砷对人胶质瘤细胞周期调控相关基因表达的影响

目的比较三氧化二砷（As2O3）对两种不同p53表型胶质瘤细胞U87MG、T98G细胞周期调控相关基因表达的影响。方法应用功能分类细胞周期基因芯片技术，筛选出As2O3作用于两种胶质瘤细胞U87MG、T98G后与细胞周期调控表达有差异的基因，并对3条有共性表达的基因进行Western印迹分析；四甲基偶氮唑蓝（MTT）法检测细胞生长抑制率；流式细胞仪分析细胞周期改变。结果MTT分析表明：As2O2对2个细胞系的生长有抑制作用，且抑制率具有浓度和时间的依赖性，砷作用72h后，U87MG和T98G细胞系的生长抑制率为50％（IC50）时，其浓度分别为1．78、3．55μmol／L。As2O3可以将U87MG和T98G细胞周期阻滞于G0／G1和G2／M期。U87MG细胞在加入As2O3后，差异表达2倍以上的基因共11条，表达上调的有5条，下调的有6条。T98G细胞在加入As2O3后，差异表达的基因共13条，表达上调的有9条，下调的有4条。Western分析显示，As2O3能上调2个胶质瘤细胞系的P53蛋白水平，同时降低细胞周期蛋白B1、D1表达。结论As2O3通过将细胞周期阻滞于G0／G1和G2／M期来抑制U87MG和T98G细胞的增殖；p53、CyclinB、CylinD等基因表达与As2O3诱导胶质瘤细胞周期阻滞的发生有关。     

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。     

沉默Survivin人耐药肺癌细胞凋亡相关基因表达变化及临床意义

目的探讨沉默Survivin基因后人耐药肺腺癌(A549DDP)细胞系凋亡相关基因表达变化及临床意义。方法应用RT-PCR法及Western印迹法比较Survivin在人肺腺癌A549细胞系及其耐药A549DDP细胞系的表达水平;通过小分子RNA干扰沉默Survivin基因,并采用微阵列PCR基因芯片(PCR-Array)技术比较沉默Survivin后A549DDP细胞凋亡相关基因表达水平变化。结果 (1)Survivin mRNA、Survivin蛋白在人耐药非小细胞肺癌细胞系呈现高表达。(2)沉默Survivin后A549DDP细胞部分抗/促凋亡基因表达发生改变,促凋亡基因TP53、CASP3、CASP7呈高表达,抗凋亡基因bcl-2、TRAF1、BFAR呈低表达。结论 (1)Survivin与部分抗/促凋亡基因共同参与了非小细胞肺癌耐药的发生。(2)封闭Survivin可改变耐药肺癌细胞的促/抗凋亡基因的表达,Survivin可能作为上游调控点参与调控部分促/抗凋亡基因组的表达。(3)Survivin可能成为逆转肺癌耐药的一个新靶点。     

ARHI基因转染PANC1细胞后对细胞凋亡和自噬相关基因表达谱的影响

目的 观察ARHI基因转染PANC1细胞后对细胞凋亡和自噬相关基因mRNA表达谱的影响.方法 采用脂质体法将表达ARHI基因的质粒pIRES2-EGFP-ARHI、空质粒pIRES2-EGFP转染胰腺癌PANC1细胞.采用基因芯片RT2ProfilerTM PCR Array行实时定量PCR,分析转染细胞的基因表达谱,包括84个与凋亡和自噬相关基因.结果 ARHI基因转染组PANC1细胞有9个基因mRNA表达下调,38个基因mRNA表达上调,37个基因mRNA表达变化无意义.在与凋亡相关的基因中有8个促凋亡基因表达显著上调（＞6倍）,主要为TNFR/TRFSR家族基因（TNFSF8、TNFRSF10B、TNFRSF11B、TNFRSF9）、CIDE家族基因（DFFA）、CASP家族基因（CASP10、CASP8）和死亡结构域家族基因（DAPK1）,其中以DAPK1上调尤为明显,达42.83倍;抗凋亡基因中3个基因（CD27、BCL2L10、BIRC4）表达显著上调（＞6倍）,3个基因（BCL2、BAD、BAG4）表达轻度上调（＞2倍）,1个基因（BCL2L1）表达轻度下调（＜-2倍）.在与自噬相关的基因中3个促自噬基因（TNFRSF10B、DAPK1、CASP10）表达显著上调（＞6倍）,4个基因（TNFRSF10A、FADD、TP53、TP53 BP2）表达轻度上调（＞2倍）;3个抑制自噬基因（BCL2、CASP8、FAS）表达轻度上调（＞2倍）,1个基因（MCL1）表达轻度下调（＜-2倍）.结论 ARHI基因显著上调细胞凋亡及自噬重要调控基因Caspase-8和DAPK1.     

