半胱天冬氨酸蛋白酶-12小干扰RNA对小鼠肝细胞凋亡的阻抑

目的观察小鼠半胱天冬氨酸蛋白酶-12（caspase-12）基因特异性小干扰RNA（siRNA）构建的表达载体pRNAT-casp12对caspase-12基因的抑制及其对内质网应激介导的小鼠肝细胞凋亡的影响。方法以小鼠肝细胞株Hepa1-6为靶细胞，利用脂质体与重组质粒pRNAT-casp-2共转染，分别在转染24、48和72h后收集细胞，采用实时荧光定量PCR分析和Western印迹检测caspase-12的表达；利用毒胡萝b素（TG）诱导细胞，建立内质网应激介导的细胞凋亡模型，通过DNA梯带凝胶电泳检测，选出适合的诱导时间；TG诱导已转染了pRNAT—casp12的干扰组细胞后，以空质粒转染组为对照，利用Western印迹检测caspase-12蛋白表达水平的变化，通过DNA梯带凝胶电泳、流式细胞仪、Hoechst33258染色等方法检测细胞凋亡，观察caspase-12siRNA对细胞凋亡的影响。结果pRNAT—casp12转染细胞24、48和72h后，caspase-12mRNA的水平分别下降了45．6％、72．5％和59．5％；caspase-12蛋白表达下降了17．1％、37．3％和60．1％；2μmol／L TG处理细胞30h后，成功诱导细胞凋亡；与对照组相比，干扰组细胞经TG诱导后，caspase-12蛋白表达水平下降了54．6％，流式细胞仪检测发现早期调亡率下调了51．4％（P〈0．01）。结论小鼠caspase-12siRNA对Hepa1—6细胞caspase-12基因的表达具有显著的抑制作用，能够明显阻抑内质网应激介导的凋亡，有望发展成为新一代抗凋亡药物。     

内质网应激在高脂血症相关性大鼠急性胰腺炎发病中的作用

目的探讨内质网应激在高脂血症大鼠合并急性胰腺炎（AP）发病中的作用。方法48只大鼠分为单纯AP组（n=18，正常大鼠腹腔内注射雨蛙肽40μg／kg×2次，间隔2h）、正常组（n=6）、高脂血症组（n=6，普通饲料＋3％胆固醇＋20％猪油）和高脂血症性AP组（n=18，高脂血症大鼠腹腔内注射雨蛙肽40μg／kg×2次，间隔2h）。单纯AP组和高脂血症性AP组大鼠均于造模后9、12、24h分批处死（n=6），测定血淀粉酶活性，评价胰腺病理变化；TUNEL法评价胰腺组织凋亡指数；免疫组化法检测胰腺内葡萄糖调节蛋白（GRP）78／Bip的蛋白表达；PT-PCR法检测胰腺组织内内质网应激过程中的重要分子GRP78／Bip、X结合蛋白（XBP）-1、CHOP／GADD153（C／EBP-homolo-gous protein，or growth arrest and DNA-damage-inducible gene 153）、caspase-12的基因动态表达；Western印迹法检测GRP78、caspase-12蛋白的动态表达。结果大鼠喂饲高脂饮食8周后，三酰甘油[（0、99±0．38）mmol／L]和胆固醇[（3．17±0．18）mmol／L]明显升高，且较正常组高281％和96％（P〈0．05）；高脂血症性AP组血清淀粉酶活性较单纯AP组高，胰腺病理变化亦较单纯AP组严重（P〈0、05），尤其在造模后9h最为显著。高脂血症AP大鼠造模后9、12、24h，胰腺腺泡细胞凋亡均较正常组明显（P〈0.05）；GRP78／Bip表达明显；CH（）P／GADD153在造膜12、24h时表达与正常对照组相比差异有统计学意义（P〈0．05）；在造模后12、24h时胰腺组织发生XBP-1和caspase-12分裂，而正常组则无。结论内质网应激参与了高脂血症性AP的发病，并可通过caspase-12通路诱导胰腺腺泡细胞发生凋亡，加重AP病程。     

内质网应激与糖尿病相关性研究进展

内质网应激(ERS)是一种特殊类型的细胞内应激,是由内质网内错误折叠与未折叠蛋白聚集以及Ca+代谢紊乱所引起.研究表明,适度ERS通过激活未折叠蛋白反应起适应性的细胞保护作用,而过高和持久的ERS则通过诱导转录因子CHOP表达、激活caspase-12和c-Jun氨基末端激酶(JNK)等导致细胞凋亡.近年的研究显示,ERS通过促使胰岛β细胞凋亡及胰岛素抵抗参与糖尿病发病,而阻断ERS诱导的凋亡通路和胰岛素抵抗则可能为糖尿病治疗提供新的手段.     

