinib组低于哮喘组(P＜0.05);PDGFR-β受体的磷酸化和MMP-9的表达在哮喘组表达较对照组为高(P＜0.01),而sunitinib干预组表达量低于哮喘组(P＜0.01).结论:sunitinib能够抑制哮喘小鼠PDGFR-β的磷酸化和MMP-9表达,减轻气道炎症反应、延缓气道重塑进程.     

蛋白酶体抑制剂乳胞素(LAC)对LPS诱导的人关节软骨细胞炎症反应的抑制作用

目的:探讨蛋白酶体抑制剂乳胞素(LAC)对LPS诱导的人关节软骨细胞炎症反应的抑制作用.方法:体外培养原代人关节软骨细胞,分为四组,即对照组、LPS组(100 ng/mL LPS处理6小时)、LAC组(5μM LAC处理1小时)和LAC+LPS组(5μMLAC预处理1小时后,给予LPS处理6小时),分别用ELISA和western blot检测和比较各组细胞内20s蛋白酶体,NF-κB,TNF-α和iNOS的含量.结果:LPS处理后的人关节软骨细胞内20s蛋白酶体,NF-κB,TNF-α和iNOS的表达均较对照组显著升高(P＜0.05),LAC+LPS组以上指标较LPS组均显著降低(P＜0.05),而LAC组和LAC+LPS组以上指标与对照组比较均无显著性差异(P＞0.05).结论:LAC可显著抑制LPS诱导的人关节软骨细胞炎症反应.     

使用透明质酸钠复合海藻酸钠水凝胶球3D培养软骨细胞抑制细胞去分化的研究

目的:验证海藻酸钠(Alginate,Alg)水凝胶球添加透明质酸钠(Sodium hyaluronate,HA)后,对大鼠膝软骨细胞体外3D培养时软骨细胞去分化的抑制情况。方法:用Ⅱ型胶原酶消化法获取2周龄SD大鼠膝关节原代软骨细胞,体外培养传代至P2。细胞分为2组,1组用2%海藻酸钠水凝胶球3D培养(Alg组),另一组用2%海藻酸钠+1%透明质酸钠的水凝胶球3D培养(Alg/HA组),采用正交法制作海藻酸钠水凝胶球。培养14天后,两组各取水凝胶球进行死活细胞染色观察细胞状态。其他水凝胶球收集后用4%多聚甲醛固定,30%蔗糖溶液脱水,OCT(Optimal cutting temperature compound)包埋剂包埋切片,采用苏木精-伊红染色法(Hematoxylin-eosin staining,HE染色)、甲苯胺蓝染色观察软骨细胞形态;采用阿利新蓝染色观察对比糖胺聚糖(Glycosaminoglycan,GAG)含量;采用免疫荧光染色对比Ⅱ型胶原(Collagen typeⅡ,ColⅡ)含量;采用蛋白质免疫印迹法(Western blot)检测软骨细胞ColⅡ和聚蛋白多糖(Aggrecan,ACAN)的表达情况。结果:死活细胞染色显示两组水凝球内软骨细胞状态良好,几乎无死细胞;阿利新蓝染色显示Alg/HA组与Alg组相比软骨细胞分泌更多的GAG;免疫荧光染色显示Alg/HA组比Alg组软骨细胞内含更多的Ⅱ型胶原;Western blot显示Alg/HA组软骨细胞比Alg组ColⅡ的蛋白表达更多。结论:含透明质酸钠的海藻酸钠水凝胶可以更好地维持软骨细胞的表型,抑制软骨细胞去分化。     

胰高血糖素样肽l对脂多糖诱导的血管内皮细胞炎性反应的影响

目的:探讨胰高血糖素样肽l(glucagon like peptide 1,GLP-1)对脂多糖(1ipopolysaccharide,LPS)诱导的血管内皮细胞(VEC)炎性反应的影响。方法:以体外培养的人动脉VEC为研究模型,将细胞分为四组(对照组、LPS刺激组、LPS+GLP-1组、GLP-1组),Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,用苏木素-伊红(HE)染色观察细胞间连接的形态特征,用示踪剂Rhodamine B isothiocyanate-Dextran检测VECs单层通透性变化改变,酶联免疫吸附实验检测细胞分泌白介素(IL)-6和IL-8的变化。结果:GLP-1(100 nM)可减少LPS(1μg/mL)刺激后细胞肌动蛋白骨架F-actin应力纤维的形成,并抑制LPS刺激后细胞间连接的中断。Rhodamine B isothiocyanate-Dextran细胞通透性检测结果显示:GLP-1可明显降低LPS刺激引起的VEC通透性增加[由(2.57±0.19)×10-5cm/s降至(2.10±0.18)×10-5cm/s,P0.05]。此外,GLP-1可抑制LPS刺激后VEC中炎性细胞因子IL-6和IL-8的表达[分别由(42130±6522)pg/ml降至(27478±5096)pg/ml和(18376±1561)pg/ml降至(14414±927)pg/ml,均P0.05]。结论:GLP-1可对抗LPS刺激引起的VEC炎症反应和细胞通透性增加,改善LPS诱导的内皮细胞炎性损伤。     

