肾母细胞瘤过度表达基因对大鼠肝星状细胞MMP-1和TIMP-1表达的作用研究

目的 构建能表达靶向肾母细胞瘤过度表达基因（NOV）的小干扰RNA（siRNA）的重组质粒，研究NOV对大鼠肝星状细胞（HSC）增殖、基质金属蛋白酶-1（MMP-1）和金属蛋白酶组织抑制因子-1（TIMP-1）表达的影响。方法 以NOV为目的基因，以质粒psiRNA-hH1 neo为载体，构建能在真核细胞中表达的靶向NOV的siRNA的重组质粒psiRNA（psiRNA1、2、3）和阴性对照重组质粒pconsiRNA。限制性酶切和测序鉴定构建成功后，脂质体介导重组质粒转染HSC，依据转染质粒的不同将HSC分为psiRNA1组、psiRNA2组、psiRNA3组、阴性对照pconsiRNA组，并以未转染重组质粒的HSC为空白对照组。半定量RT-PCR检测HSC的NOV、MMP-1、TIMP-1 mRNA表达情况，MTT法检测细胞增殖。结果限制性酶切和测序鉴定表明成功构建了NOV siRNA表达质粒；与阴性对照组相比，转染外源重组质粒psiRNA2的HSC内NOV的mRNA表达水平下降（P〈0．05），MMP-1mRNA表达水平下降不明显（P〉0．05），而TIMP-1 mRNA表达水平明显下降（P〈0．05）。与空白对照组相比，转染外源重组质粒psiRNA2的HSC增殖活性显著降低（P〈0．05）。psiRNA1组、psiRNA3组的HSC内NOV、MMP-1和TIMP-1 mRNA的表达及HSC增殖活性均无明显下降（P均〉0．05）。结论NOV可促进HSC增殖及表达TIMP-1增加，而对MMP-1表达影响不大，提示NOV可以作为肝纤维化基因治疗的一个新的靶位点。     

RNA干扰体外抑制人食管鳞癌细胞Eca-109缺氧诱导因子-1α的表达

目的:观察缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)短发夹状RNA(short hairpin RNA,shRNA)对人食管鳞癌细胞系Eca-109中 HIF-1α基因的沉默效应,筛选有效干扰靶点．方法:设计合成干扰缺氧诱导因子-1α RNA 寡核苷酸片段,经退火、连接等步骤克隆至线性化的PGCsi真核表达载体上,测序鉴定．应用LipofectamineTM2000将该质粒分别转染Eca-109细胞和293T细胞,其中Eca-109抗性细胞又经2-4 wk的G418筛选．荧光显微镜评估转染效率,荧光定量RT-PCR检测HIF-1α mRNA表达情况,western blot检测HIF-1α蛋白表达情况．结果:成功构建了3个靶点的重组质粒pGCsi- H1-shHIF,293T细胞平均转染效率为约85．4％, Eca-109细胞的平均转染效率约73．2％．荧光定量RT-PCR和Western blot显示瞬时转染这3种重组质粒的293T细胞和筛选2 wk的Eca-109 细胞HIF-1 α mRNA和蛋白表达均较对照组有不同程度的下降,其中2号和3号靶点的干扰效应尤为明显．在瞬时转染72 h后的293T 细胞中,其抑制率分别达到了78．5％、86．9％ (P=0．000,P=0．000 vs 72 h空白对照组),筛选2 wk的Eca-109细胞中,3号靶点对HIF-1α mRNA的抑制率也达到了69．7％(P=0．000 vs 2 wk空白对照组)．结论:重组质粒pGCsi-shHIF能有效抑制食管鳞癌细胞Eca-109中HIF-1α基因的表达,经不同细胞中的瞬时和稳定转染筛选出了有效干扰靶位．     

