肝脏Toll样受体4的激活与小鼠肝脏部分缺血再灌注损伤的关系

目的探讨TOLL样受体4(toll-like receptor 4,TLR4)的激活与肝缺血再灌注损伤的关系.方法以BALB/c小鼠复制肝脏部分缺血再灌注损伤模型,采用免疫组织化学法观察TLR4在肝脏的分布和表达量,同时监测血浆丙氨酸氨基转移酶(alanine aminotransferase,ALT)及门静脉血清内毒素(endotoxin,EN)和肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)的变化;再以TLR4先天性缺损小鼠C3H/Hej和野生型鼠C3H/Heouj为模型,观察ALT和门静脉血清TNF-α的变化.结果 1. BALB/c小鼠肝脏左中叶缺血1 h后,再灌注1、3 h,缺血肝叶内TLR4表达增加,且再灌注1 h最强,阳性细胞主要是枯否细胞及血管内中性粒细胞;2.门静脉血清EN在各时间点与假手术组相比无显著差异(P>0.05);3.再灌注3 h,TNF-α较假手术组升高[Hej(152±43)pg/ml vs. (18±10)pg/ml,n=6,t=5.26,P<0.01;Heouj(249±52)pg/ml vs. (25±13)pg/ml,n=6,t=7.24,P<0.01];4. Hej组小鼠肝功能损伤轻于Heouj组小鼠[再灌注1 h(662±106)pg/ml vs. (1 216±174)pg/ml,n=6,t=4.21,P<0.01;再灌注3 h(1 145±132)pg/ml vs. (2 958±187)pg/ml,n=6,t=13.72, P<0.01],且再灌注3 h其血清TNF-α水平明显低于Heouj组小鼠[(152±43)pg/ml vs. (249±52)pg/ml,n=6,t=3.94,P<0.01].结论 TLR4受体在肝脏缺血再灌注损伤过程中被激活,通过TNF-α等细胞因子介导参与了肝脏缺血再灌注损伤的过程.     

小鼠肝缺血再灌注后Toll样受体2在缺血肝组织中的激活及其意义

目的　探索小鼠肝缺血再灌注后缺血肝组织中Toll样受体 2 (TLR2 )的激活及其与肝功能损伤之间的关系。方法　缺血再灌注损伤组 (I/R组 ),假手术对照组 (S组 )均采用实时荧光定量多聚酶链反应检测肝组织中TLR2mRNA及TLR2蛋白的表达,同时检测门静脉血浆丙氨酸氨基转移酶(ALT)、肿瘤坏死因子α(TNF α)及门静脉血清内毒素 (endotoxin, EN)水平。结果　肝脏部分缺血1h再灌注 4h后,I/R组与S组小鼠缺血肝组织TLR2 mRNA的表达 (ΔCt值 )分别为 1. 0 6±0. 9 1和5. 0 8±1. 3 2, 两组间差异有显著性 (P < 0. 0 1 ),I/R组缺血肝组织TLR2 蛋白的表达 (OD值 )( 4 3 3. 9 1±2 5. 5 3 )水平较S组 ( 1 0 2. 8 6±1 3. 5 8 )显著升高 (P< 0. 0 1 )。I/R组门静脉血清TNF α[ ( 1 1 2. 5 2±1 4. 4 1 )pg/mL]较S组 [ ( 5. 9 6 ±4. 4 3 )pg/mL]显著升高 (P < 0. 0 1 );I/R组ALT[ ( 8 4 8. 3 3±2 7 1. 3 7 )U/L]较S组 [ ( 4 2. 3 9±1 4. 7 5 )U/L]显著升高 (P < 0. 0 1 );而门静脉血清内毒素水平组间差异无显著性 (P> 0. 0 5 )。结论　TLR2mRNA及蛋白在肝脏缺血再灌注过程中缺血肝组织的表达增强, 此变化伴有TNF α的升高及肝功能的损伤。     