PH797804对小鼠急性肝衰竭的保护作用及机制研究

目的:研究PH797804对脂多糖/D-氨基半乳糖氨(Lipopolysaccharide/D-galactosamine, LPS/D-Gal)诱导的急性肝衰竭(Acute liver failure, ALF)小鼠的保护作用及机制。方法:将64只小鼠随机分成四组,每组16只。腹腔注射LPS/D-Gal诱导建立小鼠ALF模型,设立生理盐水对照组(a)、LPS/D-Gal诱导模型组(b)、LPS/D-Gal诱导和PH797804干预组(c)及PH797804处理对照组(d)。观察小鼠48 h内的存活率,检测血清中谷丙转氨酶(ALT)和谷草转氨酶(AST)的含量,HE染色组织切片观察肝脏病理变化,Elisa检测血清中TNF-α和IL-6的表达水平,并对各组小鼠肝组织进行高通量转录组测序以探寻作用机制,采用Real-time PCR法验证测序结果。结果:相比于ALF模型组小鼠,PH797804处理后c组小鼠48 h存活率从20%提高至80%,小鼠肝脏组织病理学异常显著改善,血清中ALT、AST含量较ALF模型组相比显著降低(P0.01),血清中TNF-α和IL-6等促炎细胞因子含量较ALF模型组相比显著降低(P0.01)。RNA测序结果提示PH797804对LPS/D-Gal诱导小鼠ALF的保护作用主要与抑制炎症反应相关。Real-time PCR验证了测序结果的可靠性。结论:PH797804对LPS/D-Gal诱导的小鼠ALF具有保护作用,其作用机制可能与抑制炎症反应有关。     

原儿茶酸对脂多糖诱导的急性肺损伤小鼠的保护作用

目的:探索原儿茶酸(protocatechuicacid,PCA)对脂多糖(lipopolysaccharide,LPS)诱导的急性肺损伤(acute lung injury,ALI)小鼠的保护作用,探讨其保护机制。方法:将40只昆明小鼠按随机数字表法均分为空白对照组(NC组)、LPS模型组、原儿茶酸预处理组(PCA+LPS组)、地塞米松阳性对照组(Dex+LPS组),每组10只,模型组以5mg·kg-1脂多糖腹腔内注射诱导急性肺损伤。6h后处死小鼠,HE染色观察肺组织病理学变化;BCA法检测肺泡灌洗液中总蛋白浓度;ELISA检测肺泡灌洗液炎症因子TNF-α、IL-1β含量;Western Blot检测肺组织中p38MAPK、p-p38MAPK、p-ATF2蛋白的表达水平。结果:与对照组相比,模型组小鼠肺损伤明显,肺泡内出血、水肿、炎细胞浸润,肺泡灌洗液中TNF-α、IL-1β的含量及总蛋白浓度增加,肺组织中p38MAPK/p-p38MAPK、p-ATF2表达均明显增加(均P0.01)。与模型组相比,原儿茶酸预处理组、地塞米松阳性对照组肺组织病理损伤程度明显减轻,肺泡灌洗液中TNF-α、IL-1β的含量及总蛋白浓度、肺组织中p38MAPK/p-p38MAPK、p-ATF2表达均明显降低(均P0.01)。结论:PCA对LPS诱导的急性肺损伤有保护作用,其作用机制可能与其抑制p38MAPK-p-ATF2信号通路的活化、降低肺组织炎症反应有关。     

