成纤维细胞胶原合成及细胞内STAT6蛋白磷酸化与白细胞介素13的促进作用

目的:观察重组白细胞介素13对成纤维细胞3T3的胶原合成作用,及其在3T3细胞胞内的信号转导途径,探讨白细胞介素13促纤维化的作用机制。方法:实验于2005-03/2006-03在江西医学科学研究所江西省医学生物高技术重点实验室进行。将3T3细胞分为实验组和对照组,在进行实验前,两组细胞均用无血清DMEM培养12～16h,实验组加重组白细胞介素13(100 μg/L),作用24,48,72h后,应用胶原纤维特殊染色观察白细胞介素13作用下3T3细胞体外合成胶原纤维情况;上清液用羟脯氨酸试验检测细胞分泌的总胶原含量;用逆转录-聚合酶链反应检测3T3细胞白细胞介素13受体的表达;用免疫印迹技术检测白细胞介素13作用0,1,2,4h后STAT6蛋白磷酸化情况,组间比较采用t检验。结果:①实验组细胞胶原纤维合成增加及上清液分泌总胶原含量高于对照组(P<0.05)。②逆转录-聚合酶链反应检测到3T3细胞表达白细胞介素13受体α1。③免疫印迹技术检测到白细胞介素13作用1,2,4h均有磷酸化STAT6蛋白表达,且白细胞介素13作用2h磷酸化STAT6蛋白表达最强,4h衰减。结论:白细胞介素13受体α1在3T3细胞上表达,重组白细胞介素13促进3T3细胞胶原合成和STAT6蛋白磷酸化,可能在纤维化发病机制中发挥重要作用。     

胰岛素在严重烧伤大鼠急性肺损伤中的保护作用

目的:观察胰岛素对严重烧伤所致的急性肺损伤的保护效应。方法:实验于2006-01/08在解放军第四军医大学西京医院全军烧伤中心实验室完成。取成年雄性SD大鼠36只,随机分成3组,每组12只:①烫伤 胰岛素组:水浴锅94℃,20s,制备30%总体表面积全层皮肤烫伤模型,伤后即刻腹腔注射生理盐水40mL/kg,并皮下注射胰岛素3U/kg。②烫伤组:造模和腹腔注射同烫伤 胰岛素组,皮下注射等体积生理盐水。③假烫组:用室温水浴模拟烫伤过程,伤后不给予补液。烫伤24h后,收集动脉血测定超氧化物歧化酶活性,收集支气管肺泡灌洗液测定蛋白含量,取肺组织进行苏木精-伊红染色观察病理变化,并测定髓过氧化物酶活性,同时电镜观察胸主动脉血管内皮细胞变化情况。结果:36只大鼠进入结果分析。①肺病理变化:烫伤 胰岛素组肺脏渗出和水肿较烫伤组明显减轻。②肺泡灌洗液中蛋白的质量浓度:烫伤组和烫伤 胰岛素组高于假烫组[(702.9±169.5),(486.5±149.2),(240.5±140.7)mg/L,P<0.05],烫伤 胰岛素组低于烫伤组(P<0.05)。③肺脏髓过氧化物酶活性:烫伤 胰岛素组低于烫伤组[(36.01±8.17),(59.51±12.50)nkat/g,P<0.05],与假烫组比较差异不显著。④血清超氧化物歧化酶活性:烫伤组和烫伤 胰岛素组低于假烫组[(2.27±0.18),(2.63±0.19),(2.81±0.21)mkat/L,P<0.01,0.05],烫伤 胰岛素组高于烫伤组(P<0.01)。⑤电镜下可见烫伤 胰岛素组内皮细胞损伤较烫伤组轻。结论:严重烧伤早期外源性胰岛素干预后可减轻急性肺损伤,具有明显的肺脏保护作用,这种作用可能与胰岛素的内皮细胞保护效应有关。     