鱼藤酮诱导多巴胺能细胞α-突触核蛋白聚集和ERK1/2通路活化的研究

目的探讨鱼藤酮对多巴胺能细胞α-突触核蛋白聚集和细胞外信号调节激酶-1／2（ERK1／2）通路活化的影响。方法用鱼藤酮处理神经元样分化的PCI2细胞，四甲基偶氮唑盐法检测细胞活力，免疫荧光法检测α-突触核蛋白聚集，免疫细胞化学法检测ERK1／2通路活化情况。结果鱼藤酮处理后细胞活力呈时间依赖性下降；磷酸化ERK1／2水平逐渐升高（P均〈0．01）；处理16h后细胞内出现α-突触核蛋白聚集体，24h后进一步增多。结论鱼藤酮可诱导α-突触核蛋白聚集和ERK1／2通路活化，参与多巴胺能神经元损伤。     

冠心病不稳定斑块凋亡相关基因表达紊乱的机制研究

目的探讨冠心病不稳定斑块中凋亡相关基因的表达情况及调控机制。方法86例冠心病患者，其中不稳定组42例，稳定组44例。取两组冠脉斑块标本，Northern Blot和Western Blot检测Bax、caspase-3和凋亡相关蛋白激酶（DAPK）的表达水平，并与正常对照组进行比较。结果对照组和稳定组的caspase-3表达水平基本类似（P〉0．05），不稳定组约为其他组的2倍（P〈0．01）；不稳定组的Bax和DAPK表达达到对照组的3倍和6倍（P〈0．01）。对照组冠脉平滑肌细胞（VSMC）置于LDL中培养，细胞凋亡率显著上升（P〈0．01）；caspase-3的表达上升幅度为正常状态的1．5倍（P〈0．05），Bax和DAPK的上升均达到正常细胞的3倍（P〈0．01）。结论冠状动脉粥样硬化（CAS）组织中，高浓度LDL诱发凋亡相关基因表达紊乱是导致细胞凋亡失衡的重要原因，其中Bax和DAPK可能是这一调控途径中的关键分子。     

外源性Nurr1基因过表达促进6-羟基多巴胺诱导的SK-N-SH细胞凋亡

目的 研究Nurr1基因过表达在6-羟基多巴胺（6-OHDA）选择性诱导多巴胺（DA）能神经元特异性损伤中的作用。方法 ①不同浓度6-OHDA作用不同时间，比较细胞形态；②细胞经6-OHDA作用后，经流式细胞术（FCM）检测细胞周期分布比例；③不同浓度6-OHDA作用不同时间，经AnnexinV／PI双染后，运用共聚焦显微镜观察细胞凋亡情况，获得毒素诱导凋亡的最适剂量（75μmoL／L）和时间（12h），运用FCM检测两株细胞早期凋亡比例。结果 ①经6-OHDA处理后，倒置相差显微镜观察，结果显示SK-N-SH／Nurrl细胞形态损伤早于SK-N-SH细胞，且损伤程度明显；②细胞周期结果显示，6-OHDA诱导SK-N-SH细胞G2／M期细胞比例下降，而SK-N-SH／Nurr1细胞S期细胞比例下降；③经Annexin V／PI双染联合FCM检测早期凋亡比例结果显示，SK-N-SH／Nurr1细胞凋亡比铡明显高于SK-N-SH细胞（P〈0．05）。结论外源性Nurr1基因过表达促进了6-OHDA诱导的SK-N-SH／Nurr1细胞凋亡，Nurr1可能与SK-N-SH／Nurr1细胞对6-OHDA损伤敏感性增强有关。     

脑出血神经元凋亡及P53、caspase-3蛋白表达与中风闭脱证的关系

目的 探讨脑出血（ICH）中风闭脱证与细胞凋亡及相关基因P53、caspase-3蛋白表达和临床神经功能缺损积分值的关系。方法 采用TUNEL技术观测细胞凋亡形态学变化及细胞凋亡率；采用免疫组化法观察P53、caspase-3蛋白表达变化；按照《脑卒中临床神经功能缺损程度评分标准》评分。结果 ICH组血肿周围组织细胞凋亡率及P53、caspase-3蛋白表达升高，与正常对照组比较差异有统计学意义（P〈0．01）；ICH中风阴闭、阳闭、脱证各组之间血肿周围组织细胞凋亡率及P53、caspase-3蛋白表达、临床神经功能缺损积分差异有统计学意义（P〈0．01）。结论脑组织细胞凋亡及P53、caspase-3蛋白表达与ICH有关，能反映ICH脑实质的损害程度，表明细胞凋亡参与了脑出血后继发性神经细胞损伤，P53、caspase-3表达对细胞凋亡具有调控作用。     