缺血后处理对猪心肌细胞Fas基因蛋白表达及Caspase-3活性的影响

目的探讨缺血后处理对猪心肌细胞Fas基因蛋白表达及Caspase-3活性的影响。方法24只小型约克猪(体重35～40kg)被随机分为4组。组1(ACC组,n=6)：于CPB开始后并行循环45 min,阻断主动脉90 min,开放主动脉后心脏再灌注120 min；组2(pre-con组,n=6)：升主动脉阻断前进行心脏缺血预处理(阻断升主动脉5 min、开放10 min,重复3次),余处理与组1相同；组3(post-con组,n=6)：并行循环45 min,阻断主动脉90 min,开放主动脉后心脏再灌注120 min；再灌注开始进行缺血后处理(阻断升主动脉30 s后开放30 s,重复3次,共3 min)；组4(pre-con +post-con组,n=6)：升主动脉阻断前进行心脏缺血预处理(阻断升主动脉5 min、开放10 min,重复3次),阻断主动脉90 min,开放主动脉心脏再灌注120 min,再灌注开始进行心肌缺血后处理(阻断升主动脉30 s、开放30 s,重复3次共3 min)。在再灌注结束后取左心室全层心肌适量并固定。用原位化学法(TUNEL)观察各组心肌细胞凋亡,流式细胞法检测Fas基因蛋白表达及Caspase-3的活性。在CPB前、缺血90 min、再灌注30、60、120 min采静脉血检测血MDA、SOD水平。结果原位化学法测得心肌细胞凋亡率组2(10．46±0．91)%、组3(9．68±0．59)%和组4(11．35±1．37)%显著低于组1(19．75±1．81)%(P＜0．05)；流式细胞法测得Fas,Caspase-3荧光表达指数(FI),组2(1．24±0．13和1．32±0．13)、组3(1．27±0．07和1．33±0．08)和组4(1．27±0．14和1．31±0．12)显著低于组1(1．74±0．11和1．99±0．12)(P＜0．05)；与组1相比,MDA血浆浓度组2、组3和组4显著低于组1,而SOD浓度却显著高于组1(P＜0．05)。组2、组3和组4上述指标差异无统计学意义(P＞0．05)。结论Fas、Caspase-3表达改变参与了心肌细胞凋亡及缺血再灌注损伤过程；缺血后处理可以明显减少心肌细胞凋亡,抑制缺血再灌注损伤。心肌细胞凋亡的减少与Fas基因蛋白的下调、抑制Caspase-3活性及氧化应激有关；缺血后处理与缺血预处理相比可以同等程度的减少心肌细胞凋亡。本实验未观察到缺血预处理和缺血后处理的叠加作用。     

人ⅠκBα突变体对大鼠肝移植缺血再灌注中炎性因子的影响

目的探讨人IκBα突变体(IκBαM)对大鼠肝移植缺血再灌注中炎性因子的影响。方法SD大鼠随机分4组：组Ⅰ为假手术组,组Ⅱ为对照组,组Ⅲ为PcDNA 3．0组,组Ⅳ为PcDNA 3．0-I⒘BαM组。应用免疫组织化学、逆转录-聚合酶链反应(RT-PCR)测定肝组织中肿瘤坏死因子-α(TNF-α)、细胞黏附分子-1(ICAM-1)的表达,酶联免疫吸附(ELISA)检测血清中TNF-α的表达,同时检测肝脏酶学的变化。结果Ⅱ、Ⅲ组与Ⅳ组比较：术后12 h TNF-α免疫组织化学阳性率分别为84%、80%、55%,ICAM-1免疫组织化学阳性率分别为74%、76%、47%,差异有统计学意义(P＜0．05)；术后2、12 h TNF-α和术后12 h ICAM-1mRNA的表达差异有统计学意义(P＜0．05)；术后2、12 h血清TNF-α表达差异有统计学意义,以2 h为显著(258．50±46．19 vs 147．45±36．04；244．83±18．08 vs 147．45±36．04,P＜0．05)；肝脏酶学指标(ALT)在各时点差异有统计学意义(P＜0．05)。结论IκBαM通过抑制炎性因子的表达减轻大鼠肝移植缺血再灌注损伤。     

