热休克蛋白70对肠上皮细胞缺氧/复氧后线粒体功能及能量代谢的影响

目的 了解Ad-热休克蛋白70(HSP70)对缺氧/复氧损伤后肠上皮细胞线粒体功能及能量代谢的影响.方法 取肠上皮细胞株IEC-6分别转染Ad-HSP70腺病毒载体和空腺病毒载体,蛋白质印迹法观察HSP70的表达.取IEC-6细胞分为正常对照组(不作任何处理)、缺氧/复氧组(给予缺氧/复氧处理)、Ad-HSP70转染组(转染Ad-HSP70腺病毒载体后,给予缺氧/复氧处理).采用噻唑蓝比色法检测细胞内线粒体脱氧酶的活性,高效液相色谱分析法测定细胞能量代谢.结果 与转染空腺病毒载体相比,转染Ad-HSP70腺病毒载体可显著增加细胞HSP70的表达.缺氧/复氧组细胞内线粒体脱氢酶的活性明显低于正常对照组(P＜0.01),Ad-HSP70转染组该指标明显高于缺氧/复氧组(P＜0.01).缺氧/复氧组细胞内腺苷三磷酸含量较正常对照组显著下降,而腺苷二磷酸、腺苷-磷酸含量显著升高;Ad-HSP70转染组细胞能量指标与正常对照组相似(P＞0.05),较缺氧/复氧组有明显改善(P＜0.05或P＜0.01).缺氧/复氧组细胞能荷为0.615±0.060,明显低于正常对照组(0.748±0.012,P＜0.01)、Ad-HSP70转染组(0.736±0.028,P＜0.01).结论 Ad-HSP70腺病毒载体转染肠上皮细胞可诱导HSP70表达增加,显著提高细胞缺氧/复氧后胞内腺苷三磷酸的含量及细胞能荷,保护线粒体整体功能,提示线粒体是HSP70保护肠上皮细胞缺氧/复氧损伤的主要靶细胞器之一.     

ANG-1基因重组腺病毒载体的构建及其转染骨髓问充质干细胞的实验研究

目的构建携带大鼠血管生成素．1（angiopoietin-1,ANG．1）基因的重组腺病毒载体,并检测其转染对大鼠骨髓间充质干细胞（bone marrow mesenchymal stemcells,BMSCs）活力的影响。方法RT-PCR法获取大鼠ANG-1基因并亚克隆至穿梭质粒pAdTrack-CMV,经测序无误后与骨架质粒pAdEasy-1在BJ5183中同源重组。重组质粒pAdEasy-1-ANG-1经鉴定后转染293细胞进行病毒包装扩增。体外转染BMSCs,检测转染后BMSCs中ANG-1的表达。MTT法评估常氧及缺氧环境下ANG-1对BMSCs的影响。结果重组腺病毒载体pAdEasy-1-ANG-1经测序及酶切鉴定正确;BMSCs经转染ANG-1基因后表达ANG-1。MTT法检测提示常氧及缺氧条件下转染前后BMSCs活性的差异均无统计学意义（缺氧组与缺氧下转染组相比,P〉0-05;常氧组与常氧下转染组相比,P〉0-05）。结论成功构建大鼠ANG-1基因重组腺病毒载体,且其转染在体外不影响BMSCs的活性。     

腺病毒介导的p16基因转染对膀胱癌细胞生长影响的研究

目的　探讨复制缺陷型腺病毒介导的 p16基因转染对人膀胱癌细胞生长的影响。 　方法　将 p16重组腺病毒 (Ad p16 )感染p16蛋白表达阴性的人膀胱移行细胞癌T2 4细胞株 ,Ad LacZ、X gal染色法检测重组腺病毒的转染效率 ;Westernblot法检测 p16蛋白的表达 ;MTT法及流式细胞术评估Ad p16对T2 4细胞增殖的影响。　结果　在感染复数 (MOI)为 5 0时 ,重组腺病毒对T2 4细胞的转染率达 97% ;此Ad p16在T2 4细胞中可获高效表达 ;与转染Ad LacZ组及未转染组相比 ,转染Ad p16组T2 4细胞生长受到明显抑制 (P <0 .0 5 ) ,细胞周期分析表明生长抑制主要发生在G1 S节点。　结论　腺病毒载体可有效地将外源 p16基因导入T2 4细胞 ;p16基因重组腺病毒载体转染能抑制T2 4细胞的生长。     