HSF1基因剔除对HSR抗内毒素血症的影响

利用内毒素(LPS)血症小鼠模型,观察HSF1基因剔除对热休克反应(HSR)保护作用的影响.采用腹腔注射LPS建立内毒素血症小鼠模型,HSR采用肛温42℃维持15 min,室温恢复24 h,利用RT-PCR、苏木素-伊红(HE)染色、丙二醛测定以及死亡率,计算和分析重要脏器组织中炎症介质基因的表达、脏器损伤程度及小鼠存活率.注射LPS 15mg/kg 72 h后HSR LPS(HSF1 / )组存活率(7/15)显著高于LPS(HSF1 / )组(0/15)、LPS(HSF1-/-)组(0/14)和HSR LPS(HSF1-/-)组(0/14),而注射LPS 14 mg/kg 72 h后,LPS(HSF1 / )组存活率(5/15)显著高于LPS(HSF1-/-)组(0/13)和HSR LPS(HSF1-/-)组(0/13).在注射LPS 12 h后LPS(HSF1 / )组、LPS(HSF1-/-)组和HSR LPS(HSF1-/-)组的心、肺组织丙二醛含量显著升高,但HSR LPS(HSF1 / )组不升高.肺组织炎症介质基因IL-IB、IL-6、TNF-α、CCL-2、SOCS3、MCSF、GCSF、IL-15在LPS(HSF1-/-)组和LPS(HSF1 / )组表达上调,HSR LPS(HSF1-/-)组除IL-15较低外其他上调更甚,HSR LPS(HSF1 / )组除IL-1β和TNF-α较高外其他显著下调.注射LPS后LPS(HSF1 / )组和LPS(HSF1-/-)组的肺、肝、肾病理形态改变明显,HSR LPS(HSF1 / )组改变较轻,HSR LPS(HSF1-/-)组改变更加严重.HSF1基因剔除能显著消减HSR对内毒素血症小鼠的保护作用.     

虫草素对786-O和ACHN肾癌细胞系增殖、迁移和凋亡机制探究

探究肾癌细胞系经虫草素给药刺激后,细胞凋亡及迁移的机制。体外培养肾癌786-O细胞系和肾癌ACHN细胞系,采用MTT法、细胞迁移实验、HE染色、免疫荧光染色及蛋白免疫印记法;检验不同浓度虫草素处理肾癌细胞系增殖抑制率、细胞迁移情况、细胞形态变化和细胞核形态差异,检验凋亡相关基因蛋白表达情况,探究虫草素诱导肾癌细胞的凋亡机制。随着浓度剂量提高,虫草素能够显著促进肾癌细胞系凋亡,抑制迁移。形态学研究表明,HE染色观察发现癌细胞数量明显降低,并且细胞核明显变大。免疫荧光染色发现MMP-2、MMP-9和BCL-2蛋白表达显著降低,Bax、Casepase-3和Casepase-7蛋白表达显著增加,肾癌细胞可通过AKT/mTOR信号通路诱导肾癌细胞凋亡。     

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1β-induced inflammation in articular chondrocytes via the JNK signaling pathway

Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.     

Pioglitazone inhibits advanced glycation end product-induced matrix metalloproteinases and apoptosis by suppressing the activation of MAPK and NF-κB

Apoptosis and degeneration coming mainly from chondrocytes are important mechanisms in the onset and progression of osteoarthritis. Specifically, advanced glycation end products (AGEs) play an important role in the pathogenesis of osteoarthritis. Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist has a protective effect on cartilage. This study aims to evaluate the effect of pioglitazone on AGEs-induced chondrocyte apoptosis and degeneration and their underlying mechanism. The in vitro study shows that AGEs induce cleavage of caspase-3 and PARP, up-regulate MMP-13 expression, enhance chondrocyte apoptosis and down-regulate PPARγ expression in human primary chondrocytes, which is reversed by pioglitazone. Furthermore, AGEs activate phosphorylation of Erk, JNK, and p38, and pioglitazone reverses AGEs-induced phosphorylation of Erk and p38. AGEs-induced degradation of IκBα and translocation of nuclear NF-κB p65 is reversed by pioglitazone. Pretreatment of chondrocytes with SB202190 (p38 inhibitor), SP600125 (JNK inhibitor) and BAY-11-7082 (NF-κB inhibitor) inhibit AGEs-induced apoptosis and degeneration. In vivo experiments suggest that pioglitazone reverses AGEs-induced cartilage degeneration and apoptosis in a mouse model, as demonstrated by HE and Safranin O staining, immunohistochemical analyses of Type II collagen (Col II), metalloproteinases (MMPs) and caspase-3. These findings suggest that pioglitazone, a PPARγ agonist, inhibits AGEs-induced chondrocytes apoptosis and degeneration via suppressing the activation of MAPK and NF-κB.     

