日本血吸虫童虫信号序列捕捉cDNA文库的构建及初步筛选

目的建立日本血吸虫童虫信号序列捕捉cDNA(SST-cDNA)文库,筛选日本血吸虫病潜在的疫苗候选分子。方法抽提日本血吸虫童虫mRNA,并转录合成cDNA。将纯化的cDNA插入到载体pAPtag2中,构建SST-cDNA文库。将cDNA文库质粒转染到COS7细胞中进行蛋白表达,通过检测转染细胞中胎盘碱性磷酸酶(PLAP)的活性来判定插入的血吸虫童虫cDNA片段中是否带有信号序列。测定阳性克隆中外源性cDNA的核苷酸序,并通过Blast与Gen-Bank、EST数据库进行比对确定其性质。采用快速扩增cDNA未端序列方法获得基因的全长cDNA序列。用SignalP3.0、TMPred、TargetP服务器对带有信号序列基因的特征进行分析和预测。结果日本血吸虫童虫cDNA被插入到真核表达载体pAPtag2中,成功构建日本血吸虫童虫SST-cDNA文库。限制性内切酶分析表明SST-cDNA文库的容量为5×106cfu。SST-cDNA文库质粒转染到COS7细胞中后,通过近缘筛选获得21个带有信号肽cDNA序列,其中4个为未知新基因序列,5个为已知功能基因,12个与血吸虫EST序列同源。获得了8个基因的全长cDNA序列,演绎氨基酸序列特征分析表明,其中5个为膜结合蛋白基因,3个为外分泌蛋白基因。结论本研究成功构建了日本血吸虫童虫SST-cDNA文库,初步筛选获得了8个带信号肽的外分泌或膜结合蛋白分子基因。     

土耳其斯坦东毕吸虫磷酸丙糖异构酶基因的克隆及其序列分析

目的 克隆土耳其斯坦东毕吸虫磷酸丙糖异构酶的全长基因。方法 根据日本血吸虫和曼氏血吸虫磷酸丙糖异构酶基因的保守区设计引物，利用RT—PCR扩增出土耳其斯坦东毕吸虫磷酸丙糖异构酶基闪的大片段，再结合RACE技术分别得到磷酸丙糖异构酶基因的3’端和5’端，将3部分序列拼接后获得磷酸丙糖异构酶基因全长cDNA序列，并提交GenBank。结果 成功克隆了土耳其斯坦东毕吸虫磷酸丙糖异构酶基因全长cDNA序列并提交GenBank，登录号为DQ092331。结论 土耳其斯坦东毕吸虫磷酸丙糖异构酶基因全长cDNA的克隆为进一步表达及其生物学性能的分析提供了理论基础。     

日本血吸虫酪氨酸羟化酶基因的克隆和序列分析

目的 克隆并鉴定日本血吸虫酪氨酸羟化酶(TH)基因，探讨其他信号蛋白在信号传导中对TH的调节及其之间的分子作用机制，从而为血吸虫新型疫苗的设计和药物开发开辟新的途径。方法 以曼氏血吸虫的TH cDNA为模板设计引物，以日本血吸虫成虫mRNA为模板逆转录合成cDNA链，将扩增出的日本血吸虫TH蛋白编码基因序列，克隆入pGEM-T easy载体，用单酶双切法和DNA测序进行鉴定，并与曼氏血吸虫的TH基因序列进行同源性比较。结果 自日本血吸虫RNA经逆转录得到一长度为480bp的DNA片段，经测序与曼氏血吸虫TH的同源性为87％。结论 成功扩增出日本血吸虫TH的中间编码区域，为进一步扩增日本血吸虫TH基因全长以及后续实验奠定了基础。     

cDNA文库基础上运用热启动聚合酶链反应末端延伸快速分离全长cDNA序列

建立一种cDNA文库基础上运用热启动聚合酶链反应末端快速扩增分离人类新基因全长cDNA序列的新技术。以文库载体序列与待扩增目的基因片段为模板各设计引物进行热启动聚合酶链反应扩增，从人胎肝cDNA文库中获得氧化型低密度脂蛋白诱导U937细胞泡沫化过程中差异表达序列标签片段（FRG4）的全长cDNA序列，聚合酶链反应产物克隆到pGEM-T载体中，并测序鉴定，从而成功获得FRG4的全长cDNA序列，该技术是一种快速有效的分离人类新基因全长cDNA序列的方法。     