克隆并应用计算机软件分析小鼠LASS1基因启动子区的特征

目的:克隆并应用计算机分析小鼠LASS1基因启动子,为进一步研究在细胞衰老过程中LAG1基因的转录调控奠定基础。方法:实验于2006-03/07在汕头大学医学院细胞衰老实验室完成。培养EC109细胞株,预测小鼠LASS1基因启动子所在区域,用PCR技术扩增启动子区序列,构建重组质粒pGEM-T-501、pGEM-T-181、pGEM-T-26,并转化JM109感受态菌,并分别克隆入荧光素酶报告基因载体pGL3-Basic及增强型绿色荧光蛋白报告基因载体pEGFP-1,酶切pGEM-T-501、181、26重组质粒及pGL3-Basic、pEGFP-1载体,制备插入片段和载体,构建重组表达质粒pGL3-501、pGL3-181和pGL3-26及pEGFP-501、pEGFP-181和pEGFP-26,转化JM109感受态菌。用脂质体介导的方法瞬时转染EC109细胞,测定荧光素酶表达活性及观察绿色荧光蛋白的表达情况。结果:①在预测启动子区构建了3种荧光素酶报告基因表达体系及3种增强型绿色荧光蛋白报告基因表达体系,分别为pGL3-501(-501bp～ 106bp)、pGL3-181(-181bp～ 106bp)、pGL3-26( 26～ 106)、pEGFP-501、pEGFP-181和pEGFP-26。②pGL3-501表达载体与pGL3-181表达载体荧光素酶表达活性相近,pGL3-26表达载体荧光素酶表达活性极低,与阴性对照活性相近, 26bp～ 106bp无启动子活性。绿色荧光蛋白的表达情况也类似,pEGFP-501和pEGFP-181有明显的绿色荧光蛋白表达,pEGFP-26和空载体pEGFP-1无表达活性。结论:-181bp～ 26bp区域含有小鼠LASS1基因转录所必需的基本启动子序列。其中2个SP1保守序列是小鼠LASS1基因启动子所必需的。     

筛选心肌缺血再灌注大鼠差异表达基因

目的：利用抑制性消减杂交技术，筛选出大鼠心肌缺血再灌注的差异表达基因，以期通过基因线索探讨其损伤机制。方法：实验于2006—03／10在中山大学中山医学院完成。①实验分组：Sprague-Dawley雄性大鼠40只，随机分为手术组，对照组，每组20只。②实验干预：手术组结扎左冠状动脉，心电图出现急性心肌梗死改变90min后，去除结扎线，再灌注60min，建立心肌缺血再灌注大鼠模型。对照组仅穿线不结扎，余同手术组。③取缺血区心肌，提取Total RNA，构建cDNA文库，利用抑制性消减杂交技术筛选差异表达基因，测序，登录Genbank寻找同源性基因。结果：共获得124个阳性结果，56个为高表达基因，68个为低表达基因，其中发现5个新的cDNA片段。其中能量代谢、物质运输、信号转导相关差异表达基因分别占所有差异表达基因的39．25％，15．89％，15．89％，并且主要变化为下调。结论：抑制性消减杂交技术是一种高效的筛选差异基因的方法。心肌缺血再灌注后基因变化涉及多种功能的基因，以能量代谢、物质运输、信号转导相关基因下调明显。     

河北省4个地区广泛焦虑症的流行病学调查

目的:了解广泛焦虑症的患病率及人口学特点。方法:于2004-10/2005-03随机抽取河北省邯郸、保定、秦皇岛、承德4个地级市18岁以上人口进行全省精神疾病流行病学现场抽样调查工作,总样本24000人。调查筛选工具采用改编后的一般健康问卷12项,以《DSM-Ⅳ-TR轴Ⅰ障碍定式临床检查》病人版为调查的诊断工具。根据被调查者一般健康问卷12项总分,把被调查者分为高危人群、中危人群、低危人群3类。根据预试验调查结果确定三段危险人群的分界分:总分≥4分属于高危人群,高危人群全部进行DSM-Ⅳ-TR轴Ⅰ障碍定式临床检查;总分为2分或3分即属中危人群,中危人群约40%需进行DSM-Ⅳ-TR轴Ⅰ障碍定式临床检查,总分为0分或1分即属低危人群,低危人群中10%需进行DSM-Ⅳ-TR轴Ⅰ障碍定式临床检查。改编后的一般健康问卷12项分数及内容不变,另外增加8个问题均为高危因素,并进行DSM-Ⅳ-TR轴Ⅰ障碍定式临床检查。结果:①实际完成调查20716人,其中男10343人(49.9%),女10373人(50.1%)。共诊断焦虑症患者127例。②按高中低危因素调整后时点患病率为7.69/1000(95%CI6.50/1000～8.88/1000)。城市患病率4.87/1000,农村患病率8.10/1000,两者差异无显著性意义(u=1.78,P>0.05)。女性患病率明显高于男性,差异有显著性意义(分别为10.42/1000,4.97/1000,u=4.49,P<0.01),男女患病率比例为1:2.10。按不同年龄阶段的人口计算出各年龄段的时点患病率,20～29岁患病率较低(3.17/1000),50～59岁患病率较高(15.56/1000)。③通过12个因素的Logistic回归分析发现,影响广泛焦虑症的危险因素有年龄50～59岁(OR=1.713);保护性因素男性(OR=0.431),年龄20～29岁(OR=0.393),收入10001～20000元(OR=0.568),收入20001～40000元(OR=0.117)。结论:广泛焦虑症的流行病学特征为女性、中老年人患病率较高,男性、青年、收入中等者患病率较低。     