circPPP1R12A激活p53信号通路调控骨关节炎中软骨细胞增殖和凋亡

摘要 目的：探讨circPPP1R12A（circ_0000423）调控p53信号通路对骨关节炎（osteoarthritis,OA）中软骨细胞增殖和凋亡的影响。方法：采用qRT-PCR检测circPPP1R12A在OA软骨细胞中的表达水平。在OA软骨细胞中分别转染oe-circPPP1R12A和sh-circPPP1R12A后,采用CCK-8检测细胞增殖情况;免疫荧光检测Ki-67阳性细胞表达率;流式细胞术检测细胞凋亡情况;qRT-PCR检测Ki-67和p53表达水平;Western Blot检测Cleaved-caspase3、P53、BCL-2和BAX的表达水平。结果：OA软骨细胞中circPPP1R12A的表达水平明显高于正常软骨细胞。过表达circPPP1R12A能够抑制OA软骨细胞增殖和促进细胞凋亡,通过上调p53表达激活p53信号通路,低表达circPPP1R12A能够促进OA软骨细胞增殖和抑制细胞凋亡,通过下调p53表达阻滞p53信号通路。在OA软骨细胞中同时低表达circPPP1R12A和过表达p53能够反转单独低表达circPPP1R12A对OA软骨细胞增殖和凋亡的影响。结论：circPPP1R12A在OA软骨细胞中明显高表达,circPPP1R12A能够通过激活p53信号通路抑制骨OA软骨细胞增殖和促进软骨细胞凋亡。circPPP1R12A可能成为OA治疗的干预靶点。     

虫草素对四氯化碳所致小鼠急性肝损伤的保护作用

本文探究蛹虫草活性成分虫草素对四氯化碳（CCl₄）造成的小鼠急性肝损伤的保护作用及其分子机制。首先建立四氯化碳致小鼠急性肝损伤的动物模型,通过检测血清生化指标、肝功指标的变化及HE染色观察组织切片病理的病变情况,评价虫草素的保肝效果,进一步通过Western blot检测虫草素能否通过激活Nrf-2/Keap1信号通路及其下游抗氧化因子（HO-1、NQO-1）的表达来提高机体抗氧化损伤能力以及抑制炎症因子（TNFα、TNFβ、IL-6、IL-10）的表达。对比模型组结果显示,虫草素能极显著降低（P<0.01）小鼠血清中ALT、AST及肝脏中MDA水平,并能极显著提高肝脏中SOD水平（P<0.01）;HE染色结果显示虫草素能有效降低改善受损肝组织中的炎细胞浸润及纤维组织增生;Western blot结果表明虫草素能够通过激活Nrf-2信号通路,促进下游抗氧化因子及抗炎因子的表达,从而降低炎症反应。虫草素对CCl₄致小鼠急性肝损伤具有一定的保护作用,其机制与Nrf-2信号通路相关,实验结果为后续蛹虫草及虫草素的开发应用奠定基础。     

miR-382-3p靶向RASA1基因调控骨关节炎软骨细胞增殖和凋亡

目的:探讨mi R-382-3p对骨关节炎软骨细胞增殖和凋亡的影响及其机制。方法:用100 ng/mL的脂多糖(LPS)处理软骨细胞,记为LPS组,以正常培养的软骨细胞作为正常对照(NC)组。mi R-NC、mi R-382-3p、anti-miR-NC、anti-miR-382-3p转染至软骨细胞中,记为mi R-NC组、mi R-382-3p组、anti-miR-NC组、anti-miR-382-3p组;将mi R-NC、mi R-382-3p、si-NC、si-RASA1转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-NC+LPS组、mi R-382-3p+LPS组、si-NC+LPS组、si-RASA1+LPS组;将mi R-382-3p分别与pcDNA-NC、pcDNA-RASA1共转染至软骨细胞后再用100 ng/mL的LPS处理,记为mi R-382-3p+pcDNA-NC+LPS组、mi R-382-3p+pcDNA-RASA1+LPS组。实时荧光定量PCR(RT-qPCR)检测mi R-382-3p和Ras p21蛋白活化因子1(RASA1)m RNA表达水平;蛋白质印迹(Western blot)法检测RASA1、细胞周期蛋白D1(CyclinD1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-caspase-3)蛋白表达;四甲基偶氮唑盐比色法(MTT)检测细胞存活率;流式细胞术检测细胞凋亡;荧光素酶报告实验检测mi R-382-3p和RASA1的靶向关系。结果:LPS诱导的软骨细胞中mi R-382-3p表达水平显著降低,RASA1表达水平显著升高,CyclinD1表达水平显著降低,Cleaved-caspase-3表达水平显著升高,细胞存活率显著降低,细胞凋亡率显著升高(P0.05)。过表达mi R-382-3p和敲减RASA1,LPS诱导的软骨细胞中CyclinD1表达水平显著升高,Cleaved-caspase-3表达水平显著降低,细胞存活率显著升高,细胞凋亡率显著降低(P0.05)。mi R-382-3p靶向调控RASA1,高表达RASA1部分逆转了mi R-382-3p高表达对LPS处理的软骨细胞增殖和凋亡的影响。结论:过表达mi R-382-3p促进软骨细胞增殖,抑制LPS诱导的软骨细胞凋亡,其机制可能与RASA1有关。     

