TNF-α对人牙囊细胞表达单核细胞趋化蛋白-1的影响及其信号传导通路

目的研究不同浓度肿瘤坏死因子α(TNF-α)对体外培养人牙囊细胞表达单核细胞趋化蛋白-1(MCP-1)的影响及其信号传导通路。方法取第5代人牙囊细胞,分别与浓度为0(对照组)5、1、02、55、0、100ng/ml的TNF-α共孵育6h,夹心ELISA法检测上清液中MCP-1的含量,RT-PCR法检测MCP-1 mRNA表达的变化。另取第5代人牙囊细胞,分别加入25μmol/L SB203580、50μmol/L PD98059、15μmol/L SP600125,以阻断p38丝裂原活化蛋白激酶(p38MARK)、细胞外信号调节激酶(ERK)J、un氨基端激酶(JNK),培养30min后加入10ng/ml TNF-α,孵育6h后,RT-PCR检测MCP-1 mRNA含量。结果 ELISA结果显示,与对照组比较,TNF-α浓度为10～100ng/ml时,可显著增强MCP-1的分泌(P<0.05);RT-PCR结果显示,与对照组比较,TNF-α浓度为10～50ng/ml时,MCP-1表达增强,且此作用可被阻滞剂SP600125阻断。结论 TNF-α可增强MCP-1的基因表达、蛋白合成和分泌,此作用通过JNK信号转导途径实现。     

海马神经元损伤及保护的纳米级三维构像变化研究

目的：观察海马神经元细胞表面形态的三维构像变化，探讨抑制p38 MAPK信号通路对减轻红藻氨酸(KA)毒性作用引起大鼠海马神经元所造成损伤的作用和机制。方法：原代培养海马神经元给予SB203580(0．2μmol／L)预处理，30min后再予不同浓度(0，25和250μmoL／L)红藻氨酸分别作用10min和100min，利用原子力显微镜(AFM)对胞膜表面结构进行纳米级水平扫描和观测。结果：正常海马神经元表面光滑，起伏均匀、规律；红藻氨酸作用后神经元呈损伤性改变，表现为胞体肿胀，胞膜表面粗糙，出现“孔洞”样结构，并且其变化程度分别与作用时间和红藻氨酸浓度呈量-效关系；预先给予SB203580处理，以上变化有所减轻。结论：抑制p38MAPK信号通路，对红藻氨酸诱导海马神经元胞膜的损伤起一定保护作用。     

神经生长因子对小鼠脑突触体游离钙的调节作用

本文报导在离体情况下,NGF对青年(1月龄)和老年(18月龄)小鼠海马突触体内游离钙浓度的影响,以及在不同钙浓度条件下,NGF对Ca~(2 )的调节作用。结果表明:①不同浓度的NGF对青年小鼠海马突触体内的游离钙离子均无明显作用;②只有浓度为10μg/ml的NGF,可显著降低老年小鼠海马突触体内的游离钙离子浓度(P<0.05);③当胞内钙水平较低时,NGF具有升高钙离子浓度的作用,反之,则具有降低钙离子浓度的作用。提示NGF对Ca~(2 )的调节作用呈双向性。     

低分子肝素抑制bFGF诱导犬动脉平滑肌细胞增殖的体外实验研究

目的　明确低分子肝素 (LMWH)对碱性成纤维细胞生长因子 (bFGF)诱导培养的犬动脉平滑肌细胞增殖的抑制作用。进一步探讨LMWH作用的最佳有效浓度。材料与方法　在 10ng/mlbFGF刺激下 ,一定数量的第 5代犬动脉平滑肌细胞 (5× 10 3 /孔 )含 0、10、5 0、10 0、2 0 0、40 0、80 0、10 0 0、2 0 0 0antiXaIU/ml各浓度组LMWH的DMEM +10 %FBS细胞培养液中孵育 72h ,MTT比色计量法测出各组吸收光密度 (OD)值 ,计算抑制率 ,选择最佳抑制浓度。结果　LMWH对bFGF诱导平滑肌细胞增殖的抑制与其浓度呈线性正相关。 10 0 0antiXaIU/ml浓度的抑制率达 5 0 %以上 ,为显著有效。结论　进一步验证了LMWH对PTA术后多种细胞因子 (尤其是bFGF)局部刺激下的平滑肌细胞增殖的抑制作用。并为今后局部应用有效浓度的LMWH防治PTA术后再狭窄提供了实验依据     