腰神经根受压模型大鼠神经根局部炎症因子对温和灸的反应

目的:观察温和灸对腰神经根受压模型大鼠受压神经根局部炎症因子白细胞介素1和白细胞介素6的调节作用,并与灌胃给药莫比可相比较。方法:实验于2005-09/2006-04在上海第六人民医院实验中心完成。①分组:将40只SD大鼠随机分为4组,正常对照组,模型对照组,温和灸组及莫比可组,每组10只。②方法:温和灸组、莫比可组、模型对照组通过手术建立大鼠腰神经根压迫模型,正常对照组做与以上各组相同的捆绑固定,不予手术。温和灸组穴位选取受压神经根相应节段右侧的夹脊穴,采用精制清艾条以穴位为中心、半径为5mm的区域进行温和灸,20min/次,1次/d,7次为1个疗程,共治疗14次。莫比可组按3.75mg/kg灌胃莫比可(10mL生理盐水含药物5mg),每天灌胃1次。正常对照组及模型对照组:生理盐水灌胃10mL/kg,每日灌胃1次。于治疗14d后麻醉下处死取受压神经根组织。③评估:于光镜下观察受压神经根组织形态学变化;用免疫组织化学技术检测神经根局部炎症因子白细胞介素1、白细胞介素6水平。结果:40只大鼠均进入结果分析。光镜下观察温和灸组神经节细胞、神经纤维结构基本正常,见极少量炎细胞。与模型对照组相比,温和灸组与莫比可组神经根局部炎症因子白细胞介素1、白细胞介素6的含量有不同程度的减少(P<0.05);温和灸治疗组白细胞介素1、白细胞介素6的水平低于莫比可组(P<0.01)。结论:温和灸及西药莫比可均能有效抑制神经根局部炎症因子分泌,其中温和灸作用迅速,其效果明显优于莫比可。     

溃疡性结肠炎的外科治疗指南

0引言此治疗指南是在Medline,PudMed以及循证医学Cochrane中心数据库中进行的一次回顾性资料的有组织查询分析．其中关键词包括溃疡性结肠炎、回肠肛管储袋式吻合、回肠造瘘术、结直肠肿物、外科手术、回肠直肠吻合术和一些相关的主题．循证医学水平和推荐等级见表1．     

Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.     

Colorectal cancer biomarkers: To be or not to be? Cautionary tales from a road well travelled

Colorectal cancer(CRC)is the second most common cause of cancer-related death worldwide and places a major economic burden on the global health care system.The time frame for development from premalignant to malignant disease typically spans 10-15 years,and this latent period provides an ideal opportunity for early detection and intervention to improve patient outcomes.Currently,early diagnosis of CRC is hampered by a lack of suitable non-invasive biomarkers that are clinically or economically acceptable for populationbased screening.New blood-based protein biomarkers for early detection of CRC are therefore urgently required.The success of clinical biomarker discovery and validation studies is critically dependent on understanding and adjusting for potential experimental,analytical,and biological factors that can interfere with the robust interpretation of results.In this review we outline some important considerations for research groups undertaking biomarker research with exemplars from our studies.Implementation of experimental strategies to minimise the potential effects of these problems will facilitate the identification of panels of biomarkers with the sensitivity and specificity required for the development of successful tests for the early detection and surveillance of CRC.     

Critical Appraisal on the Role and Outcome of Emergency Colectomy for Uncomplicated Right-sided Colonic Diverticulitis

Background Emergency colectomy is well accepted for treating complicated right-sided colonic diverticulitis. However, the role of colectomy for uncomplicated diverticulitis is not well defined. The aim of this study was to evaluate the short-term and long-term surgical outcome of uncomplicated right-sided diverticulitis in our locality. Patients and Methods Retrospective chart review of patients operated for right-sided diverticulitis over a 20-year period was conducted. Recurrent attacks of right-sided diverticulitis, re-operation rate and re-hospitalisation rate were the long-term parameters of interest. An updated telephone interview was carried out for all surviving patients. Results Seventy-four patients (35 males and 39 females), median age 35.5 (range 16–70) years, were operated for uncomplicated diverticulitis. Thirty patients underwent colectomy, whereas the others underwent appendectomy with diverticulectomy (n = 8) or appendectomy alone (n = 36). All short-term parameters were less favourable for the colectomy group, including higher complication rate, slower return of gastrointestinal function, higher requirement of parenteral analgesic and longer hospital stay. Without colectomy, only 2 patients developed recurrent diverticulitis necessitating hospitalisation, both of whom resolved on conservative treatment. On the other hand, 1 patient required re-operation after colectomy because of intestinal obstruction. The overall re-hospitalisation rate was comparable between the colectomy and the non-colectomy group (16.7% vs. 13.6%). Conclusions Emergency colectomy can eradicate suspicious lesions and eliminate risk of recurrent diverticulitis but at the expense of higher morbidity rates. As the natural course of uncomplicated right-sided colonic diverticulitis is usually benign, conservative treatment with minimal surgery may be a better therapeutic option.