Survivin靶向RNA干扰对人结肠癌HCT116细胞生长影响的研究

目的设计和构建survivin特异性的siRNA真核表达载体,观察survivin siRNA重组体对人大肠癌HCT116细胞株生长的影响,为大肠癌的基因治疗提供理论依据。方法针对survivin mRNA序列设计合成编码siRNA的DNA模板,构建2个survivin RNAi真核表达载体;实验分为survivin siRNA重组质粒转染组、空载体组和HCT116组,转染大肠癌细胞HCT116细胞,采用RT-PCR法检测survivin mRNA的表达,观察重组质粒对转染的HCT116细胞survivin基因表达的影响;用四甲基偶氮唑盐（MTT）法测量细胞生长情况;用流式细胞术检测细胞凋亡情况。结果测序表明survivin干扰序列完全正确,RT-PCR结果显示2条survivin siRNA真核表达质粒均能有效抑制survivin mRNA的转录表达。MTT比色法检测显示,与阴性对照组及未转染组细胞比较,干扰组细胞增殖率明显下降（P〈0．05）。流式细胞术检测的转染重组质粒组细胞凋亡率高于脂质体对照组和空质粒对照组（P〈0．05）。结论成功构建了survivin干扰真核表达载体,siRNA重组体能有效抑制人大肠癌细胞survivin mRNA表达,并抑制结肠癌细胞生长,促进其凋亡。     

白藜芦醇对人食管癌细胞Eca-109增殖的影响及其机制探讨

目的观察白藜芦醇（Res）对人食管癌细胞Eca-109增殖的影响,并探讨其机制。方法将Eca-109细胞分别与0、10、20、40μmol/L的Res共同培养,采用MTT法测定Res对Eca-109细胞的生长抑制率,采用RT-PCR法检测Eca-109中的VEGF mRNA。结果随着Res浓度增加、作用时间延长,Eca-109细胞生长抑制率增加（P均〈0.05）,VEGF mRNA表达量降低（P均〈0.05）。结论Res可抑制Eca-109细胞增殖,其作用可能与抑制VEGF表达有关。     

活性氧在COPADG诱导食管癌Eca-109细胞凋亡中的作用

不同浓度2-（3-羧基-1-丙酰氨基）-2-脱氧-D-葡萄糖（COPADG）作用于人食管癌Eca-109细胞24h。结果显示随COPADG浓度的升高Eca-109细胞的抑制率增加，Eca-109细胞凋亡率与COPADG浓度呈正相关，r=1．0，P〈0．01；Eca-109细胞内活性氧（ROS）水平与Eca-109细胞凋亡率呈正相关，r=1.0，P〈0．01。认为COPADG可能通过提高Eca-109细胞内ROS水平诱导Eca-109细胞凋亡。     

针对P基因的siRNA对乙肝病毒S表达稳定性的影响

目的以乙型肝炎病毒（HBV）P基因为靶点，构建表达小干扰RNA（siRNA）的质粒载体psRNA1、psRNA2、psRNA3、psRNA4及对照psiRNA0，体外观察针对P基因的siRNA对HBs表达的影响。方法设计并合成针对HBV-P基因的siRNA寡核苷酸，经退火形成双链后克隆入psiRNA-H1neo质粒中，通过鉴定将构建成功的5个psiRNAs真核表达质粒瞬时转染HepG2．2．15细胞，用半定量RT-PCR对筛选到的位点进行了试验观测抑制效果。结果表明针对P基因的siRNA能下调HBs的表达，其中psiRNA1、psiRNA2的抑制HBs基因表达的效果最强。结论针对P基因的siRNA可以有效的抑制HBs的表达，抑制的效果和siRNA的靶位点有关。     