急性高容量血液稀释下肝下下腔静脉阻断对血流动力学的影响

目的观察琥珀酰明胶行急性高容量血液稀释(AHHD)对Pringle法联合肝下下腔静脉阻断行肝部分切除患者血液动力学的影响。方法选择ASAI～Ⅱ级择期行肝切除术的患者20例,麻醉诱导后20～30 min内输入琥珀酰明胶(20 ml/kg体重),行第一肝门联合肝下下腔静脉阻断,联合阻断和开放过程根据HR、MAP和CVP调节异氟醚吸入浓度和复方氯化钠输入速率,必要时用血管活性药物。记录AHHD、阻断和开放三个过程不同时间点的HR、MAP和CVP的变化。结果与AHHD前比较,MAP在AHHD完成即刻[(84±10)mm Hg]和AHHD后10 min[(85±9) mm Hg]均有明显升高(P＜0．05)；CV P在AHHD 10 min[(9．7±2．0)cm H_2O]、HHD完成即刻[(12．6±2．3)cm H_2O]和AHHD后10 min[(12．4±1．9)cm H_2O]均有显著性升高(P＜0．05,P＜0．01．P＜0．01)。与联合阻断前比较,MAP在阻断后5min[(69±10)mm Hg]、10min[(72±11)mm Hg]下降显著(P＜0．05)；CVP在阻断后1min[(7．2±2．4)cm．H_2O]、5 min[(4．8±2．8)cm H_2O]和10min[(5．0±2．3)cm H_2O]均明显下降(P＜0．05,P＜0．01,P＜0．01)；与阻断开放前比较,MAP在阻断开放后5min[(79±12)mm Hg]、10min[(78±10)mm Hg]上升显著(P＜0．05)；CVP在阻断开放后1min[(9．8±2．4)cm H_2O]、5min[(11．2±2．8)cm H_2O]和10min[(11．5±2．5)cm H_2O]均明显上升(P＜0．05,P＜0．01,P＜0．01)。在整个手术过程中HR的变化差异无统计学意义。结论术前急性高容量血液稀释,阻断过程中必要时再辅以小剂量血管收缩药,可安全地用于Pringle法联合肝下下腔静脉阻断行肝部分切除术。     

骨化三醇诱导锌指蛋白A20抑制下游基因表达延长肝移植大鼠生存期

目的探讨骨化三醇延长大鼠肝移植后受体生存期的机理。方法观察肝移植后受体大鼠应用骨化三醇前后锌指蛋白A20及其下游基因表达的变化。结果原位肝移植后预防性应用骨化三醇可以抑制急性排斥反应(RAI 3．0±1．0 vs 8．7±0．6,P＜0．05),保护移植物功能[AST：(334±40)U/L vs(2572±537)U/L,P＜0．01；BIL：(38±11)mmol/L vs(159±47)mmol/L,P＜0．01],延长移植后受体生存期[100 d的生存率(83．33±29．81)%vs(66．67±37．65)%,P＜0．01]。同时,预防性应用骨化三醇后移植物局部Th1类细胞因子浓度明显降低[IL-2浓度(80．61±18．63) ng/ml vs(770．99±92．92)ng/ml,P＜0．01；IFN-γ浓度(230．05±47．42)ng/ml vs(984．19±166．16) ng/ml,P＜0．01]。其上游调控因子NF-(?)B核内表达水平明显降低,抑制蛋白I_(?)B和锌指蛋白A20表达水平明显增强。结论原位肝移植后预防性应用骨化三醇可以诱导“细胞保护性”基因A20表达,从而抑制蛋白I_(?)B的降解,抑制NF-_(?)B活化,并阻断损伤性细胞因子的表达,抑制急性排斥反应,延长移植后受体生存期。     

