电离辐射对肺腺癌细胞A549线粒体的影响

目的 研究电离辐射对肺腺癌细胞A549线粒体的影响。方法 0、0.5、3和8 Gy ⁶⁰Co γ射线照射A549细胞后,利用JC-1探针检测照射后肺腺癌细胞A549线粒体膜电位的变化;使用ATP试剂盒检测辐射对A549细胞ATP生成的影响;利用实时定量PCR方法检测线粒体拷贝数。结果 在照射后24 h,各剂量组A549细胞膜电位出现明显改变（F=243.44,P<0.05）,与0 Gy照射组相比,0.5和3 Gy照射的A549细胞线粒体膜电位显著升高（t=-10.12、-5.59,P<0.05）,而8 Gy照射的线粒体膜电位显著降低（t=15.22,P<0.05）,照后48 h各照射组线粒体膜电位基本恢复正常（F=10.36,P<0.05）;照射后24 h ATP水平与膜电位变化趋势一致（F=97.08,P<0.05）,其中0.5和3 Gy A549细胞中ATP水平升高（t=1.66、7.27,P<0.05）,8 Gy照射组降低（t=-8.24,P<0.05）;照射后48 h,ATP水平也发生变化（F=39.49,P<0.05）,0.5和3 Gy照射后A549细胞中ATP水平显著高于0 Gy组（t=4.60、8.53,P<0.05）。照射后24 h线粒体拷贝数显著增加,其中0.5 Gy mtDNA拷贝数增加至对照组的12倍（t=0.02,P<0.05）,3和8 Gy组mtDNA拷贝数分别增加至对照组的7和10倍（t=9.68、15.10,P<0.05）。结论 大剂量电离辐射会导致A549细胞线粒体膜电位降低,从而影响ATP的生成,中等剂量以下的照射会导致A549细胞线粒体膜电位代偿性增高,从而使ATP的生成增多,8 Gy以内的电离辐射可使mtDNA拷贝数代偿性升高。     

放射性肺炎方抗大鼠放射性肺纤维化作用机制的研究

目的 探讨放射性肺炎方(ARPD)干预放射性肺纤维化的作用机制。方法 将105只雄性SD大鼠采用随机数字表法分为中药(6 MV X射线,15 Gy单次照射+ARPD,10 ml ·kg^-1 ·d^-1)组、单纯照射组(6 MV X射线,15 Gy单次照射)和对照组,每组35只,分别于第15、30、60、75、90、105、140天各收集5只大鼠肺组织及血液样本,肺组织行常规病理学检查;Western blot及RT-PCR法检测转化生长因子-β1(TGF-β1)、纤溶酶原激活物抑制因子-1(PAI-1)、Ⅲ型胶原(collagen type Ⅲ,CⅢ)蛋白及基因在组织中的动态表达;ELISA法检测血清TGF-β1及血浆PAI-1含量;酸水解法及碱水解法分别检测组织及血清羟脯氨酸(HYP)。结果 受照肺组织病理学检查在各时间点均见炎性反应改变,60 d后出现纤维化,中药组早期炎性反应、后期纤维化均显著轻于单纯照射组。中药组30 d后TGF-β1、PAI-1、C Ⅲ蛋白及基因在肺组织中表达水平低于同期单纯照射组(蛋白:t=2.49~3.74,t=2.63~4.57,t=2.76~3.83;基因:t =2.59~4.33,t=2.83~4.62,t=2.83~3.96,P<0.05),15 d后血浆PAI-1、血清TGF-β1水平低于同期单纯照射组(t =2.85~6.27,t=3.69~5.27,P<0.05),60 d后肺组织及血清HYP水平低于同期单纯照射组(t=3.65~4.40,t=6.56~3.75,P<0.05),且肺组织TGF-β1分别与PAI-1及CⅢ的蛋白(r=0.604、0.759,P<0.05)和基因(r=0.519、0.816,P<0.05)变化水平呈显著正相关。结论 ARPD可通过下调TGF-β1、PAI-1,减少CⅢ的合成干预放射性肺纤维化。     