环维黄杨星D对急性分离大鼠心肌细胞内游离钙浓度的影响

目的研究环维黄杨星D（CVB-D）对急性分离大鼠心肌细胞内游离Ca^2＋浓度的影响。方法急性分离大鼠心肌细胞,应用特异性荧光指示剂Fluo^-3/AM负载细胞,用激光共聚焦显微镜检测胞内游离钙的变化。结果在台氏液中,CVB-D浓度为10^-7、10^-6、10^-5mol·L^-1时,心肌细胞内[Ca^2＋]i逐步升高,其峰值分别为（52.78±1.41）、（59.86±4.17）、（62.99±2.66）,并呈剂量依赖性;在有外钙和无外钙时,环维黄杨星D对胞内钙的升高有显著性差异（P〈0.05）;在无外钙并加入内罗啶孵育后,环维黄杨星D引起的胞内[Ca^2＋]i轻度升高,但与无钙组相比无显著性差异（P〉0.05）。结论环维黄杨星D具有升高心肌细胞内游离钙离子浓度的作用,[Ca^2＋]i的升高既源于内钙释放又源于外钙内流。     

PI3K/AKT/mTOR信号传导通路在姜黄素抑制人肝癌细胞Cox-2表达中的作用

目的探讨MAPK和PI3K信号传导通路在姜黄素调节的环氧合酶2(Cox-2)表达中的作用。方法分别加入25、50μmol/L LY294002,10、20μmol/L U0126,5、10ng/ml西罗莫司(雷帕霉素,rapamycin)处理人肝癌细胞BEL-7402,30min后加入10μmol/L姜黄素,对照组单独加入0、10μmol/L姜黄素,培养6h后,采用RT-PCR和Western blotting方法检测BEL-7402细胞中Cox-2 mRNA和蛋白的表达。以不同浓度(0、2.5、5、10、15、20μmol/L)姜黄素或25μmol/L LY294002处理BEL-7402细胞,培养6h后,采用Western blotting检测总AKT蛋白和磷酸化AKT蛋白的表达;以不同浓度(0、2.5、5、10、15、20μmol/L)姜黄素或10μmol/L U0126处理BEL-7402细胞,培养6h后,采用Western blotting检测总ERK蛋白和磷酸化ERK蛋白的表达。结果与仅加入10μmol/L姜黄素的BEL-7402细胞比较,分别加入25、50μmol/L LY294002,5、10ng/ml西罗莫司后,BEL-7402细胞中的Cox-2 mRNA和蛋白表达均明显降低,差异有统计学意义(P<0.01),而加入10、20μmol/L U0126后表达无明显变化(P>0.05)。采用不同浓度姜黄素或25μmol/L LY294002处理后,BEL-7402细胞磷酸化AKT蛋白表达逐渐降低,与对照组相比差异有统计学意义(P<0.01),但总AKT蛋白表达与对照组相比无明显变化(P>0.05)。不同浓度姜黄素或10μmol/L U0126处理后,BEL-7402细胞磷酸化ERK蛋白和总ERK蛋白表达与对照组相比均无明显变化(P>0.05)。结论姜黄素可能通过PI3K/AKT/mTOR信号传导通路抑制人肝癌细胞BEL-7402中Cox-2的表达。     

ERK1/2信号通路在小鼠库普弗细胞-内毒素反应中的活化规律及其作用

目的：研究内毒素诱导下小鼠库普弗细胞（KC）ERK1／2活化规律、肿瘤坏死因子α（TNFα）分泌规律以及ERK1／2信号通路在TNFα分泌中的作用，探讨防治内毒素血症的新方法。方法：磷酸化ERK时效组以含有100ng/mlLPS 的培养基分别培养KC0，5，10，20，30，45，60，120min；磷酸化ERK量效组分别用含有0，0．1，1，10，100，1000，10000ng/mlLPS的培养基孵育KC30min，免疫印迹杂交检测内毒素诱导KC ERPK1／2活化规律，酶联免疫分析检测内毒素诱导KC释放TNFα规律。以0，0．5，1，10，25，50μmol/L PD98059（ERK信号通路特异性阻断剂）预处理KC1h，酶联免疫分析检测PD98059对LPS诱导TNFα分泌的抑制作用。结果：100ng/ml内毒素刺激后，KC同ERK1／2被迅速活化，30min达到高峰，2h后基本恢复至正常水平；在10pg/ml-100ng/ml范围内，内毒素对ERK1／2激酶具有剂量依赖性的激活作用；内毒素诱导KC TNFα释放显著增加，呈显著的剂量依赖关系；随着PD98059剂量的增加，其对LPS诱导KC TNFα分泌的抑制作用较大。结论：内毒素可诱导KC ERK1／2活化和TNFα分泌，ERK1／2对内毒素诱导KC分泌TNFα具有显著的调节作用，ERK1／2可能是治疗内毒素血症的新靶位。     

