ZEB1通过上调ATM表达调控胃癌细胞AGS的放射敏感性

目的 探究锌指结构E-box结合蛋白1（ZEB1）对胃癌细胞AGS放射敏感性的影响并探究其可能的作用机制。方法 通过不同剂量（0、2、4、6、8 Gy）X射线照射AGS细胞,蛋白质印迹法（Western blot）实验观测细胞中ZEB1的表达量;取对数生长期AGS细胞,将过表达ZEB1、ZEB1干扰表达质粒以及相应的对照质粒（pcDNA3.1）和阴性对照干扰质粒转染至AGS细胞中,分别记为过表达ZEB1组、沉默ZEB1组、对照组和阴性对照组,细胞克隆实验检测过表达和沉默ZEB1对AGS细胞存活率的影响;流式细胞术实验检测细胞的凋亡率;Western blot检测细胞损伤相关组蛋白H2A（H2AX）、磷酸化H2AX（γ-H2AX）以及毛细血管扩张突变基因（ATM）表达量。结果 ZEB1在AGS细胞中的表达对放射剂量具有依赖性（F=58.57,P<0.05）;过表达ZEB1能够提高AGS细胞的存活,抑制γ-H2AX表达（t=12.18,P<0.05）,并阻碍细胞的凋亡（t=7.27,P<0.05）,并随时间上调ATM表达量（F=165.70,P<0.05）;沉默ZEB1降低AGS细胞的存活,增加γ-H2AX表达（t=12.88,P<0.05）和细胞的凋亡（t=8.36,P<0.05）,随时间下调ATM表达量（F=44.80,P<0.05）。结论 ZEB1通过上调ATM表达调控胃癌AGS细胞放射敏感性。     

DDX46基因对结肠癌细胞电离辐射敏感性的影响及机制研究

目的 探讨DDX46基因对结肠癌细胞的电离辐射敏感性的影响及其作用机制。方法 DDX46基因RNA干扰慢病毒转染的SW480细胞为实验组,空载质粒慢病毒转染的SW480细胞为对照组。两组细胞转染72 h后,分别进行0和4 Gy X射线照射,采用CCK-8方法检测两组的细胞活力;实验组联合4 Gy X射线照射24 h后,采用免疫荧光技术和Western blot法检测细胞γ-H2AX foci数量以及DNA损伤修复通路关键蛋白的表达水平,确定DDX46与辐射诱导的DNA损伤修复的关系。结果 4 Gy X射线照射24 h后,实验组的细胞活力比照射前降低（15.02±3.92）%（t=-4.696,P<0.05）,比对照组降低（17.43±1.83）%（t=4.844,P<0.01）;而对照组照射前后比较差异无统计学意义（P>0.05）。同时,实验组γ-H2AX foci数量相比于对照组增加了（43.03±17.6）%（t=-3.108,P<0.05）,ATM的蛋白水平显著升高（t=7.530,P<0.01）,而ATM的活性形式p-ATM以及ATM的下游靶蛋白Rad50的蛋白水平则明显降低（t=4.260、4.260、P<0.05）,同时γ-H2AX的蛋白表达水平也有一定程度的升高（t=-3.090,P<0.05）,DNA-PK在两组细胞内的蛋白水平变化不大。结论 沉默DDX46可以提高结肠癌细胞系SW480的辐射敏感性,其机制可能是DDX46表达沉默抑制结肠癌细胞系SW480中ATM的活化,从而抑制辐射诱导的DNA损伤的修复,提高SW480的辐射敏感性。     

DNA-PKcs在细胞辐射超敏感性中的作用研究

目的 研究DNA-PKcs在细胞辐射超敏感性中的作用。方法 X射线照射M059K和M059J细胞后,克隆形成法实验检测其存活分数;微核分析法和γ-H2AX焦点形成实验检测DNA损伤;Western blot实验检测M059K,M059J细胞中磷酸化Chk1、Chk2和总Chk1、Chk2蛋白的表达。结果 在X射线照射剂量<1 Gy时,M059K细胞呈现出辐射超敏感性;DNA损伤水平不能用于表征低剂量区的HRS/IRR;0.2 Gy X射线照射后,M059K细胞中pChk1/Chk1在20~60 min内显著高于M059J细胞（t=14.157、13.661、14.177、11.317、14.512, P<0.05）;0.2 Gy X射线照射后,M059K细胞中pChk2/Chk2在20~50 min内显著高于M059J细胞（t=13.182、13.868、14.155、14.477, P<0.05）。结论 DNA-PKcs野生型的人胶质瘤M059K细胞中存在着低剂量辐射超敏感性现象,其发生可能与DNA-PKcs介导的G₂/M期检验点相关蛋白激活有关。     