氯化钴对RNA干扰食管鳞癌细胞HIF-1α基因表达及功能的影响

目的研究氯化钴模拟缺氧对RNA干扰的缺氧诱导因子-1α（HIF-1α）基因表达及功能的影响，观察在人食管鳞癌细胞系Eca-109中RNA干扰抑制HIF-1α的效果。方法选择4组细胞分别为食管癌Eca-109细胞和3株稳定转染HIF-1αSiRNA的Eca-109细胞（本实验室编号分别为H2／14号、H3／15号、H2／20号细胞），分别用200、400、600、800μmol／L氯化钴模拟缺氧，缺氧培养时间分别为12、24．48、72h，采用Western印迹和逆转录聚合酶链反应（RT-PCR）检测HIF—1α蛋白及mRNA表达，同时Western印迹检测血红素加氧酶（HO-1）、基质金属蛋白酶-2（MMP-2）、葡萄糖转运体-1（Glut-1）、P53等相关基因的表达。结果4组细胞HIF-1α蛋白表达均随氯化钴浓度加大而增加，常氧与400μmol／L氯化钴处理24h后筛选出H3／15号细胞HIF-1α蛋白表达最少，与其它3组相比差异有统计学意义（P〈0．05），mRNA检测差异无统计学意义；干扰细胞常氧下HO-1、MMP-2、Glut-1、P53表达不同程度减少，缺氧后HO-1、P53表达增加，MMP-2、Glut-1表达无明显变化。结论RNA干扰能抑制Eca-109细胞的HIF-1α基因表达，从而不同程度减少HO-1、MMP-2、Glut-1、P53等相关基因表达；经氯化钴模拟缺氧筛选出干扰效果较好的一株细胞，为进一步研究HIF-1α的功能奠定了基础。     

NF-κB/p65特异的RNAi腺病毒载体的构建及其对p65表达的抑制效应

目的构建NF-κB p65亚基特异的RNA干扰（RNAi）腺病毒表达载体，并验证其对p65亚基的基因沉默效应。方法设计合成三对针对p65 mRNA不同位点的短发夹RNA（shRNA）编码序列，克隆到穿梭载体中，通过体外同源重组将短干扰RNA（siRNA）表达盒转移到腺病毒骨架质粒，构建RNAi腺病毒表达载体；在HEK293A细胞中包装并扩增病毒、空斑实验法进行病毒滴度测定；腺病毒感染人脐静脉内皮细胞株ECV304细胞，Western印迹法和免疫细胞化学法验证构建的RNAi腺病毒对p65蛋白表达的抑制效应。结果成功构建了NF-κB p65亚基特异的RNAi腺病毒表达载体，获得高滴度的腺病毒液；RNAi腺病毒感染ECV304细胞后可以高效抑制p65蛋白的表达，且对p65蛋白表达的抑制作用可持续6d以上。结论应用RNAi腺病毒表达载体能有效阻断目的基因的表达。     

JNK MAPK信号通路在食管癌细胞系Eca-109细胞中的作用机制

目的探讨c-jun氨基末端激酶1/2（c-jun N-teuninal kinase,JNK 1/2）信号通路在食管癌细胞系Eca-109细胞中的作用。方法体外培养Eca-109细胞,以特异性JNK信号转导通路抑制剂SP600125处理Eca-109细胞;RT-PCR的方法检测JNK1和JNK2基因的表达,Western blot法检测JNK和p-JNK蛋白的表达,MTT（3-（4,5-Dimethylthiazol-2-yl）-2,5-di-phenyltetrazolium bromide）法检测细胞增殖,流式细胞术检测细胞凋亡。结果 Eca-109细胞经SP600125分别处理24h和48 h后,分别与对照组比较,JNK1 mRNA的表达无统计学差异（均P〉0.05）,JNK2 mRNA的表达也无统计学差异（均P〉0.05）,但活化的JNK即P-JNK1/2蛋白的表达显著减少,细胞的增殖显著被抑制,细胞的凋亡率有统计学差异（均P〈0.05）。结论 JNK信号通路可能在Eca-109细胞的发生发展中发挥重要作用。     