小鼠肝缺血/再灌注损伤时肝脏Kupffer细胞中TLR2的表达

目的　观察小鼠肝部分缺血 /再灌注损伤时肝脏Kupffer细胞表面TLR2mRNA的表达及蛋白质的合成情况。方法　制作小鼠肝部分缺血 /再灌注损伤模型。采用原位灌注消化法分离并纯化Kupffer细胞 ,用大鼠抗小鼠TLR 2IgG和异硫氰酸荧光素 (FITC )的二抗进行染色 ,流式细胞仪 (FCM )测定阳性细胞数 ;并测定Kupffer细胞的TLR2mRNA含量。结果　缺血 60min ,再灌注 4h时 ,实验组小鼠肝脏Kupffer细胞表面TLR2mRNA表达及蛋白的合成量均明显高于假手术组 ,且缺血叶高于非缺血叶。结论　小鼠肝缺血 /再灌注损伤时 ,肝脏Kupffer细胞表面的TLR2mRNA表达明显增高 ,其蛋白质的水平也明显升高     

TLR2/4蛋白在小鼠全肝缺血再灌注损伤肝脏的表达及意义

目的观察Toll样受体2／4蛋白在小鼠全肝缺血再灌注损伤中肝脏的表达，并分析其与肝功能损伤的关系。方法通过夹闭BALB／c小鼠肝门，复制小鼠全肝缺血再灌注损伤模型。采用Western blot方法定量检测缺血肝叶中TLR2／4蛋白的表达变化，并检测门静脉血浆丙氨酸氨基转移酶（pALT）、肿瘤坏死因-α（TNF-α）及门静脉血清内毒素（endotoxin，EN）水平。结果与假手术组（sham-operated group，SH组）相比：（1）全肝缺血20min并行再灌注后，缺血再灌注组（ischemic／reperfusion group，I／R组）血清ALT在再灌注1h即明显升高，且在再灌注3h时较1h时明显升高；（2）I／R组缺血肝脏TLR2／4蛋白的表达（OD值）明显升高，TLR2蛋白的表达在再灌注3h较1h明显高；而TLR4蛋白的表达以再灌注1h时水平最高。（3）I／R组中门静脉血清TNF-α在再灌注1h即开始高，在再灌注3h达高峰。（4）门静脉血清内毒素水平明显升高（与SH相比，P〈0．01），但I／R组在不同灌注时间点之间无显著性差异（P〉0．05）。结论TLR2／4蛋白表达的上调参与了小鼠全肝脏缺血再灌注中肝脏的损伤。     

小鼠肝内源性损伤下TLRs在Kupffer细胞和肝窦内皮细胞中的表达

目的　观察在小鼠内源性损伤模型下肝脏Kupffer细胞 (Kupffercell,KC)和肝窦内皮细胞 (sinusoidalendothelialcells,SEC)表面Toll样受体 2 (TLR2 )蛋白及mRNA表达。方法　在肝部分缺血 再灌注损伤模型下 ,采用原位灌注消化法分离并纯化KC和SEC ,用大鼠抗小鼠TLR2 IgG和异硫氰酸荧光素 (FITC)的二抗进行染色 ,流式细胞仪 (FCM)测定阳性细胞数 ,并用实时定量PCR(Real TimeRT PCR)检测两种细胞中TLR2 mRNA含量。结果　损伤组KCTLR2 的表达明显高于假手术组 ,蛋白质表达为 ( 9.19± 1.0 7) %vs ( 1.5 2± 0 .2 1) % ,P<0 .0 1;mRNA表达为 0 .5 4± 0 .77vs 2 .6 2± 2 .19,P<0 .0 5。SEC差异并无显著性意义。结论　在肝缺血 再灌注损伤中 ,小鼠肝KCTLR2 在蛋白质和mRNA水平的表达明显增高     