塞来昔布对嘌呤氨基核苷诱导的足细胞凋亡的影响

以条件永生性小鼠足细胞株为研究对象，分为正常对照组（CON组）、嘌呤霉素氨基核苷组（PA组）、地塞米松组（DEX组）、塞来昔布组（CELE组）、地塞米松＋嘌呤氨基核苷组（DEX＋PA组）、塞来昔布＋嘌呤氨基核苷组（CELE＋PA组）。在0、8、24和48h检测足细胞凋亡水平、caspase-3蛋白酶活性水平及凋亡过程中p53水平。结果显示，与CON组比较，CELE、DEX组凋亡率无明显变化，PA组凋亡率明显增高（P〈0．01），而CELE＋PA、DEX＋PA组凋亡率较PA组明显下降（P均〈0．05）。PA组24h时，p53表达较CON组明显增多（P〈0．01），用CELE、DEX干预后明显降低（P均〈0．05）。认为塞来昔布可以抑制PA诱导的足细胞凋亡，且p53参与了PA诱导的足细胞凋亡过程。     

绿茶多酚对帕金森病大鼠黑质多巴胺能神经元的保护作用及机制

目的探讨绿茶多酚对偏侧帕金森病（PD）大鼠黑质多巴胺能神经元的保护作用及机制,为其临床应用提供依据。方法将SD大鼠随机分为对照组、6-羟多巴胺（6-OHDA）组和绿茶多酚组各12只,后两组于纹状体内立体定向注射6-OHDA制作PD模型,绿茶多酚组在造模前5d予绿茶多酚（400mg／kg）灌胃,共2周。造模4周后免疫组化法观察黑质多巴胺能神经元数量,比色法观察中脑氧化应激水平。结果绿茶多酚组、6-OHDA组损毁侧黑质致密部多巴胺能神经元数量显著减少,但绿茶多酚组显著高于6-OHDA组（P〈0．01）;6-OHDA组损毁侧氧化应激水平明显高于绿茶多酚组（P〈0．01）。结论绿茶多酚对偏侧PD大鼠模型的多巴胺神经元损伤有明显保护作用,其机制可能为抑制氧化应激反应。     

血管紧张素Ⅱ激活内质网应激促进大鼠血管平滑肌细胞凋亡的研究

目的:探讨内质网应激(ERS)在血管紧张素Ⅱ(Ang Ⅱ)诱导大鼠血管平滑肌细胞(VSMC)凋亡中的作用。方法:Ang Ⅱ诱导大鼠VSMC凋亡模型,并应用ERS抑制剂干预,采用流式细胞仪检测VSMC凋亡率;Western Blot检测ERS相关凋亡因子的表达变化。结果:不同浓度Ang Ⅱ(1、10、50μM)均可诱导VSMC凋亡,其总凋亡率(23.1±5.5)%、(30.0±2.5)%、(55.4±3.3)%与对照组(10.9±2.5)%相比显著升高(P0.05);Ang Ⅱ可诱导大鼠VSMC细胞ERS相关凋亡因子表达升高,与对照组相比,CHOP及Caspase12水平分别升高3.15倍和2.35倍(P0.05);与Ang Ⅱ刺激组相比,应用PERK通路抑制剂可显著降低Ang Ⅱ诱导的CHOP和Caspase12表达水平(P0.05),并降低Ang Ⅱ诱导的总凋亡率((14.6±2.7)%,P0.05)。结论:Ang Ⅱ可通过激活内质网应激CHOP和Caspase12通路诱导大鼠VSMC凋亡。     

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.     

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.     

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.     

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.     

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。     

Study of constancy of hydrogen-consuming flora of human colon

The constancy of the hydrogen consuming flora of the human colon was studied in 15 healthy subjects via two measurements obtained 18 to 36 months apart. Hydrogen disappearance rate and the major products of H₂-consuming bacteria, methane and sulfide, were measured during incubation of fecal homogenates with excess hydrogen and sulfate. In 11/15, the hydrogen consumption rate and the predominant hydrogen-consuming pathway (methanogenesis, sulfate reduction, or neither) remained constant. However, major shifts in these pathways were observed in four subjects, with two losing and two gaining the ability to produce methane. Methanogenesis was associated with the highest hydrogen consumption rate. This study demonstrates that clinically unrecognizable, major alterations of the colonic flora occur in healthy subjects. Understanding of the factors responsible for these alterations might allow for therapeutic manipulation of the colonic flora.Supported in part by the Department of Veterans Affairs and NIDDKD RO1 DK 13309-25.