转化生长因子βⅠ型受体基因的靶向小干扰RNA抑制肝星状细胞合成胶原的体外研究

目的 观察大鼠转化生长因子βⅠ型受体(TβR Ⅰ)基因的靶向小干扰RNA(siRNA)表达质粒对肝星状细胞(HSC)合成胶原的影响.方法 根据大鼠Tβ R Ⅰ的基因序列,设计并构建3个大鼠TβR Ⅰ基因的靶向siRNA表达质粒,以脂质体作转染试剂,将siRNA表达质粒分别转染至HSC-T6细胞中,RT-PCR和Western印迹技术分析TβRⅠ mRNA及蛋白表达水平,噻唑蓝(MTT)法检测细胞增殖,放射免疫法测HA、PCⅢ含量.采用LSD法进行统计学处理.结果 酶切证实siRNA的目的 基因片段已成功克隆入载体中.与空白对照组比较,转染siRNA表达质粒后,3组HSC-T6细胞TβR Ⅰ mRNA表达水平均抑制,其中以psiRNA2组抑制作用最强(psiRNA1组:t=7.354,P＜0.01;psiRNA2组:t=9.214,P＜0.01;psiRNA3组:t=5.967,P＜0.01).3组TβRⅠ蛋白表达水平均降低,以psiRNA2组降低最明显(psiRNA1组:t=6.324,P＜0.01;psiRNA2组:t=8.741,P＜0.01;psiRNA3组:t=4.128,P＜0.01).3组HSC-T6细胞增殖活性均下降,合成胶原均减少,以psiRNA2组最明显;而转染无关siRNA组无明显变化.结论 构建的TβR Ⅰ siRNA表达质粒可抑制HSC-T6细胞合成胶原,为肝纤维化基因治疗提供新的靶点.     

胰岛素瘤的解剖分布特点分析

目的胰岛素瘤是最常见的胰腺神经内分泌肿瘤，因其临床表现多样，导致诊断困难。影像学诊断尤其是超声内镜(EUS)在胰岛素瘤的诊断中起着重要作用，拥有较高的敏感性和特异性。本研究拟通过明确胰岛素瘤的解剖分布特点，以期有助于提高影像学的诊断准确率和降低漏诊率，尤其是在教育和培训实践中对于EUS的学习者更具有指导价值。 方法回顾性分析解放军总医院第一医学中心病案资料数据库1993年1月至2019年11月经外科手术、病理确诊为胰岛素瘤的患者的临床资料，检索方法采取搜索术后病理诊断为"胰岛素瘤"的病例，通过查阅病例的方法，提取出胰岛素瘤的大小和解剖分布等数据，进一步分析其特点。 结果共检索到确诊为胰岛素瘤的患者116例，其中，男45例、女71例，年龄13～76岁，平均年龄(44.4±14.85)岁。胰岛素瘤单发110例(94.8%)、多发6例(5.2%)。位置分布：头颈部46例(39.7%)，单发45例、多发1例；体尾部68例(58.6%)，单发65例、多发3例；全胰腺多发2例(1.7%)。病变大小特点：最大径0.4～3.4 cm，平均大小(1.53±0.58)cm。≤1 cm 29例、>1 cm而≤1.5 cm41例、>1.5 cm而≤2.0 cm28例，≤3 cm 15例，>3 cm 3例。年龄与肿瘤的大小相关，≤44岁患者肿瘤平均大小为(1.36±0.51)cm、>44岁患者肿瘤平均大小为(1.70±0.60)cm，P<0.05。头颈部的肿瘤大于体尾部的肿瘤，头颈部肿瘤平均大小(1.66±0.63)cm，体尾部(1.42±0.52)cm，P<0.05。 结论胰岛素瘤在胰腺体尾部较头颈部更好发；绝大多数单发，但可以全胰腺多发；多数小于1.5 cm，肿瘤的大小与患者年龄和肿瘤的解剖分布相关。     