miR-27b-3p通过靶向PLK2促进乳腺癌细胞的辐射抵抗

目的 研究miR-27b-3p对乳腺癌细胞辐射抵抗的影响。方法 通过检索基因表达（GEO）数据库，筛选分析miR-27b-3p在正常乳腺组织和乳腺癌组织中的表达水平。利用实时荧光定量PCR技术分析其在不同乳腺癌细胞系中的表达水平。通过细胞克隆形成、免疫荧光和5-乙炔基-2''-脱氧尿苷（EDU）检测技术评价miR-27b-3p对乳腺癌细胞辐射抵抗作用。采用荧光素酶报告基因技术确定miR-27b-3p的靶基因PLK2，并在乳腺癌细胞中进一步验证miR-27b-3p的辐射抵抗作用。结果 与正常乳腺组织和细胞相比，miR-27b-3p在乳腺癌组织（t=2.99，P<0.01）和乳腺癌细胞中表达水平显著上调（t=21.21、32.88，P<0.05）。尤其在抗辐射细胞MCF-7R中升高更加明显（t=25.63，P<0.05）。过表达miR-27b-3p增强了MCF-7细胞的克隆形成能力（t=10.32，P<0.05），且随着辐照剂量的增加，miR-27b-3p对MCF-7增殖能力的保护效应逐渐凸显（t=8.77、8.26、8.03，P<0.05）；干扰miR-27b-3p则抑制MCF-7R细胞克隆形成数（t=40.00，P<0.05），且随着辐照剂量的增加，进一步削弱了MCF-7R细胞的增殖能力（t=8.54、8.32、8.23，P<0.05）。报告基因实验结果表明，PLK2是miR-27b-3p的直接靶标。过表达PLK2抑制了miR-27b-3p介导的乳腺癌细胞辐射抵抗（MCF-7：t=9.66，P<0.05；MCF-7R：t=6.42，P<0.05）。结论 miR-27b-3p可通过靶向PLK2提高乳腺癌细胞的辐射抗性。     

miR-141过表达增强人食管癌细胞的放射敏感性

目的 研究miR-141对食管癌细胞放射敏感性的影响及可能的作用机制。方法 将miR-141 mimic或miR-对照转染到食管癌KYSE-150细胞中,分别使用qRT-PCR和Western blot技术检测miR-141和增殖相关蛋白Ki67、凋亡相关蛋白Bax和Bcl-2的表达。CCK-8,流式细胞术及克隆形成实验检测放射处理后细胞的放射敏感性变化。结果 随放射剂量的增加,食管癌细胞中miR-141表达逐渐降低（t=2.57~8.96,P<0.05）。miR-141过表达抑制了细胞的增殖和克隆形成能力,促进了KYSE-150细胞凋亡,使得放射敏感性增高（t=3.24,P<0.05）。此外,与对照组相比,miR-141过表达显著抑制KYSE-150细胞中的Ki67和Bcl-2蛋白表达（t=6.56、8.24,P<0.01）,并促进Bax蛋白表达（t=3.24,P<0.01）,进一步证实了miR-141增强食管癌细胞的放射敏感性。结论 miR-141通过调控细胞增殖和凋亡相关蛋白表达增强食管癌细胞的放射敏感性。     