兔放射性肺损伤模型的建立及鉴定

目的 建立适合放射性肺损伤(RILI)CT灌注成像研究的兔动物模型。方法 健康新西兰大白兔48只,随机分为对照组(12只,做单肺假性照射)和实验组(36只,高能X射线单侧肺单次照射25 Gy),分别于照射后第1、6、12、24、48和72小时及第1、2、4、8、16和24周被处死,取两肺中带上、中、下野6处标本,分别进行HE染色光镜、电镜和局部肺组织TNF-α和TGF-β₁的检测。结果 实验组所有兔受照射肺均产生了RILI,早期以急性炎性反应为主,晚期以进行性肺纤维化为特征。实验组受照射1 h后局部肺组织TNF-α表达、72 h后TGF-β₁表达与对照组差异均有统计学意义(t=3.04~14.95,P＜0.05)。光镜下,实验组受照射12 h后肺泡壁厚度、6 h后肺间质面积密度、24 h后间质内纤维母细胞和纤维细胞数量与对照组差异均有统计学意义(t=4.44~39.78,P＜0.05),并分别与照射后的时间直线相关(r=0.82、0.87、0.68)。电镜下,实验组各时间点之间胶原纤维相对含量差异有统计学意义(F=100.31,P=0.000),对照组各时间点之间的差异无统计学意义。实验组受照射72 h后肺内胶原纤维相对含量与对照组差异均有统计学意义(t=3.07~45.18,P＜0.05),并与照射后时间直线相关(r=0.99)。结论 25 Gy单肺单次高能X射线照射能制作稳定、可靠的兔RILI模型,较好地模拟了RILI的发生、发展的演变过程。      

低剂量X射线对人树突状细胞体外迁移能力的影响及其机制

目的 研究低剂量X射线对人树突状细胞(DC)体外迁移能力影响并探讨其机制。方法 分离人外周血单个核细胞(PBMC),以人GM-CSF和IL-4共同诱导分化为DC,于培养的第5天加入TNF-α促进成熟,在培养的第6天用X射线照射DC,受照剂量分别是0、0.05、0.1、0.2、0.5 Gy,照射后48 h收获DC;采用RT-PCR法及Western blot法分别检测CCR7 mRNA及蛋白表达水平;Transwell迁移实验法检测DC的体外迁移能力。结果 与0 Gy相比,0.2和0.5 Gy照射后,CCR7 mRNA的相对表达量显著高于其他剂量(t=14.72、4.72,P<0.05);0.2 Gy照射后,CCR7蛋白表达量高于其他剂量(t=4.46,P<0.05),DC迁移能力显著高于其他剂量(t=2.95,P<0.05);以抗CCR7单克隆抗体封闭CCR7蛋白活性,在接受同等剂量照射时,DC细胞迁移能力显著降低(t=4.63~8.96,P<0.05)。结论 受到0.2 Gy X射线照射的DC,体外迁移能力显著增强,其机制可能与受照后DC表达CCR7 mRNA及蛋白水平升高有关。     

137Cs γ射线诱导人外周血线粒体DNA缺失的时间和剂量-效应关系

目的 研究¹³⁷Cs γ射线诱导的人外周血线粒体DNA 4934 bp和4977 bp缺失的时间和剂量-效应,并探讨其在电离辐射受照者剂量估算中的应用意义。方法 采集5名健康成人外周血进行¹³⁷Cs γ射线离体照射,取其中1份血样给予5 Gy照射后分别培养2、24、48和72 h,另4份血样均经6等分后分别给予0、0.5、1、2、5和10 Gy照射,孵养2 h后采用实时荧光定量PCR方法和凝胶电泳,检测人外周血线粒体DNA 4934 bp(mtDNA 4934 bp)和4977 bp(mtDNA 4977 bp)缺失的表达水平,并用Curve Expert 1.4软件拟合剂量-效应曲线。结果 ¹³⁷Cs γ射线照射离体人血后,诱发的mtDNA 4934 bp 缺失和mtDNA 4977 bp 缺失在照后2 h即升高,mtDNA 4934 bp缺失水平在照后2 h和48 h有相对表达高点(t=10.782和8.966, P<0.05);而mtDNA 4977 bp缺失水平在照后48 h表达最高(t=7.433, P<0.05)。0.5~10 Gy的¹³⁷Cs γ射线照射诱发的mtDNA 4934 bp 缺失(t=2.895~8.105, P<0.05)和mtDNA 4977 bp 缺失(t=3.006~7.715, P<0.05)均随照射剂量增加。其中,mtDNA 4977 bp缺失在相同照射剂量下变化更大,尤其是在10 Gy剂量照射时,二者差异更明显(t=2.919, P<0.05),即对于大剂量照射,mtDNA 4977 bp缺失可能更为灵敏,但是个体差异较mtDNA 4934 bp缺失大。拟合的剂量-效应曲线回归方程分别为Ŷ₁=1.178+0.1219D(R²=0.9269, mtDNA 4934 bp缺失)和Ŷ₂=1.2578+0.1933D(R²=0.9016, mtDNA 4977 bp缺失)。结论 ¹³⁷Cs γ射线诱发的线粒体DNA片段缺失与辐射剂量有良好的数学回归关系,有可能为照射后生物剂量快速估算和预后评估提供依据。     