Pathogenesis of carcinoma of the papilla of Vater

Most adenomas and carcinomas of the small intestine and extrahepatic bile ducts arise in the region of the papilla of Vater. In familial adenomatous polyposis (FAP) it is the main location for carcinomas after proctocolectomy. In many cases symptoms due to stenosis lead to diagnosis at an early tumor stage. In about 80%, curative intended resection is possible. Operability is the most relevant prognostic factor. Most ampullary carcinomas resp. carcinomas of the papilla of Vater develop from adenomatous or flat dysplastic precursor lesions. They can be sited in the ampulloduodenal part of the papilla of Vater, which is lined by intestinal mucosa. They also can develop in deeper parts of the ampulla, which are lined by pancreaticobiliary duct mucosa. Intestinal-type adenocarcinoma and pancreaticobiliary-type adenocarcinoma represent the main histological types of ampullary carcinoma. Furthermore, there exist unusual types and undifferentiated carcinomas. Many carcinomas of intestinal type express the immunohistochemical marker profile of intestinal mucosa (keratin 7?, keratin 20+, MUC2+). Carcinomas of pancreaticobiliary type usually show the immunohistochemical profile of pancreaticobiliary duct mucosa (keratin 7+, keratin 20?, MUC2?). Even poorly differentiated carcinomas, as well as unusual histological types, may conserve the marker profile of the mucosa they developed from. These findings underline the concept of histogenetically different carcinomas of the papilla of Vater which develop either from intestinal- or from pancreaticobiliary-type mucosa of the papilla of Vater. Molecular alterations in ampullary carcinomas are similar to those of colorectal as well as pancreatic carcinomas, although they appear at different frequencies. In future studies, molecular alterations in ampullary carcinomas should be correlated closely with the different histologic tumor types. Consequently, the histologic classification should reflect the histogenesis of ampullary tumors from the two different types of papillary mucosa.     

Demonstration of the mechanism of action of biguanides

Summary Palmitic acid oxidation in rat diaphragm homogenate is depressed by biguanide concentrations that are still incapable of inhibiting oxidative phosphorylation. Glucose oxidation is not directly effected by the same biguanide concentrations: however, the inhibitory effect of palmitic acid on glucose oxidation is partly removed by biguanides. Inhibition of fatty acid oxidation, which accounts for most of the metabolic effects caused by these drugs, can be regarded as the fundamental mechanism of action of biguanides. There is some evidence suggesting that these drugs might interact with carnitine, thus preventing long-chain fatty acids from being transported across the mitochondrial membrane to the site of oxidation. Traduzione a cura degli AA.     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.     

氯硝柳胺悬浮剂的毒性评价

目的评价氯硝柳胺悬浮剂的毒性，为现场大规模应用灭螺提供依据。方法按照中华人民共和国国家标准GB 15670-1995《农药登记毒理学试验方法》和鱼类毒性试验方法进行。结果经口、经皮肤的LDso雌、雄性大鼠均>5 000 mg/kg，经呼吸道的LCso雌、雄性大鼠均>5 000mg/m3，该药经口、经皮肤、经呼吸道毒性均属微毒类药物；兔眼用药后，观察期内无不良反应，对眼无刺激性；皮肤用药后对皮肤无刺激性。与氯硝柳胺原药、氯硝柳胺乙醇胺盐原药和氯硝柳胺乙醇胺盐可湿性粉剂相比，氯硝柳胺悬浮剂对鱼急性毒性最低。结论氯硝柳胺悬浮剂属微毒类药物，对鱼的毒性低于其乙醇胺盐可湿性粉剂，适合于现场应用。     

血吸虫童虫生长发育及其机制的研究进展

血吸虫童虫是宿主免疫系统攻击的重要靶标,包括皮肤型、肺型和肝门型童虫。宿主分子对童虫生长发育具有重要作用。童虫生长发育机制包括免疫调节、信号转导、性别发育及凋亡等。肌动蛋白、组织蛋白酶、烯醇化酶和葡萄糖基转移酶等分子为血吸虫童虫生长发育的重要分子。本文对血吸虫童虫生长发育及其机制的研究进展做一综述。     