不同非小细胞肺癌细胞对碳离子放射敏感性的影响

目的 分析不同非小细胞肺癌（NSCLC）细胞对碳离子照射放射敏感性的影响,并探讨其相关的分子机制。方法 采用¹²C⁶⁺对肺腺癌A549细胞、鳞癌H520细胞和大细胞癌PGCL3细胞进行照射,吸收剂量分别为0、2、4和6 Gy。通过克隆实验检测3种细胞系的存活率,流式细胞术检测细胞的周期分布,利用Hoechst 33258染色检测细胞的凋亡率,实时RT-PCR方法检测细胞内DNA-PKcs基因的表达。结果 2、4、6 Gy照射后,A549、H520和PGCL3细胞的存活率明显下降,其中A549细胞辐射敏感性最低,其次是PGCL3细胞,H520细胞辐射敏感性最高。受照射后细胞均出现G₂/M期阻滞与凋亡,且随剂量的升高呈现上升的趋势。受照射H520与PGCL3细胞的阻滞率（t=4.813、20.738、25.654和t=2.790、2.977,P<0.05）和凋亡率（t=8.579、14.289、15.244和t=3.785、5.098、8.105,P<0.05）明显高于A549细胞。受照射细胞DNA-PKcs表达明显上调,A549细胞最高,其次是PGCL3细胞,H520细胞最低;并且随着剂量的升高,DNA-PKcs的表达呈现下降趋势。2、4 Gy照射后H520和PGCL3细胞DNA-PKcs的表达明显低于A549细胞（t=7.782、3.689和t=3.889、2.814,P<0.05）。结论 不同NSCLC细胞对¹²C⁶⁺离子的放射敏感性不同,H520鳞癌细胞最高,A549腺癌细胞最低。肺腺癌A549细胞放射敏感性较低可能与高表达的DNA-PKcs参与NHEJ途径修复DNA双链断裂有关。     

Tip60基因沉默对细胞DNA双链断裂修复和辐射敏感性的影响

目的 探讨Tip60对细胞辐射敏感性的影响及相关机制。方法 采用siRNA和Tip60乙酰转移酶抑制剂漆树酸,抑制U2OS细胞中Tip60的表达或乙酰转移酶活性;用克隆形成率分析细胞对⁶⁰Co γ射线的敏感性;采用γ-H2AX原位免疫荧光集簇点法,检测DNA双链断裂损伤修复;用免疫共沉淀检测蛋白质的相互作用。结果 siRNA沉默Tip60表达明显提高了U2OS细胞对1、2 Gy中、低剂量γ射线的敏感性(t=3.364、3.979,P＜0.05),但对4 Gy大剂量照射的细胞存活率无明显影响。γ-H2AX集簇点检测结果表明,照射后1、4和8 h,Tip60失活导致细胞DNA双链断裂修复能力降低(t=3.875、3.183和3.175, P<0.05)。细胞在受到电离辐射损伤后,Tip60与DNA修复蛋白DNA-PKcs发生相互作用,漆树酸能抑制DNA-PKcs的T2609位点的磷酸化。结论 Tip60通过与DNA-PKcs相互作用,调控细胞DNA双链断裂修复机制,对细胞辐射敏感性产生影响。     

下调血管内皮生长因子A对食管癌ECA-109细胞的辐射增敏作用

目的 下调食管癌ECA-109细胞的血管内皮生长因子A（VEGFA）表达，观察其辐射敏感性的改变并初步探讨其发生机制。方法 将食管癌ECA-109细胞分成VEGFA转染组、空载组、X射线组和VEGFA转染组联合X射线组。运用qPCR法检测VEGFA基因的表达；Western blot法检测VEGFA蛋白的表达；细胞增殖实验（CCK8法）测定细胞的增殖变化；克隆形成法分析不同照射剂量（0、2、4、6、8 Gy）细胞的辐射敏感性；流式细胞术分析细胞凋亡变化；免疫荧光法检测γ-H2AX焦点的数量。结果 ECA-109细胞VEGFA转染组与空载组比较，VEGFA基因表达明显减少（t=11.98，P<0.05）、VEGFA蛋白表达显著下调（t=12.38，P<0.05）；ECA-109转染组细胞相较于空载组细胞，其增殖（A₄₅₀值）明显受到抑制（t=2.78、7.25、21.93、13.21，P<0.05）；与空载组比较，VEGFA转染组ECA-109细胞的D₀、D_q、SF₂值下降（t=5.83、8.56、7.68，P<0.05），放射增敏比SER_D0、SER_Dq分别为1.41、2.09，凋亡比例明显增加、且联合X射线后ECA-109细胞的凋亡比例进一步增加（t=17.63、22.48、33.87，P<0.05）；ECA-109细胞VEGFA转染组和空载组在X射线照射后2 h内细胞核形成的γ-H2AX焦点数量均明显增加，24 h后空载组细胞核的γ-H2AX焦点数量恢复照射前水平，而转染组中的γ-H2AX焦点数量仍高于照射前水平（t=7.00，P<0.05）。结论 下调VEGFA能抑制食管癌细胞的增殖，减少细胞集落形成并促进其凋亡，增加食管癌ECA-109细胞的辐射敏感性，其机制与DNA损伤修复密切相关。     