虫草素对肺癌细胞生长及迁移的影响

探讨虫草素对非小细胞肺癌细胞株H1781细胞凋亡及迁移的影响及作用机制。培养H1781细胞并分组,对照组用不含药物的培养基处理,虫草素组用含有10、20、30和40 μmol/L虫草素处理,处理24 h后测定细胞活力,通过显微镜观察细胞形态学,HE染色观察虫草素对细胞整体的影响,细胞免疫荧光技术检测细胞中MMP-9和DAPI核染色情况观察细胞凋亡,Western blotting检测凋亡等相关蛋白表达。与对照组相比,虫草素处理24 h后,H1781细胞系活力显著降低;细胞数量明显减少;HE染色观察发现随着虫草素浓度增加,细胞数量及细胞集团明显变少,免疫荧光技术检测发现药物处理后细胞凋亡明显促进;划痕实验发现虫草素明显降低细胞迁移能力;Western blotting实验中Bax、cleaved caspase-3蛋白表达明显上调,MMP-9、Bcl-2蛋白表达明显下调。虫草素对肺癌H1781细胞迁移有抑制作用、对凋亡有促进作用,推测其作用机制为上调促凋亡蛋白表达、下调抗凋亡蛋白表达。     

A role for age-related changes in TGFβ signaling in aberrant chondrocyte differentiation and osteoarthritis

Transforming growth factor beta (TGFβ) is a growth factor with many faces. In our osteoarthritis (OA) research we have found that TGFβ can be protective as well as deleterious for articular cartilage. We postulate that the dual effects of TGFβ on chondrocytes can be explained by the fact that TGFβ can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFβ not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFβ on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFβ signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFβ signaling in OA development.     

17β-estradiol reduces expression of MMP-1, -3, and -13 in human primary articular chondrocytes from female patients cultured in a three dimensional alginate system

Clinical observations have suggested a relationship between osteoarthritis and a changed sex-hormone metabolism, especially in menopausal women. This study analyzes the effect of 17β-estradiol on expression of matrix metalloproteinases-1, -3, -13 (MMP-1, -3, -13) and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, -2) in articular chondrocytes. An imbalance of matrix metalloproteinases (MMPs) specialized on degradation of articular cartilage matrix over the respective inhibitors of these enzymes (TIMPs) that leads to matrix destruction was postulated in the pathogenesis of osteoarthritis. Primary human articular chondrocytes from patients of both genders were cultured in alginate beads at 5% O(2) to which 10(-11)M-10(-5)M 17β-estradiol had been added and analyzed by means of immunohistochemistry, immunocytochemistry and real-time RT-PCR. Since articular chondrocytes in vivo are adapted to a low oxygen tension, culture was performed at 5% O(2). Immunohistochemical staining in articular cartilage tissue from patients and immunocytochemical staining in articular chondrocytes cultured in alginate beads was positive for type II collagen, estrogen receptor α, MMP-1, and -13. It was negative for type I collagen, MMP-3, TIMP-1 and -2. Using real-time RT-PCR, it was demonstrated that physiological and supraphysiological doses of 17β-estradiol suppress mRNA levels of MMP-3 and -13 significantly in articular chondrocytes of female patients. A significant suppressing effect was also seen in MMP-1 mRNA after a high dose of 10(-5)M 17β-estradiol. Furthermore, high doses of this hormone led to tendentially lower TIMP-1 levels whereas the TIMP-2 mRNA level was not influenced. In male patients, only incubations with high doses (10(-5)M) of 17β-estradiol were followed by a tendency to suppressed MMP-1 and TIMP-1 levels while TIMP-2 mRNA level was decreased significantly. There was no effect on MMP-13 expression of cells from male patients. Taken together, application of 17β-estradiol in physiological doses will improve the imbalance between the amounts of MMPs and TIMPs in articular chondrocytes from female patients. Downregulation of TIMP-2 by 17β-estradiol in male patients would not be articular cartilage protective.