124例耐多药结核分枝杆菌基因突变特征分析

目的对临床分离的耐多药结核分枝杆菌相关基因的突变特征进行分析。方法对124例耐多药结核分枝杆菌以及50株敏感株的耐药相关基因(包括异烟肼inh A、kat G、oxyR-ahp C间隔区以及利福平rpo B)进行序列测定,分析其基因突变情况。结果异烟肼耐药inh A基因突变率为14.5%;kat G基因突变率为70.2%(87/124),主要位于315位;oxyR-ahp C间隔区突变率为15.3%;inh A、kat G两种基因同时突变率75.0%,三种基因同时突变率为89.5%。利福平rpo B基因突变的检出率高达95.2%,突变主要发生在531、526、516位点。结论我省耐多药菌异烟肼耐药相关基因最常见突变为kat G 315、inh A C-T(-15)、axyR-ahp C间隔区(-10)C-T,利福平为rpo B531、526、516。结合MDR-TB耐药相关基因的特征分析,可以建立一种快速、准确、特异的适合于我省的检测结核菌耐多药性的新方法。     

Assessment of quality of life of parents of children with juvenile chronic arthritis

The aim of the study was to assess the quality of life (QOL) and the psychological status of parents of children with juvenile chronic arthritis (JCA). The QOL, anxiety and depression of the parents of 28 children with JCA were evaluated and compared to those of the parents of 28 healthy children. Mothers of JCA children and mothers of healthy children reported similar QOL. The reported anxiety and depression levels were similar for mothers and fathers in both groups. The parents of children with pauciarticular-type JCA reported lower QOL and higher levels of anxiety and depression than the parents of children with other types, namely polyarticular and systemic JCA. These findings may be explained by the fact that the pauciarticular patients had shorter disease duration and were less frequently seen in the outpatient clinic. The QOL of mothers of children with JCA was found to be slightly impaired in the group of children with pauciarticular JCA. Future larger studies are needed to confirm these results, as the number of subjects in the three groups was rather low. Received: 26 September 2001 / Accepted: 8 February 2002     

Numerical Simulation of the Effect of Rate of Change of Glucose on Measurement Error of Continuous Glucose Monitors

Background

A 5-day in-patient study designed to assess the accuracy of the FreeStyle Navigator® Continuous Glucose Monitoring System revealed that the level of accuracy of the continuous sensor measurements was dependent on the rate of glucose change. When the absolute rate of change was less than 1 mg•dl⁻¹•min⁻¹ (75% of the time), the median absolute relative difference (ARD) was 8.5%, with 85% of all points falling within the A zone of the Clarke error grid. When the absolute rate of change was greater than 2 mg•dl⁻¹•min⁻¹ (8% of the time), the median ARD was 17.5%, with 59% of all points falling within the Clarke A zone.

Method

Numerical simulations were performed to investigate effects of the rate of change of glucose on sensor measurement error. This approach enabled physiologically relevant distributions of glucose values to be reordered to explore the effect of different glucose rate-of-change distributions on apparent sensor accuracy.

Results

The physiological lag between blood and interstitial fluid glucose levels is sufficient to account for the observed difference in sensor accuracy between periods of stable glucose and periods of rapidly changing glucose.

Conclusions

The role of physiological lag on the apparent decrease in sensor accuracy at high glucose rates of change has implications for clinical study design, regulatory review of continuous glucose sensors, and development of performance standards for this new technology. This work demonstrates the difficulty in comparing accuracy measures between different clinical studies and highlights the need for studies to include both relevant glucose distributions and relevant glucose rate-of-change distributions.     

治疗高血压药物的经济学评价

重视高血压治疗中的经济学评价,对利用我国有限的卫生资源来遏制高血压对人民群众的危害有着重要的现实意义。药物经济学对于药物治疗的成本和治疗的结果给予同样的关注。因为治疗高血压的费用,不仅涉及药物价格,还包括患者的危险水平,降压疗效和对临床终点事件的影响,以及治疗的依从性和安全性。因此药物经济学更强调整体成本和价-效比。低危病人,若非药价低廉,治疗的价-效比不够理想。而在高危的患者,价-效比越小越经济而不是药费越便宜越好。