沉默细胞周期检测点激酶1(Chk1)对宫颈癌HeLa细胞放射敏感性的影响

目的 探讨siRNA沉默细胞周期检测点激酶1（Chk1）基因对宫颈癌HeLa细胞放射敏感性的影响。方法 合成Chk1基因的小分子干扰RNA（si-Chk1）,将si-NC或者si-Chk1分别转染到宫颈癌HeLa细胞中,0、2、4、6、8、10 Gy剂量的X射线照射细胞,RT-qPCR和Western blot检测转染后Chk1的mRNA水平和蛋白水平的表达,MTT方法检测细胞增殖能力,流式细胞仪检测细胞凋亡,克隆形成实验检测转染后HeLa细胞放射敏感性,Western blot检测转染后P21蛋白和抗凋亡基因Bcl-2蛋白的表达水平。结果 si-Chk1转染到HeLa细胞后,HeLa细胞Chk1基因的mRNA和蛋白表达水平均显著下降（t=2.12~5.86,P<0.05）,沉默Chk1的表达抑制了宫颈癌HeLa细胞的增殖（t=3.15~6.36,P<0.05）,同时降低宫颈癌HeLa细胞克隆形成能力（t=1.58~6.36,P<0.05）,上调P21（t=4.35,P<0.05）、下调Bcl-2蛋白（t=2.37,P<0.05）的表达水平。结论 siRNA沉默Chk1表达能够增强HeLa细胞放射敏感性。     

γ-H2AX用于辐射生物剂量计研究的动物模型评价

目的 通过比较γ射线照射诱导大鼠和人淋巴细胞DNA双链断裂(DSBs)修复动力学的变化,评价大鼠用于γ-H2AX焦点辐射生物剂量计动物模型的可行性. 方法 采用γ射线外照射Sprague-Dawley (SD)雄性大鼠和健康成人外周血淋巴细胞以及整体照射大鼠,分别于照射后不同时间采用免疫荧光技术检测DSBs分子标志物γ-H2AX焦点的变化,检测修复蛋白pATM(S1981)和pDNA-PKcs (T2609) 焦点的形成,以及它们与γ-H2AX焦点的共定位情况.结果0.5 Gy γ射线照射诱发大鼠和人淋巴细胞γ-H2AX焦点形成和消除动力学相一致,表现为受照后30 min,γ-H2AX焦点形成达到最大值(t_大鼠=62.64,t_人=28.52,P<0.05),6 h内快速下降 (t_大鼠=45.96,t_人=14.80,P<0.05),至受照后24 h残留焦点数为最大值的3%~8%.γ射线照射后30 min,大鼠和人淋巴细胞pATM (S1981)和pDNA-PKcs (T2609)焦点形成数较未照射对照组显著增加 (t_大鼠=21.05、25.80,t_人=11.07、29.52,P<0.05),分别与γ-H2AX焦点共定位比例亦显著升高 (t_大鼠=5.34、9.14,t_人=18.32、51.28,P<0.05),占26%~32%.离体照射和整体照射诱导大鼠淋巴细胞γ-H2AX焦点形成数相一致,而且γ-H2AX焦点形成与照射剂量之间呈良好的线性关系.结论 大鼠为γ-H2AX用于辐射生物剂量计研究提供了很好的动物模型,ATM与DNA-PKcs共激活在辐射诱导大鼠和人淋巴细胞DSBs的修复中发挥重要的作用.     

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.