基因芯片技术研究原发性干燥综合征患者唇腺基因表达

目的应用基因芯片技术筛选原发性干燥综合征（SS）患者与健康对照唇腺组织中的差异表达基因，探讨其可能的发病机制。方法实验组和对照组各3例，分别制成eRNA探针，与基因芯片杂交，筛选出差异表达基因。结果实验组与对照组显著差异表达基因181个，其中上调表达128个，下调表达53个。涉及细胞因子及受体相关基因、凋亡相关基因等。结论SS的发生发展受多种基因调控，差异基因表达谱的建立较全面地反映了SS的分子生物学概貌。     

IC53基因在血管及动脉粥样硬化斑块中的表达谱分析

目的 IC53基因是我室1998年从成人主动脉文库克隆到的新基因，前期研究发现该基因与心衰发展相关。为进一步分析IC53基因与血管性疾病的关系，我们分析了IC53基因在人血管及动脉粥样硬化斑块中的表达。方法 利用本实验室自备的IC53单克隆抗体，通过免疫组化分析IC53基因的表达情况。结果 IC53基因主要在血管内皮表达，在平滑肌中未检测到信号，在外膜低表达。在动脉粥样硬化斑块中，IC53基因在巨噬细胞中表达。结论 IC53基因可能通过内皮细胞及巨噬细胞在心血管疾病中发挥作用。     

C/EBPα基因在大鼠肝星状细胞内的表达及意义

目的 初步探讨CCAAT增强子结合蛋白α(C／EBPα)在肝星状细胞(HSC)激活过程中的作用。方法 采用免疫细胞化学、western blot及RT-PCR检测原代培养不同时间段HSC内C／EBPd蛋白和mRNA的表达，以及α平滑肌肌动蛋白(α SMA)、结蛋白、基质金属蛋白酶-2(MMP2)mRNA、Ⅰ型前胶原(α1)mRNA的表达；Lipofect AMINE2000介导的瞬时转染方法将pcDNA3．1(-)-C／EBPα真核表达质粒转染入激活的HSC内，采用免疫细胞化学方法鉴定转染成功及转染后HSC内增殖细胞核抗原(PCNA)的表达；在相差显做镜下观察HSC在原代培养过程中形态的改变。结果 原代正常HSC中可以检测到C／FBPα mRNA和蛋白的表达，蛋白位于细胞质和细胞核内，但主要位于细胞质内，且除新鲜分离(0d)组以外，随着HSC培养至2、4、7、10d，C／EBPα蛋白和mRNA的表达呈逐步下降的趋势，而α-SMA、MMP2和Ⅰ型前胶原(α1)的表达则逐步增强；转染后24h，目的基因转染组HSC内C／EBPα的表达明显强于空载体对照组，而PCNA的阳性细胞数较空载体对照组明显减少；转染后36h，目的基因转染组细胞几乎全部死亡，残存的细胞形态变细，体积缩小，而空载体对照组细胞仍存括。结论 C／EBPα基因可能参与HSC的激活调控机制，且C／EBPα过表达对HSC的增殖存在抑制作用。     

NF-E2对红系造血相关基因表达的影响

目的探讨红系特异性转录因子NF—E2对红系造血相关基因表达的影响。方法应用基因芯片方法检测转染p45NF—E2基因后红白血病细胞株HB22．2中基因表达的变化；通过改变培养条件诱导红白血病细胞株HB60—5向成熟细胞分化，检测诱导分化过程中NF—E2等红系造血相关基因表达变化。结果p45NF-E2缺陷细胞HB22．2转染外源p45NF-E2后，血红蛋白基因及Gata-1、EDRF基因表达上调，Fli基因表达下调；HB60-5向成熟细胞分化过程中，随着p45NF—E2的表达上调，同样出现Gata-1和EDRF表达增强，FIi表达减弱。结论NF—E2能对多种红系相关基因表达调控。从而促进红系分化。     

小鼠卵透明带3基因疫苗体外瞬时表达的研究

运用基因重组技术，将小鼠ZP3 cDNA片段克隆到真核表达载体pCDNA3上，成功构建了pCDNA3-ZP3真核表达质粒，转染HeLa细胞后，ELISA法检测到pCDNA3-ZP3真核表达质粒在HeLa细胞瞬时表达系统中能够特异性表达ZP3蛋白，结果表明pCDNA3-ZP3真核表达质粒能够作为基因疫苗用于小鼠免疫接种的研究。     

胸腺肽α1对人淋巴细胞抗感染相关的信号转导分子的表达

目的 用基因微矩阵技术研究胸腺肽α1作用于人外周血淋巴细胞后，细胞内与抗感染相关的信号转导分子的基因表达谱变化。方法 取正常外周血600m1分离淋巴细胞，实验组加Tα1(0．1μg／m1)刺激，对照组不加Tα。培养48h后，收获细胞、抽提细胞总RNA并纯化mRNA。逆转录反应将对照组和实验组的cDNA分别标记上荧光分子Cy3和Cy5，与信号转导表达谱基因微矩阵杂交后激光扫描、图像分析。结果 筛选出61种Cy5／Cy3比值大于2或小于0．5的差异表达基因，其中与感染相关的有cAML，P13-K，TSP2，HM74，PIAS，PTP，PSTP等7种基因表达上调，MEK2，MAP3K，Lok等3种基因表达发生下调。结论 Tα1的直接抗感染作用与这些信号转导分子的基因表达变化有关。     

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.     

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。     

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。     

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.     

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.