日本血吸虫酪氨酸羟化酶基因的克隆和序列分析

目的　克隆并鉴定日本血吸虫酪氨酸羟化酶(TH)基因,探讨其他信号蛋白在信号传导中对TH的调节及其之间的分子作用机制,从而为血吸虫新型疫苗的设计和药物开发开辟新的途径。　方法　以曼氏血吸虫的 TH cDNA为模板设计引物,以日本血吸虫成虫mRNA为模板逆转录合成cDNA链,将扩增出的日本血吸虫TH蛋白编码基因序列,克隆入 pGEM T easy载体,用单酶双切法和DNA测序进行鉴定,并与曼氏血吸虫的TH基因序列进行同源性比较。　结果　自日本血吸虫RNA经逆转录得到一长度为 480 bp的 DNA片段,经测序与曼氏血吸虫 TH的同源性为 87%。结论　成功扩增出日本血吸虫TH的中间编码区域,为进一步扩增日本血吸虫TH基因全长以及后续实验奠定了基础。     

基于cDNA末端快速扩增的刚地弓形虫核仁G蛋白-1的克隆

目的　克隆弓形虫核仁G蛋白- 1(NOG1)基因的全长cDNA序列。方法　根据NCBIGeneBank中登录的弓形虫NOG1基因的唯一EST序列设计引物,利用cDNA 5’-末端快速扩增法(5’-RACE)和cDNA 3’-末端快速扩增法(3’-RACE)分别对已知序列进行延伸,将扩增获得的3’-端和5’-端未知序列与位于中间位置的已知序列进行拼接,然后经Blast检验全长序列的正确性。结果　5’-RACE扩增获得3个不同长度的片断,最长片断全长12 87bp ,去除引物和夹杂的已知序列后,通过5’-RACE在5’端扩增获得未知序列为1196bp。3’-RACE扩增后得到一条特异性条带,位于大约1.7kb左右,测序全长为16 34bp ,去除引物和已知序列,经3’-RACE扩增获得的3’-端未知序列为12 0 4bp。Blast全长为316 7bp的3段拼接序列,发现其覆盖NOG1基因cDNA的完整ORF或者CDS序列(6 5 0bp 2 80 9bp)。推导翻译出的蛋白质一级结构包含719个氨基酸(aa) ,在所有已发现物种的NOG1中,弓形虫NOG1基因的cDNA和相应的aa序列都是最长的。该序列已登录NCBIGeneBank ,核酸序列登录号AY6 86 734,对应蛋白质的aa序列登录号为AAT94 2 90。结论　本研究首次克隆获得了弓形虫NOG1基因的全长cDNA序列,并对相应的aa序列进行了推导翻译和简单分析。     

日本血吸虫(中国大陆株)磷酸丙糖异构酶(SjC-TPI)基因克隆与序列分析

根据已知日本血吸虫菲律宾株TPI序列，设计合成一对5’端分别带有BamH1，Sal1酶切位点的引物P1和P2，制备日本血吸虫成虫（中国大陆株）mRNA，采用反转录聚合酶链反应技术，从mRNA中扩增出日本血吸虫中国大陆株TPI基因片段，序列分析表明，日本血吸虫（中国大陆株）TPI基因开放阅读框架全长为759bp，与曼氏血吸虫TPI基因的同源性为84％。与日本血吸虫（菲律宾株）TPI基因的同源性为99.7％。     