人肺腺癌A549细胞低剂量辐射超敏感性及其机制的研究

目的 观察A549细胞的低剂量辐射超敏感性现象,探讨其发生的机制。方法 A549细胞接受0~2 Gy的⁶⁰Coγ射线照射后,流式细胞仪对其分选计数,克隆形成法检测细胞存活分数,Western blot法检测ATM1981Ser-P蛋白表达,Hoechst 33258荧光染色法、AnnexinⅤ-FITC/PI双染流式细胞仪检测细胞凋亡,PI单染流式细胞仪检测细胞周期。结果 细胞在0~0.3 Gy表现出单位剂量杀伤增强,在0.3~0.5 Gy表现出一定的辐射抗性,0.5 Gy后的区域存活分数随辐射剂量的增加而降低。照射后1 h,ATM激酶在0.2 Gy时开始活化,0.5 Gy时活化达高峰(t=7.96,P<0.05);与0.5 Gy相比1.0和2.0 Gy的活化水平无明显变化(t=0.69、0.55,P>0.05)。照射后24 h,部分细胞发生凋亡,其凋亡曲线与存活曲线相吻合。与未照射组相比,0.1和0.2 Gy组在各时间点(照射后6、12和24 h)的细胞周期无明显变化,而0.3、0.4和0.5 Gy 组,照射后6和12 h细胞发生G₂/M期阻滞(t＝2.87、2.88、4.92和3.70、3.12、8.11,P<0.05),照射后24 h G₂/M期细胞比例下降(t＝3.87、4.77、3.01,P<0.05)。结论 A549细胞存在HRS/IRR现象,其发生可能与ATM激酶、细胞周期变化有关,凋亡是细胞死亡的主要方式。     

电离辐射对小鼠脾脏ICOS/ICOSL及相关信号通路的影响

目的 观察不同剂量X射线照射后小鼠脾细胞可诱导共刺激分子及其配体（ICOS/ICOSL）与核因子NF-κB、细胞因子IL-10表达量的变化。方法 健康ICR小鼠按随机数字表法分为低剂量照射组（0.05、0.075和0.2 Gy）、高剂量照射组（1、2、4和6 Gy）和假照组。假照组于假照后16 h处死,高、低剂量照射组分别于照后0、4、8、16、24、48、72 h处死小鼠,分离小鼠脾脏制成单细胞悬液,提取脾组织总蛋白,采用流式细胞术检测ICOS/ICOSL,Western blot法检测NF-κB蛋白水平,采用ELISA法检测IL-10分泌量的变化。结果 在0.05、0.075 Gy低剂量照射后,ICOS/ICOSL双阳性细胞百分比显著低于假照组（t=4.743、4.120,P<0.01）;在4、6 Gy高剂量照射后 ,则上调其表达（t=-4.950、-7.310,P<0.01）。核因子NF-κB变化趋势与ICOS/ICOSL表达变化相似。IL-10在0.075、0.2 Gy低剂量照射后明显低于假照组（t=5.277、2.854,P<0.05）,但在6 Gy照射后明显高于假照组（t=7.196,P<0.01）。结论 低剂量电离辐射抑制ICOS/ICOSL、NF-κB和IL-10的表达,而高剂量辐射上调ICOS/ICOSL表达,并激活核因子NF-κB,进而导致IL-10分泌量升高。     