Bcl-2基因修饰的肝细胞移植对肝脏缺血再灌注损伤的影响

目的观察人Bcl-2(hBcl-2)基因修饰的肝细胞移植对肝脏缺血再灌注损伤的影响。方法将包含hBcl-2基因阅读框架(0·7kb)的核苷酸亚克隆至Psectag2A载体,脂质体转染balb/c小鼠胎肝细胞(BNL.CL2),经门静脉移植入balb/c小鼠(2×106细胞)。移植72h后左肝后叶常温下缺血45min后再灌注8h,比较测定凋亡细胞,血清ALT、AST、LDH,以及组织学的改变。结果转染hBcl-2基因的balb/c小鼠在肝脏缺血再灌注后ALT(P<0·05或0·01)、AST(P<0·05)、LDH(P<0·05)、凋亡细胞(P<0·05)均较对照组显著降低,组织学改变减轻。进行缺血再灌注的各组细胞损伤均以细胞凋亡为主(P<0·05或0·01)。结论hBcl-2基因修饰的肝细胞移植对肝脏缺血再灌注损伤具有保护作用,主要通过抑制缺血再灌注后的细胞凋亡。     

2015年乳腺癌辅助化疗进展

【摘要】〓乳腺癌是危害我国女性健康的头号杀手,尽管近年来辅助化疗的研究进展突飞猛进,但临床中仍有不少问题未能明确,如辅助化疗的合适人群、化疗的开始时间、蒽环及紫杉类的地位和用法、强化维持治疗的作用、疗效及预后的生物标志物等。本文结合乳腺癌辅助化疗在临床上的常见问题和2015年各大乳腺癌会议阐述乳腺癌辅助化疗的最新进展。     

Alterations in T-Cell Subset Frequency in Peripheral Blood in Obesity

Background: Obesity affects the regulation of immune and inflammatory responses. This study characterizes differences in peripheral blood lymphocyte phenotype in obese humans. Methods: Frequencies of lymphocyte subsets among peripheral blood mononuclear cells were compared between 10 obese (BMI ≥35) and 10 lean subjects, as determined by antibodies directed against cluster differentiation (CD) markers. Results: Obese patients demonstrated an increased frequency of CD3+CD4+ T-cells (mean difference 12%, P=0.004), a decreased frequency of CD3+CD8+ T-cells (mean difference 9.4%, P=0.016) and an increased frequency of CD3+CD8+CD95+ T-cells (mean difference 13.3%, P=0.032). No other differences among T-cell or monocyte subsets were noted. Conclusions: Obesity is associated with alterations in frequencies of peripheral CD4+ and CD8+ T-cells and aberrations in the expression of CD95 among CD8+ T-cells. These data suggest both CD4+ and CD8+ T-cell compartments, as well as the regulation of CD95 expression on CD8+ T-cells, as targets for further study into obesity's effects on the immune system.     

西宁地区烧伤病人动脉血气分析观察

对高海拔地区的27例烧伤病人动脉血气变化进行了分析和观察。结果证明:无论是存活病人还是死亡病人伤后均存在有低氧血症问题。并且在死亡病人和烧伤合并吸入性损伤病人其低氧血症的发生早于单纯烧伤病人。提示:吸入性损伤病人应立即行气管切开术以保障氧气供给,单纯烧伤病人可常规吸氧以维持正常血 PaO_2,ARDS 均发生在合并吸入性损伤的病人,高频喷射通气技术对纠正低氧血症有一定效果。     

Controversies in Fistula in Ano

Managing a complex fistula in ano can be a daunting task for most surgeons; largely due to the two major dreaded complications—recurrence & fecal incontinence. It is important to understand the anatomy of the anal sphincters & the aetiopathological process of the disease to provide better patient care. There are quite a few controversies associated with fistula in ano & its management, which compound the difficulty in treating fistula in ano. This article attempts to clear some of those major controversies.     