重组腺病毒载体Ad5-CCL20的构建及其表达

目的:构建表达趋化因子CCL20的重组腺病毒载体Ad5-CCL20,检测体外转染小鼠结肠癌细胞CT-26后的产物表达。方法:通过EcoR I/Sal I双酶切CCL20质粒和pDC316质粒,将获取编码CCL20的基因片段连接到pDC316重组穿梭质粒。将测序正确的穿梭质粒pDC316-CCL20与骨架质粒pBHGlox_E1,3Cre共同转染293T细胞,构建重组腺病毒载体Ad5-CCL20。予Ad5-CCL20体外转染结肠癌细胞CT-26,Western blotting和Elisa检测转染后不同时间段CCL20的表达情况,并以含CCL20的上清分别对mDC、iDC进行趋化实验。结果:成功构建表达趋化因子CCL20的重组腺病毒载体Ad5-CCL20;Western blotting和Elisa均检测到CCL20表达,且CCL20表达量随病毒转染CT-26细胞的时间延长而逐渐增加;趋化实验表明,趋化因子CCL20对iDC、mDC都有趋化作用,但对iDC的趋化作用更加明显(P0.05)。结论:重组腺病毒载体Ad5-CCL20的构建及获取为后续开展肿瘤的免疫治疗提供实验基础。     

大鼠galectin-9基因重组腺病毒载体的构建和鉴定

目的克隆大鼠galectin-9基因全长cDNA,构建含大鼠galectin-9基因的重组腺病毒载体,并予以鉴定。方法从大鼠的肝脏组织中用RT-PCR的方法克隆扩增大鼠galectin-9基因,再定向插入到带NotⅠ和HindⅢ酶切的pDC316-GFP穿梭质粒中,获得穿梭质粒pDC316-GFP-galectin-9。经PCR、NotⅠ和HindⅢ酶切及测序鉴定后,用脂质体将穿梭质粒pDC316-GFP-galectin-9与腺病毒骨架质粒pBHGlox△E1.3Cre共转染HEK-293细胞。经位点特异性重组获得含目的基因的重组腺病毒Ad5-galectin-9,行PCR鉴定,经HEK-293细胞扩增及纯化制备高滴度病毒液,用细胞培养方法测定病毒TCID50滴度。结果 PCR、酶切及测序证实穿梭质粒构建正确,PCR鉴定证实大鼠galectin-9基因重组腺病毒载体构建正确,病毒的感染滴度为1.4×109U/ml。结论成功构建了含大鼠galectin-9基因的重组腺病毒表达载体,为进一步研究大鼠galectin-9基因的功能奠定了基础。     

转染血红素氧合酶—1基因减轻人肝细胞体外缺氧—复氧损伤

目的 探讨血红素氧合酶-1重组腺病毒载体(Ad-HO-1)体外转染人肝细胞的转染效果及其对肝细胞缺氧-复氧损伤的影响.方法 取人肝细胞系L-02细胞,滴加低温保存的Ad-HO-1,分别培养24 h、48 h和72 h(24 h组、48 h组和72 h组),并以加入空载体腺病毒共培养的L-02细胞为空白对照.采用逆转录聚合酶链反应法检测各组肝细胞HO-1 mRNA的表达水平;以间接免疫荧光标记法检测各组肝细胞的HO-1表达率;在倒置荧光显微镜下观察转染24 h和72 h的肝细胞中绿色荧光蛋白(EGFP)的表达.取空白对照组肝细胞(培养48 h)和48 h组肝细胞,缺氧培养4 h后再有氧培养8 h,采用四甲基偶氮唑盐法测定两组肝细胞存活率.结果 24 h组、48 h组和72 h组HO-1 mRNA表达水平明显高于空白对照组,且随着转染时间的延长,HO-1 mRNA的表达水平逐渐升高.空白对照组HO-1的表达率为2.0%,24 h组为29%,48 h组为85.6%,72 h组为84.6%.基因转染后24 h和72 h,可以观察到L-02细胞中EGFP的表达.经历缺氧-复氧实验后,空白对照组肝细胞的存活率为(37.7±3.5)%,48 h组肝细胞的存活率为(89.4±5.2)%,二者相比较,差异有统计学意义(P<0.01).结论 Ad-HO-1在体外能有效的转染人肝细胞;与未转染者相比,转染肝细胞的缺氧-复氧损伤程度较轻.     