日本血吸虫(大陆株)卵壳蛋白编码基因的克隆和序列测定

目的：克隆和鉴定日本血吸虫虫卵卵壳蛋白（SjEP）编码基因，以寻找血吸虫新的候选疫苗和诊断分子。方法：设计合成引物，分别以日本血吸虫雌、雄成虫cDNA第一链为模板，用PCR法从中扩增出SjEP基因编码序列，将其克隆入pGEM-T载体，用双酶切、以质粒为模板进行PCR扩增和测序进行鉴定。结果：PCR法从雌虫cDNA中扩增出大小为624bp SjEP基因编码序列，重组质粒pGEM-SjEP经双酶切、以质粒为模板进行PCR扩增，均可获得一条与PCR产物一致的DNA片段，序列测定结果表明具有一个长度为624bp的完整开放阅读框，与日本血吸虫（菲律宾株）虫卵卵壳蛋白核苷酸序列有高度同源性（99．9％）。结论：本实验成功地克隆了SjEP编码基因，并进行了序列测定，为进一步研究提供了条件。     

中华按蚊防御素基因 cDNA序列和基因组序列的克隆及鉴定

目的 克隆中华按蚊防御素基因全长cDNA序列及基因组序列,并对其进行鉴定和生物信息学分析。方法 根据已发表的埃及伊蚊和冈比亚按蚊等的防御素基因序列设计引物,提取中华按蚊总RNA并构建其基因组文库,分别进行多轮RT-PCR和巢式PCR扩增,将所得片段进行克隆、测序,并应用相关生物信息学软件对序列进行鉴定和分析。结果 从中华按蚊基因组文库中扩增出完整的防御素基因组序列(由两个外显子和一个内含子组成)以及5′端和3′端的非编码序列(UTR)片段,总长度为2 256 bp;从中华按蚊总RNA中扩增出大小为324 bp的cDNA片段,经测序证实为中华按蚊防御素基因全长cDNA序列,其开放阅读框共编码107个氨基酸,成熟肽部分具有40个氨基酸残基。结论 首次克隆出中华按蚊防御素基因全长cDNA序列及基因组序列,为进一步的功能研究奠定了基础。     

肝细胞癌高表达基因片段P02的全长RACE扩增和序列分析

目的扩增肝细胞癌高表达基因基因P0 2的全长cDNA序列,并利用生物信息学知识对其进行分析,为进一步研究该基因在肝细胞癌发生、发展中的作用奠定基础。方法运用SMARTRACE(RapidAmplificationofcDNAEnds)技术扩增P0 2的5’和3’末端cDNA ,对RACEPCR产物作TA克隆,碱裂解法抽提阳性克隆质粒,酶切和PCR验证目的片断的插入,Sanger双脱氧链中止法测序,利用BLAST、基因探索者等生物学软件对序列进行分析、拼接,获得全长cDNA ,利用在线预测软件预测该基因的生物学功能。结果获得865bp的全长cDNA序列,其开放读码框长172个氨基酸,与TPT1基因高度同源,是一个生理功能尚未完全明了、与生长相关的蛋白质,蛋白质结构和功能预测提示该基因产物可能是一个位于细胞膜的与生长相关的裂解酶。结论在肝细胞癌组织中成功克隆扩增了长865bp的P0 2全长cDNA序列,为进一步功能研究奠定基础。     

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.     

Angiographic diagnosis of the degree of serosal invasion of carcinoma of the colon

Angiography using Prostaglandin El^® was performed on 38 patients with carcinoma of the colon in order to diagnose the degree of serosal cancer invasion. The findings at angiography were classified into four groups:1) AG-S3, abnormal change (irregularity and/or encasement) up to marginal vessels; 2) AG-S2, abnormality up to vasa recta; 3) AG-S1, abnormality of penetrating branches of vasa recta within the wall of the colon; and 4) AG-S0, no distinct findings of abovementioned vessels. These angiographic findings were compared with both macroscopic and microscopic serosal cancer invasion. Angiographic diagnosis is in accord with the macroscopic findings in 84.2 percent of cases. Angiographic diagnosis is in accord with the microscopic findings in 32.4 percent of cases. Macroscopic findings confirm the angiographic diagnosis precisely but the conflict with microscopic findings should not be overlooked. This may be the result of inflammatory change, adhesion, and fibrosis around carcinoma of the colon.     

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.