β-半乳糖苷酶在体内外成骨细胞中的表达

目的 研究β—半乳糖苷酶(β—gal)在成骨细胞中的表达状况，为阐明MorquioB综合征的发病机制提供依据。方法 裸鼠各器官和骨组织标本行X-gal染色检测。抽取羊和人骨髓行骨髓基质细胞(BMSCs)培养，分为4组：I：Adv-hBMP-2转染组；Ⅱ：Adv—β—gal转染组；Ⅲ：未转染组；Ⅳ：地塞米松诱导组。分别行X-gal染色和RT-PCR检测β—gal的表达。结果 裸鼠骺板两侧、骨膜内面及松质骨的成骨细胞和破骨细胞可见多量β—gal的表达。未转染BMSCs组有少量β—gal的表达，其他3组细胞的β—gal表达增高。结论成骨细胞和破骨细胞可表达多量β—gal，该两种细胞的β—gal缺乏可能是MorquioB综合征骨骼异常的直接原因。     

Smoking-Attributable mortality in Spain in 2016

IntroductionSmoking-attributable mortality (SAM) is a valuable indicator that can be used to characterize the course and health burden of the smoking epidemic. The aim of this paper was to estimate SAM in Spain in 2016 in the population aged 35 and over, using the best available evidence.MethodsA smoking prevalence-dependent analysis based on the estimation of population-attributable fractions was performed. Smoking prevalence (never, former, and current smokers) was calculated from a combination of the Spanish Health Survey (2016) and the European Health Survey (2014); the relative risk of death among current and former smokers was taken from the follow-up of various cohorts; and mortality rates were obtained from National Center for Statistics data. SAM estimates are presented globally, and by sex, age groups, and major disease categories: cancer, cardiometabolic diseases and respiratory diseases.ResultsIn 2016, 56,124 deaths were attributed to tobacco consumption, 84% in men (47,000), and 50% in the population aged over 74 (27,795). Overall, 50% of SAM was due to cancer (28,281), 65% of which was lung cancer. One in 4 attributable deaths (13,849) occurred before the age of 65.ConclusionsOne in 7 deaths in Spain in 2016 were attributable to smoking. This estimation of SAM clearly highlights the great impact of smoking on mortality in Spain, mainly due to lung cancer and chronic obstructive pulmonary disease.     

MicroRNAs in hepatic pathophysiology in diabetes

MicroRNAs(miRNAs or miRs) are small approximately 22 nucleotide RNA species that are believed to regulate diverse metabolic and physiological processes.In the recent past,several reports have surfaced that demonstrate the role of miRNAs in various biological processes and numerous disease states.For a disease as complex as diabetes,the emergence of miRNAs as key regulators leading to the disease phenotype has added a novel dimension to the area of diabetes research.On the other hand,the liver,a metabolic hub,contributes in a major way towards maintaining normal glucose levels in the body as it can both stimulate and inhibit hepatic glucose output.This equilibrium is frequently disturbed in diabetes and hence,the liver assumes special significance considering the correlation between altered hepatic physiology and diabetes.While the understanding of the mechanisms behind this altered hepatic behavior is not yet completely understood,recent reports on the status and role of miRNAs in the diabetic liver have further added to the complexities of the knowledge of hepatic pathophysiology in diabetes.Here,we bring together the various miRNAs that play a role in the altered hepatic behavior during diabetes.     

Fluid-phase transcytosis in the primate epididymis in vitro and in vivo

Ligated tubules from the corpus epididymidis of men and monkeys were incubated in medium containing horseradish peroxidase (HRP) as a marker for fluid-phase endocytosis. HRP was localized by light and electron microscopy after 0, 15, 30 and 60 min of incubation. Movement between the cells was prevented by tight junctions, but bypass of this barrier was apparently achieved by an intracellular vesicular mechanism leading to a time-dependent appearance of HRP in the lumen. Uptake of HRP into basal cells and capture by the lysosomal apparatus of principal cells were also observed. HRP-filled vesicles also appeared in the basal, mid and apical cytoplasm of epithelial cells in the caput 1 h after injection of the tracer into the epididymal circulation of the monkey, suggesting that this pathway also operates in vivo.