脂肪来源干细胞促进放射损伤骨骼肌血管生成的实验研究

目的 观察新西兰兔脂肪来源干细胞(adipose-derived stem cells,ASCs)植入放射损伤骨骼肌后的微血管密度改变,分析组织中血管内皮生长因子(VEGF)和碱性成纤维生长因子(bFGF)的变化,探讨ASCs移植对兔放射损伤骨骼肌血管生成的影响.方法 64只实验动物单侧臀部予9 MeV电子线一次性照射80 Gy,采用随机数字表法分为ASCs组和磷酸缓盐冲溶液(PBS)组,每组为32只.照射后24 h,ASCs组和PBS组照射侧分别肌肉注射1 ml含5×10⁷个ASCs的细胞悬液及 PBS.免疫组织化学法检测照射后1、4、8和26周骨骼肌组织中CD31阳性的微血管、VEGF和bFGF的表达.蛋白印迹法检测骨骼肌组织中VEGF、bFGF蛋白表达.ELISA法检测ASCs在条件培养基中分泌的VEGF、bFGF浓度.结果 ASCs组受照射骨骼肌组织中CD31染色阳性的微血管密度较PBS组在照射后4、8和26周增高(t=8.14、7.44、18.58,P<0.05).ASCs组受照射骨骼肌组织VEGF、bFGF的累计光密度值(IOD)较PBS组在照射后1、4、8和26周增高(VEGF:t=3.13、4.66、2.19、6.79,P<0.05;bFGF:t=3.14、3.84、4.28、3.61,P<0.05).ASCs组VEGF蛋白的表达水平较PBS组在照射后1、4、8和26周增高(t=4.28、5.02、4.24、4.56,P<0.05).ASCs组bFGF蛋白的表达水平较PBS组在照射后4、8和26周增高(t=3.36、3.16、5.77,P<0.05).ASCs在放射损伤肌肉匀浆上清条件培养基中较在正常肌肉匀浆上清条件培养基中分泌的VEGF浓度在48、72和96 h增高(t=2.81,9.96,4.75,P<0.05),分泌的bFGF浓度在72和96 h增高(t=3.80,4.33,P<0.05).结论 ASCs能上调骨骼肌放射损伤组织VEGF和bFGF分泌、增加新生血管形成,可能是修复骨骼肌放射损伤的部分机制.     

电离辐射联合自噬和凋亡抑制剂或诱导剂对MCF7细胞的影响

目的 探讨电离辐射联合自噬抑制剂、诱导剂和凋亡抑制剂对MCF7细胞自噬和凋亡的影响。方法 MCF7细胞经0 Gy、4 Gy、4 Gy+rapamycin、4 Gy+3-MA、4 Gy+z-VAD-fmk不同方法处理后,采用四甲基偶氮唑蓝(MTT)检测细胞的生长倍增时间;采用Western blot检测MCF7细胞经不同方法处理后自噬特异蛋白LC3和beclin1的表达,并比较蛋白表达量的差异;流式细胞仪(FCM)检测不同处理方法MCF7细胞凋亡百分率。结果 经4 Gy照射后,MCF7细胞生长倍增时间明显长于未经照射的细胞生长倍增时间(t= 4.41,P<0.05);而4 Gy+rapamycin处理后,明显长于单纯4 Gy照射组(t= 4.35, P<0.05);经4 Gy + 3-MA和4 Gy+z-VAD-fmk处理后,均明显短于4 Gy+rapamycin组 (t= 4.32, P<0.05)。Western blot检测结果表明,Beclin1和LC3-Ⅱ蛋白表达4 Gy+rapamycin组为最高,4 Gy+3-MA组为最低,除4 Gy+3-MA组与4 Gy+z-VAD-fmk组比较差异无统计学意义外,其余各处理组间比较差异有统计学意义(t= 3.91~4.78, P<0.05)。结论 电离辐射联合自噬诱导剂可促进MCF7细胞凋亡;联合自噬抑制剂抑制自噬发生,进而延缓肿瘤细胞凋亡。     

电离辐射对成骨细胞核因子κB受体活化因子配体和骨保护素mRNA及其蛋白表达的影响

目的 研究电离辐射对成骨细胞RANKL和OPG表达的影响,探讨辐射导致骨损伤的分子机制。方法 采用MC3T3-E1细胞诱导分化为成骨细胞,经0、2和4 Gy¹³⁷Cs γ射线照射后,采用实时定量PCR及Western blot方法检测电离辐射对于成骨细胞RANKL和OPG mRNA及其蛋白水平表达的影响。结果 4 Gy照射可以导致成骨细胞RANKL mRNA(t=5.41,P<0.05)及其蛋白(t=68.37,P<0.01)表达水平上调;2和4 Gy照射可以导致成骨细胞OPG mRNA(t=5.20、7.02,P<0.05)及其蛋白(t=7.78、9.45,P<0.05)表达水平下调。结论 2和4 Gy电离辐射可以导致成骨细胞中RANKL/RANK/OPG通路发生改变,促进破骨细胞的分化和成熟,进而促进破骨细胞的骨吸收作用。     

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.