转染转化生长因子β1基因减轻非协调性异种心脏移植急性血管性排斥反应

目的探讨移植物局部转染转化生长因子β1(TGF-β1)基因对非协调性异种心脏移植急性血管性排斥反应(AVR)的影响。方法建立豚鼠到SD大鼠的颈部心脏移植模型,移植前受鼠接受中华眼镜蛇毒因子和环孢素A预处理,供心切取后,经冠状动脉按每克心肌组织灌注5×1010PFU携带TGF-β1基因的重组腺病毒载体进行基因转染,再移植到经过预处理的SD大鼠颈部(基因转染组),另设灌注5×1010PFU腺病毒空白载体的空白载体组和对照组。术后观察各组移植物的存活情况、移植物组织学变化情况以及移植物中CD68和CD57的表达、细胞凋亡指数、TGF-β1的表达。结果基因转染组移植物的存活时间为(95±3)h,明显长于空白载体对照组的(57±2)h和对照组的(60±2)h(P<0.01);各组移植物均呈急性血管性排斥反应的病理改变,但基因转染组较轻;基因转染组移植物组织中炎症细胞浸润数、CD68和CD57的表达量及细胞凋亡指数明显低于空白载体对照组和对照组,并能检测到外源性TGF-β1的表达。结论经冠状动脉灌注重组腺病毒载体介导的TGF-β1基因转移可减轻非协调性异种心脏移植AVR,明显延长移植物的存活时间。     

重组腺病毒介导HOSM基因在胰腺癌细胞中的表达

目的 重组腺病毒介导HOSM基因转染胰腺癌细胞后，用RT-PCR方法检测外源性HOSM基因的表达情况。方法 构建了含人抑瘤素M(OSM，oncostatin M)基因的复制缺陷型重组腺病毒载体ad-HSOM，以ad-HOSM转染胰腺癌细胞株后，提取细胞的总RNA，用RT-PCR法检测HOSM基因在胰腺癌中的表达。结果 HOSM基因在转染细胞中有效的表达。结论 RT-PCR法能有效的检测外源性HOSM基因在胰腺癌中的表达，为重组腺病毒介导HOSM基因用于胰腺癌治疗的实验研究奠定了基础。     

携反义二型基质金属蛋白酶基因的重组腺病毒的构建

目的　构建携带反义二型基质金属蛋白酶 (MMP2 )基因的重组腺病毒。方法　从新鲜肝癌组织中提取总RNA ,用RT PCR法合成MMP2cDNA序列中 5′端转录起始位点附近长约 5 0 0bp的基因片段 ,将此片段反义克隆到腺病毒载体 (AdEasy)系统的多克隆位点 ,经转染 2 93细胞生成携带反义MMP2基因的重组腺病毒Ad MMP2 AS。结果　成功构建并包装携带反义MMP2基因片段的重组腺病毒Ad MMP2 AS,病毒滴度达 1× 10 8/ml。结论　构建的重组腺病毒Ad MMP2 AS可望能有效地将反义MMP2基因片段导入人肝癌细胞株 ,为进一步研究肝癌浸润和转移机理以及探讨抑制肝癌浸润和转移的方法提供实验基础。     

川芎嗪预先给药对胎鼠海马神经细胞缺氧/复氧时c-fos和HSP70表达的影响

目的 探讨川芎嗪预先给药对胎鼠海马神经细胞缺氧/复氧时c-fos和热休克蛋白70(HSP70)表达的影响.方法 胎鼠海马神经细胞培养鉴定后,随机分为5组(n=24):正常对照组(C组)、缺氧/复氧损伤组(A/R组)、不同浓度川芎嗪预先给药组(L组、M组和H组).C组不制备缺氧/复氧模型;A/R组、L组、M组和H组制备缺氧/复氧模型;L组、M组和H组加入川芎嗪,终浓度分别为60、200和800μg/ml,孵育1 h后制备缺氧/复氧模型.缺氧/复氧模型制备方法:海马神经细胞置入90%N2-10%CO2培养箱中孵育2 h诱导缺氧,然后放入37 ℃、5%CO2培养箱中复氧24 h.处理结束后测定海马神经细胞凋率、细胞活力、c-fos和HSP70的表达水平.结果 与C组比较,A/R组、L组和H组海马神经细胞活力降低,细胞凋亡率升高(P＜0.01);与A/R组比较,L组、M组和H组海马神经细胞活力升高,细胞凋亡率降低,c-fos表达下调,HSP70表达上调(P＜0.05);与L组比较,M组和H组海马神经细胞活力升高,细胞凋亡率降低,c-fos表达下调,HSP70表达上调(P＜0.05);与M组比较,H组细胞活力下降,细胞凋亡率升高,c-fos表达上调,HSP70表达下调(P＜0.01).结论川芎嗪预先给药抑制胎鼠海马神经细胞缺氧/复氧时细胞凋亡的机制可能与下调c-fos表达,上调HSP70表达有关.     

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.