白介素-1β对人牙周膜成纤维细胞MMP-13表达影响的研究

目的 探讨白介素-1β（IL-1β）对人牙周膜成纤维细胞（hPDLFs）中基质金属蛋白酶-13（MMP-13）表达影响的研究.方法 体外分离培养hPDLFs并随机分为对照组和实验组,对照组中加入等量无血清培养液,实验组加入不同浓度IL-1β（0 ng/ml、0.5 ng/ml、5 ng/ml、10 ng/ml、20 ng/ml）作用hPDLFs 24 h.采用RT-PCR检测MMP-13 mRNA的表达情况,并选取最适浓度作用24 h,采用Western blot检测MMP-13蛋白表达的变化情况.结果实验组与对照组比较,MMP-13在IL-1β浓度为10 ng/ml和20 ng/ml时mRNA表达最为明显（P＜0.05）,10 ng/ml组和20 ng/ml组MMP-13 mRNA表达相比较,无明显差异（P＞0.05）.采用10 ng/ml IL-1β作为最适浓度进行刺激,并作用hPDLFs 24 h,与对照组比较,MMP-13蛋白表达显著增加,具有显著差异性（P＜0.05）.结论 在IL-1β调控下,hPDLFs中MMP-13的表达显著增加,并呈明显的浓度依赖性,而MMP-13表达的增加可能是引起正畸治疗过程中牙根吸收的重要机制之一.     

碱性成纤维细胞生长因子及胰岛素样生长因子-1对体外兔关节软骨细胞的生物学效应

目的：了解胰岛素样生长因子－1（IGF－1）及碱性成纤维细胞生长因子（bFGF)对兔关节软骨细胞分裂增殖及功能代谢的影响，为组织工程方法修复关节软骨缺损提供实验依据。方法：取生长良好的兔正常关节软骨细胞，在含体积分数为10％新生小牛血清的DMEM条件下体外单层培养，细胞贴壁后随机分组，并分别加入不同浓度的bFGF和（或）IGF－1；培养液不加任何因子为对照组，以四唑盐（MTT）法检测细胞相对数，二苯胺显色法测各瓶细胞DNA含量，咔唑硫酸法测基质中糖醛酸含量，以间接反映蛋白多糖含量。并采用流式细胞技术进行细胞周期亚时相分析。结果：在实验浓度范围内，两种因子均促进细胞增殖，DNA合成及增加胞外基质中葡萄糖醛酸含量，且呈剂量－效应依赖关系。当IGF－1浓度≥10ng/ml,bFGF浓度≥1ng/ml时，其促进效果较显著（P＜0．05，0．01）。bFGF的促细胞增殖作用更为明显，最大为对照组的1．32倍，而IGF－1对细胞外基质葡萄糖醛酸含量的影响更显著，最大为对照组的1．78倍，bFGF能明显缩短DNA合成前期（G1期）和分裂前期及分裂期（G2M期）时间；bFGF IGF-1作用后，同样缩短G1期和G2M期时间，显著缩短G1期时间；而IGF－1仅缩短G2M期时间。结论：在本实验条件下，bFGF及IGF－1均以剂量依赖性方式影响细胞，刺激细胞增殖及功能代谢，两种因子的协同作用仅表现在对细胞的增殖作用上，对细胞功能代谢没有明显影响。两种因子促进细胞增殖是通过缩短细胞周期不同亚时相而实现的。     

阿伦膦酸钠对去势大鼠骨髓间充质干细胞成脂分化的影响

目的 探讨阿伦膦酸钠(Alendronate,Aln)对骨髓间充质干细胞(BMSCs)成脂分化的影响,并阐明丝裂原活化蛋白激酶(MAPK)信号通路在该过程中的作用. 方法 BMSCs取自9个月龄去势SD大鼠,分别暴露于0.01,0.1,1,10 μmol/L Aln.成脂诱导2周后进行油红O染色和镜下计数分析,RT-PCR检测过氧化物酶体增殖体激活受体γ2(PPARγ2)表达;使用MAPK通路特异性抑制剂并诱导2周后再观察PPARγ2表达情况,Western blot观察Aln对MAPK通路表达的影响. 结果 BMSCs成脂诱导2周后,油红O染色阳性细胞数随着药物浓度的增高而显著减少(P<0.01).PPARγ2表达随着药物浓度的增高而显著降低.分别使用ERK1/2、JNK抑制剂PD98059和SP600125并诱导2周后,PPARγ2表达上调;使用p38抑制剂SB203580并诱导2周后,PPARγ2表达下调.Western blot结果显示,Aln在5,15,30 min时上调P-ERK1/2和P-JNK的表达,分别使用抑制剂后,表达下调. 结论 Aln通过激活ERK1/2和JNK信号通路,而不是p38,发挥抑制去势大鼠来源的BMSCs成脂分化作用,其效应具有浓度依赖性.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.